Interleukin-21 peptidomimetics

Abstract

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

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 378,797, filed on October 7, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing Statement

[0004] A computer-readable form of the sequence listing is submitted electronically with this application and is incorporated herein by reference in its entirety. The sequence listing is contained in a file created on October 6, 2023, with the file name "22-1530-WO.xml" and a file size of 892,905 bytes.

[0005] Federal Funding Statement

[0006] This invention was made with government support under Grant No. R01CA240339 from the National Cancer Institute and Grant No. R01AI160052 from the National Institute of Allergy and Infectious Diseases. The U.S. Government has certain rights in this invention. Background Art

[0007] Interleukin-21 (IL-21) is a pleiotropic cytokine that plays an important role in innate and adaptive immune responses. IL-21 is primarily produced by CD4+ T cells and induces the differentiation of various immune cells. IL-21 is involved in regulating immune responses to infection, cancer, and autoimmune diseases, and has become a target of clinical research in recent years. Clinical trials involving IL-21 have been conducted for various types of cancer, including melanoma, renal cell carcinoma, ovarian cancer, and non-Hodgkin's lymphoma. In this context, IL-21 is being studied as a single agent or in combination with other immunotherapeutic agents (such as checkpoint inhibitors or cancer vaccines). Due to poor stability, low cross-reactivity with human IL-21 in mouse models, and lack of engineering feasibility, fully utilizing the potential of natural IL-21 for therapeutic purposes is challenging. The limited efficacy of human IL-21 in mouse models further hinders the predictive evaluation of the toxicity and activity of IL-21-based therapeutic candidates. Human and mouse cross-reactive IL-21 mimetic proteins provide a promising solution for cytokine-based immunotherapy using IL-21. Summary of the Invention

[0008] In one aspect, the present disclosure provides a polypeptide comprising domains X2, X3, and optionally X4, wherein:

[0009] (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933);

[0010] (b) X3 is 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 (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934) or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO:937), wherein one, two or all three of the following conditions are true relative to SEQ ID NO:937: residue 4 is Y, residue 6 is F, and / or residue 9 is D; and

[0011] (c) X4 is optional, and when present, comprises a helical structure;

[0012] wherein X2, X3 and X4, when present, can be in any order in the polypeptide;

[0013] wherein an amino acid linker may be present between any of the domains; and

[0014] The polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0015] In one embodiment, the polypeptide comprises domains X2, X3 and X4, wherein:

[0016] (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933);

[0017] (b) X3 is 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934); and

[0018] (c) X4 is 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935);

[0019] wherein X2, X3 and X4 can be in any order in the polypeptide;

[0020] wherein an amino acid linker may be present between any of the domains; and

[0021] The polypeptide is capable of binding to IL-21 receptor (IL-21Rα, CD360) and γc, CD132 receptor.

[0022] In another embodiment,

[0023] (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and

[0024] (b) X3 is 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 KRFYIFMQDLIDIVTHVKRE (SEQ ID NO:937), wherein one, two or all three of the following conditions are true relative to SEQ ID NO:937: residue 4 is Y, residue 6 is F, and / or residue 9 is D;

[0025] wherein X2 and X3 can be in any order in the polypeptide;

[0026] wherein an amino acid linker may be present between the domains; and

[0027] The polypeptide can bind to the IL-21 receptor (IL-21Rα, CD360) but cannot bind to the γc and CD132 receptors at the same time.

[0028] 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, when present, can be in any order in the polypeptide.

[0029] In another embodiment, the present disclosure provides a fusion protein comprising:

[0030] (a) the polypeptide of any embodiment herein; and

[0031] (b) one or more functional domains.

[0032] In other embodiments, the polypeptide or fusion protein comprises an amino acid sequence comprising:

[0033] (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 shown in Tables 1-6; or

[0034] (b) 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, 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, 716-717 17-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; or

[0035] (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 or

[0036] (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.

[0037] In another aspect, the present disclosure provides a conditionally active IL-21 receptor binding protein comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present simultaneously in the same fusion protein, wherein the first polypeptide component and the second polypeptide component collectively comprise domains X2, X3, and optionally X4, as defined in any one of claims 1 to 34, wherein:

[0038] (i) the first polypeptide component comprises at least one of X2, X3 and X4 (when present), but does not comprise each of X2, X3 and X4 (when present); and

[0039] (ii) the second polypeptide component comprises each of X2, X3 and X4 (when present) that are not present in the first polypeptide component;

[0040] The first polypeptide component and the second polypeptide component alone are not active receptor binding proteins, and the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein capable of binding to an IL-21 receptor (IL-21Rα, CD360).

[0041] In one embodiment,

[0042] (i) the first polypeptide component comprises at least one of X2, X3 and X4, but does not comprise each of X2, X3 and X4; and

[0043] (ii) the second polypeptide component comprises each of X2, X3 and X4 that is not present in the first polypeptide component;

[0044] The first polypeptide component interacts with the second polypeptide to form an active IL-21 receptor binding protein that can bind to the IL-21 receptor (IL-21Rα, CD360) and can bind to the γc, CD132 receptor.

[0045] In another embodiment, the conditionally active IL-21 receptor binding protein comprises a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present simultaneously in the same fusion protein, wherein the first polypeptide component and the second polypeptide component collectively comprise domains X1, X2, X3, and X4, as defined in any embodiment herein; wherein:

[0046] (i) the first polypeptide component comprises at least one of X1, X2, X3 and X4, but does not comprise each of X1, X2, X3 and X4; and

[0047] (ii) the second polypeptide component comprises each of X1, X2, X3 and X4 that are not present in the first polypeptide component;

[0048] The first polypeptide component interacts with the second polypeptide to form an active IL-21 receptor binding protein that can bind to the IL-21 receptor (IL-21Rα, CD360) and can bind to the γc, CD132 receptor.

[0049] The present disclosure also provides: a recombinant nucleic acid encoding a polypeptide, a first polypeptide, or a second polypeptide according to any embodiment of the present invention; an expression vector comprising a recombinant nucleic acid according to the present invention operably linked to a promoter; a recombinant host cell comprising a nucleic acid, an expression vector, a polypeptide, a first polypeptide, and / or a second polypeptide according to any embodiment of the present invention; a pharmaceutical composition comprising a polypeptide, a fusion protein, a first polypeptide, a second polypeptide, a recombinant nucleic acid, an expression vector, and / or a recombinant host cell according to any embodiment of the present invention and a pharmaceutically acceptable carrier; and a method for treating cancer or for regulating an immune response in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1. De novo IL-21 agonistic mimetics can bind to the IL-21 receptor (IL-21R; CD360) and the common γ receptor (γ c ; CD132) to achieve receptor heterodimerization. (A) Schematic diagram of the design, screening, optimization and characterization of IL-21 mimetics. (B) IL-21 agonist mimetics bind to hIL-21R and hγ cComposite model. (C) SDS-PAGE of human IL-21 (lane 2), mouse IL-21 (lane 3), and 21h10 (SEQ ID NO: 24) (lane 4) using the Precision Plus Protein Kaleidoscope ladder (lane 1). (D) Expression of 21h10 revealed a monomeric peak at the expected retention volume in size exclusion chromatography using an S75 Plus column. (E) Circular dichroism spectroscopy revealed the presence of a helical secondary structure in 21h10 (SEQ ID NO: 24). (F) A 222 nm wavelength scan of the thermal melt from 25°C to 95°C demonstrated the excellent thermal stability of 21h10 (SEQ ID NO: 24). The helical structure began to unfold at 65°C. (GJ) Example receptor binding affinity measurements of 21h10 (SEQ ID NO: 24) by biolayer interferometry, as described in the Methods section of the Examples. The measured Kd values ​​for this design and related designs are reported in Table 1.

[0051] Figure 2. Shuffling of helical domains for retopological and de novo cleavage of IL-21 agonistic mimetics. (A) Monomeric IL-21 mimetics can be circularly permuted or shuffled using their helical domains (H1-H4) to generate different topologies while maintaining their IL-21R (CD360) and common γ c (CD132) receptor interface. (B) can generate three split pairs, H1 / H234, H12 / H34 and H123 / H4 from the given topology of IL-21 mimetics. (C) the split fragment of IL-21 mimetics can be fused with anti-A nano antibody and anti-B nano antibody or other targeting domains (A and B are markers, and can be identical or different) respectively, to realize conditional activation only when two components are co-located, so that reconstruct active IL-21 mimetic structure. The mimetic of reorganization transmits IL-21 signal to target cell. (D) the 21h10 of three splits can be reconstructed into its original four-helix bundle structure, and by making IL-21R (CD360) and common γ c (CD132) receptors to heterodimerize to form the IL-21 receptor complex.

[0052] Figure 3. De novo IL-21 agonist mimetics can be fused with antibodies to achieve targeted cytokine delivery. (A) Schematic diagram of immunocytokines in a heavy chain:cytokine = 2:2 construct. Take atezolizumab fused to an IL-21 mimetic as an example. The stoichiometry can also be a 2:1 construct, which has knob / hole mutations in the heavy chain to achieve a specific heterodimer configuration; the heavy chain with the knob mutation fused to the cytokine can be coupled to another heavy chain with the hole mutation but without the cytokine fusion. (B) SDS-PAGE of the atezolizumab-21h10 fusion construct under non-reducing conditions, which shows a band around the expected size of 179.2 kDa. (C) The atezolizumab-21h10 fusion construct can bind to human IL-21R and human PD-L1 independently. (D) The atezolizumab-21h10 fusion construct can bind to human IL-21R and human PD-L1 simultaneously. Atezolizumab-21h10 fusion construct was at 10 nM, where human PD-L1 was immobilized to the streptavidin (SA) tip and associated with the Atezolizumab-21h10 fusion construct, and then associated with 50 nM human IL-21R, or vice versa.

[0053] Figure 4. De novo IL-21 antagonist mimetics can bind only to the IL-21 receptor (IL-21R; CD360) but not to γ c (CD132) combination. (A) five antagonistic hits (table 5, SEQ ID NO:627-631) of preliminary screening have shown that human IL-21R combines in flow cytometry. Use yeast surface display to identify five hits in conjunction with human IL-21R. Use 100nM human IL-21R to mark the yeast cells of expression design. All these hits cannot be combined with γ c Binding, and therefore classified as an antagonist. (B) Using Rosetta TM , site-saturation mutagenesis (SSM) data or ProteinMPNN to design other antagonistic mimetics from agonistic IL-21 mimetics to remove γ c interface.

[0054] Figure 5 Site-saturation mutagenesis study of the agonist 21JC15. Using yeast surface display, mutations at all positions on 21JC15 (SEQ ID NO: 8) are shown as positive (black) and negative (white) mutations that increase and decrease binding affinity to the IL-21 receptor, respectively. The darker the color, the more likely the mutation is to increase binding to IL-21R (CD360) and common γ cBinding affinity of the human IL-21 receptor (CD132). The library was sorted against the human IL-21 receptor to populate the data. This study revealed residue-level information on how mutations affect binding affinity to the receptor.

[0055] Figure 6 . Agonist design follows its binding to IL-21R or IL-21 receptor complex (IL-21R / γ c ) varies with the interface affinity of IL-21Ra binding and ternary complex binding (binding to γ ​​in the presence of IL-21Ra) c The X-axis represents the binding affinity to hIL-21R or mIL-21R, while the Y-axis represents the binding affinity to hIL-21R / hγ c or mIL-21R / mγ c The binding affinity of mIL-21R / mγ is expressed in molar units [M]. c Affinity less than 10 -4 The construct of M was filled with 10 -4 M. Numbers 1 to 21 correspond to 21A1 to 21A21, respectively (Table 1, SEQ ID NOs: 164-184). DETAILED DESCRIPTION

[0056] All references cited are incorporated herein by reference in their entirety. In this application, unless otherwise indicated, the techniques used can be found in any of several well-known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition (RI Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, EJ Murray, ed., The Humana Press Inc., Clifton, NJ) and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0057] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0058] As used herein, amino acids are referred to by the following abbreviations: 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).

[0059] 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 when absent, is not considered when determining percent identity). Similarly, any N- or C-terminal histidine tag is optional and may or may not be present, and when absent, is not considered when determining percent identity.

[0060] Unless the context clearly dictates otherwise, all embodiments of any aspect of the disclosure can be used in combination.

[0061] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense and not in an exclusive or exhaustive sense; that is, in the sense of "including, but not limited to." Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this entire application and not to any particular portions of this application.

[0062] In a first aspect, the present disclosure provides a polypeptide comprising domains X2, X3 and optionally X4, wherein:

[0063] (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933);

[0064] (b) X3 is 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 (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934) or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO:937), wherein one, two or all three of the following conditions are true relative to SEQ ID NO:937: residue 4 is Y, residue 6 is F, and / or residue 9 is D; and

[0065] (c) X4 is optional, and when present, comprises a helical structure;

[0066] wherein X2, X3 and X4, when present, can be in any order in the polypeptide;

[0067] wherein an amino acid linker may be present between any of the domains; and

[0068] The polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0069] The inventors have demonstrated that the polypeptides of this aspect of the disclosure can bind to the IL-21 receptor and that some embodiments can act as agonists and some embodiments can act as antagonists. The polypeptides are ultrastable de novo IL-21 mimetic proteins that recapitulate the interaction of native IL-21 with the receptor and the biology of native IL-21 in humans and mice, making them well-suited for therapeutic use.

[0070] In one embodiment, the polypeptide comprises domains X2, X3 and X4, wherein:

[0071] (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933);

[0072] (b) X3 is 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934); and

[0073] (c) X4 is 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935);

[0074] wherein X2, X3 and X4 can be in any order in the polypeptide;

[0075] wherein an amino acid linker may be present between any of the domains; and

[0076] The polypeptide is capable of binding to IL-21 receptor (IL-21Rα, CD360) and γc, CD132 receptor.

[0077] The inventors have demonstrated that the polypeptide of this embodiment can act as an agonist, by specifically binding to the IL-21 receptor (IL-21Rα, CD360) and the common gamma chain receptor (γc, CD132) and promoting its dimerization, activating IL-21-like signaling and inducing cell differentiation in human and mouse cells. The designed polypeptide has shown anti-tumor effects in mouse MC38 adenocarcinoma models, B16F10 melanoma models, and pancreatic cancer models, and therefore can be used, for example, to treat cancer.

[0078] Agonist IL-21 mimetics are constructs that can not only bind to the IL-21 receptor (IL-21Rα, CD360) and the common γ chain receptor (γc, CD132) individually, but can also bind to both receptors simultaneously to allow potential downstream cell signaling through receptor heterodimerization. The ability to act as an agonist is identified by confirming that the mimetics bind to both IL-21R and γ simultaneously. c Receptor binding. c The affinity between receptors is very weak, γ c Receptor binding itself is difficult to detect, so targeting γ in the presence of IL-21R c Receptor binding assays will allow for the detection of IL-21R at concentrations of 1000 nM and 1000 nM γ c Agonist variants were identified at the highest concentration of the receptor.

[0079] In one embodiment, (a) X2 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: 933;

[0080] (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: 934; and

[0081] (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.

[0082] In another embodiment, (a) X2 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: 933;

[0083] (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: 934; and

[0084] (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.

[0085] In another embodiment, (a) X2 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: 933;

[0086] (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: 934; and

[0087] (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.

[0088] In one embodiment, (a) X2 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: 933;

[0089] (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: 934; and

[0090] (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.

[0091] In another embodiment, (a) X2 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: 933;

[0092] (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: 934; and

[0093] (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.

[0094] The inventors performed extensive site saturation mutagenesis (SSM), yeast surface display assays using flow cytometry, biolayer interferometry (BLI), and other functional studies described in the methods section of the examples to identify various substitutions that retain receptor binding activity (and therefore retain agonist activity). In one embodiment, the polypeptide has an amino acid selected from the following residues relative to X2 (SEQ ID NO: 933):

[0095] Residue 1: G, A, S, T, Q, D, E, R, K, and H;

[0096] residue 2: G, A, L, S, T, N, and Q;

[0097] residue 3: A, M, Y, W, N, Q, E, R, and K;

[0098] Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E and H;

[0099] residue 5: M, L, Q, E, and K;

[0100] residue 6: V, I, L, Y, R, and K;

[0101] Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E and R;

[0102] residue 8: A, L, F, S, T, E, and H;

[0103] residue 9: A, V, I, and L;

[0104] residue 10: E, R, and K;

[0105] residue 11: V, E, K, R, and H;

[0106] residue 12: A, I, and L;

[0107] Residue 13: V and I;

[0108] residue 14: S, E, K, and R;

[0109] Residue 15: A, I, M, L and E;

[0110] residue 16: C, A, and S;

[0111] Residue 17: N and R;

[0112] residue 18: V, S, N, E, K, R, and H;

[0113] residue 19: A, V, I, and L;

[0114] Residue 20: V and I;

[0115] Residue 21: W, S, E, K, and R; and

[0116] Residue 22: M and D.

[0117] The inventors have identified that residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) are present at the binding interface with IL-21Rα (CD360). In some embodiments, the polypeptide has one, two, three, or all four of the following residues relative to X2 (SEQ ID NO: 933):

[0118] (a) X2 residue 11 is K, E, or R;

[0119] (b) X2 residue 14 is R, E, or K;

[0120] (c) X2 residue 18 is V, R, H or K; and / or

[0121] (d) X2 residue 21 is R or K.

[0122] The inventors have identified that residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) are present at the binding interface with IL-21Rα (CD360). In some embodiments, the polypeptide has one, two, three, or all four of the following residues relative to X2 (SEQ ID NO: 933):

[0123] (a) X2 residue 4 is I or W;

[0124] (b) X2 residue 7 is V, D, E, or I;

[0125] (c) X2 residue 10 is K or R; and / or

[0126] (d) X2 residue 15 is A, I, L or M.

[0127] In another embodiment, the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO:934):

[0128] Residue 1: P, N, D, E, R, K, and H;

[0129] residue 2: G, F, Y, E, R, and K;

[0130] residue 3: V, I, M, L, F, T, and Q;

[0131] residue 4: A, I, L, S, T, E, R, and K;

[0132] Residue 5: A, V, I, L and E;

[0133] residue 6: A, V, L, E, R, and K;

[0134] residue 7: M and L;

[0135] residue 8: V, L, F, Y, T, N, R, and K;

[0136] residue 9: G, A, V, Q, and E;

[0137] Residue 10: A, I, M, and L;

[0138] residue 11: A, V, I, and L;

[0139] residue 12: D, E, and K;

[0140] residue 13: A, V, I, L, and T;

[0141] residue 14: C, A, V, S, T, and E;

[0142] residue 15: A, D, E, and R;

[0143] Residue 16: A, I, L, Y, W, S, Q, E, R, K and H;

[0144] Residue 17: A, V, and I;

[0145] Residue 18: A, L, Y, Q, E, R, and K;

[0146] Residue 19: T, D, E, R, and K; and

[0147] Residue 20: Y, T, N, E, and H.

[0148] The inventors have identified that residues 2, 6, 8, 12, and 16 of X3 (SEQ ID NO: 934) are present at the binding interface with the IL-21Rα (CD360) or γc (CD132) receptors. In some embodiments, the polypeptide has one, two, three, four, or all five of the following residues relative to X3 (SEQ ID NO: 934):

[0149] (a) X3 residue 2 is R or K;

[0150] (b) X3 residue 6 is R or K;

[0151] (c) X3 residue 8 is R or K;

[0152] (d) X3 residue 12 is D or E; and / or

[0153] (e) X3 residue 16 is H or Y or W.

[0154] The inventors have identified that residues 1, 5, 9, 11, 13, and 19 of X3 (SEQ ID NO: 934) are present at the binding interface with the IL-21Rα (CD360) or γc (CD132) receptors. 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):

[0155] (a) X3 residue 1 is K or R;

[0156] (b) X3 residue 5 is I or V;

[0157] (c) X3 residue 9 is Q or G;

[0158] (d) X3 residue 11 is I or L;

[0159] (e) X3 residue 13 is I or V; and / or

[0160] (f) X3 residue 19 is R or K.

[0161] In another embodiment, the polypeptide has an amino acid selected from the following residues relative to X4 (SEQ ID NO:935):

[0162] Residue 1: R and K;

[0163] Residue 2: I, W, S, E, R and K;

[0164] residue 3: A, V, I, and L;

[0165] residue 4: C, A, and M;

[0166] residue 5: A, L, S, E, R, and K;

[0167] residue 6: M, N, Q, D, E, R, and K;

[0168] residue 7: A, L, and F;

[0169] residue 8: Y, S, E, R, and K;

[0170] residue 9: V, M, S, and T;

[0171] residue 10: A, E, and K;

[0172] residue 11: C and A;

[0173] Residue 12: Q and R;

[0174] Residue 13: A and K;

[0175] Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E and K;

[0176] residue 15: I, L, and F;

[0177] residue 16: G, F, and Y;

[0178] residue 17: S, R, and K;

[0179] Residue 18: L, F, Y, W, and E; and

[0180] Residue 19: L, F, Y and E.

[0181] The inventors have identified that residues 9 and 16 of X4 (SEQ ID NO: 935) are present at the binding interface with γc (CD132). In some embodiments, the polypeptide has one or two of the following residues relative to X4 (SEQ ID NO: 935):

[0182] (a) X4 residue 9 is S or T; and / or

[0183] (b) X4 residue 16 is G or Y.

[0184] The inventors have identified residues 8, 12, 13, 15, and 19 of X4 (SEQ ID NO: 935) as being present at 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 relative to X4 (SEQ ID NO: 935):

[0185] (a) X4 residue 8 is K or R;

[0186] (b) X4 residue 12 is Q;

[0187] (c) X4 residue 13 is K;

[0188] (d) X4 residue 15 is L or I; and / or

[0189] (e) X4 residue 19 is L, F or Y.

[0190] In one embodiment, the polypeptide comprises:

[0191] (1) relative to the following residues in X2 (SEQ ID NO: 933):

[0192] (a) X2 residue 11 is K, E, or R;

[0193] (b) X2 residue 14 is R, E, or K;

[0194] (c) X2 residue 18 is V, R, H, or K;

[0195] (d) X2 residue 21 is R or K;

[0196] (e) X2 residue 4 is I or W;

[0197] (f) X2 residue 7 is V, D, E, or I;

[0198] (g) X2 residue 10 is K or R; and

[0199] (h) X2 residue 15 is A, I, L, or M; and

[0200] (2) relative to the following residues in X3 (SEQ ID NO: 934):

[0201] (a) X3 residue 2 is R or K;

[0202] (b) X3 residue 6 is R or K;

[0203] (c) X3 residue 8 is R or K;

[0204] (d) X3 residue 12 is D or E;

[0205] (e) X3 residue 16 is H, Y, or W;

[0206] (f) X3 residue 1 is K or R;

[0207] (g) X3 residue 5 is I or V;

[0208] (h) X3 residue 9 is Q or G;

[0209] (i) X3 residue 11 is I or L;

[0210] (j) X3 residue 13 is I or V; and

[0211] (k) X3 residue 19 is R or K; and

[0212] (3) relative to the following residues in X4 (SEQ ID NO: 935):

[0213] (a) X4 residue 9 is S or T; and

[0214] (b) X4 residue 16 is G or Y; and

[0215] (c) X4 residue 8 is K or R;

[0216] (d) X4 residue 12 is Q;

[0217] (e) X4 residue 13 is K;

[0218] (f) X4 residue 15 is L or I; and

[0219] (g) X4 residue 19 is L, F or Y.

[0220] In another embodiment, the present disclosure provides a polypeptide comprising domains X2 and X3, wherein:

[0221] (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and

[0222] (b) X3 is 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 KRFYIFMQDLIDIVTHVKRE (SEQ ID NO:937), wherein one, two or all three of the following conditions are true relative to SEQ ID NO:937: residue 4 is Y, residue 6 is F, and / or residue 9 is D;

[0223] wherein X2 and X3 can be in any order in the polypeptide;

[0224] wherein an amino acid linker may be present between the domains; and

[0225] The polypeptide can bind to the IL-21 receptor (IL-21Rα, CD360) but cannot bind to the γc and CD132 receptors at the same time.

[0226] In this embodiment, polypeptide can serve as IL-21 antagonist.Antagonistic IL-21 mimetics is such construct, it can be combined with IL-21 receptor (IL-21Rα, CD360), but can not be combined with common gamma chain receptor (γc, CD132) simultaneously, may prevent the heterodimerization of IL-21 receptor (IL-21Rα, CD360) and common gamma chain receptor (γc, CD132) and the potential downstream cell signal conduction of result.Can be by first confirming to be combined with IL-21R, then confirm lack simultaneously with gamma chain receptor (γc, CD132) and be combined with IL-21R. c The activity of the designed antagonist is tested by binding to the IL-21 receptor. c The affinity between receptors is very weak, γ c Receptor binding is difficult to detect alone, so it is necessary to perform γ-antibody detection in the presence of IL-21R. c Receptor binding assay. Confirmation of IL-21R binding is crucial, as lack of IL-21R binding may also result from lack of binding to γ c Similar to agonist identification, the assay should be performed at 1000 nM IL-21R and 1000 nM γ c The highest receptor concentration was used.

[0227] The inventors have provided extensive teachings 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 capabilities: SEQ ID NO: 627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-75 1, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900 and 902-907.

[0228] Polypeptide antagonists can be used, for example, to modulate the immune response of a subject in need thereof, such as for immunosuppressive purposes against autoimmune diseases including, but not limited to, multiple sclerosis, lupus, and rheumatoid arthritis.

[0229] The X2 domain of this embodiment is identical to the X2 domain of the agonists detailed above, in that the X2 domain mediates interaction with the IL-21 receptor (IL-21Rα, CD360), but not with the γc, CD132 receptor.

[0230] In one embodiment, (a) X2 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: 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.

[0231] In another embodiment, (a) X2 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: 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.

[0232] In another embodiment, (a) X2 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: 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.

[0233] In one embodiment, (a) X2 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: 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.

[0234] 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 at least 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 937.

[0235] The inventors performed extensive site saturation mutagenesis (SSM), yeast surface display assays using flow cytometry, biolayer interferometry (BLI), and other functional studies described in the methods section of the examples to identify various substitutions that retain receptor binding activity (and therefore retain antagonist activity). In one embodiment, the polypeptide has an amino acid selected from the following residues relative to X2 (SEQ ID NO: 933):

[0236] Residue 1: G, A, S, T, Q, D, E, R, K, and H;

[0237] residue 2: G, A, L, S, T, N, and Q;

[0238] residue 3: A, M, Y, W, N, Q, E, R, and K;

[0239] Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E and H;

[0240] residue 5: M, L, Q, E, and K;

[0241] residue 6: V, I, L, Y, R, and K;

[0242] Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E and R;

[0243] residue 8: A, L, F, S, T, E, and H;

[0244] residue 9: A, V, I, and L;

[0245] residue 10: E, R, and K;

[0246] residue 11: V, E, K, R, and H;

[0247] residue 12: A, I, and L;

[0248] Residue 13: V and I;

[0249] residue 14: S, E, K, and R;

[0250] Residue 15: A, I, M, L and E;

[0251] residue 16: C, A, and S;

[0252] Residue 17: N and R;

[0253] residue 18: V, S, N, E, K, R, and H;

[0254] residue 19: A, V, I, and L;

[0255] Residue 20: V and I;

[0256] Residue 21: W, S, E, K, and R; and

[0257] Residue 22: M and D.

[0258] The inventors have identified residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) as being present at the binding interface with IL-21Rα and CD360. In some embodiments, the polypeptide has one, two, three, or all four of the following residues relative to X2 (SEQ ID NO: 933):

[0259] (a) X2 residue 11 is K, E, or R;

[0260] (b) X2 residue 14 is R, E, or K;

[0261] (c) X2 residue 18 is V, R, H or K; and / or

[0262] (d) X2 residue 21 is R or K.

[0263] The inventors have identified residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) as being present at the binding interface with IL-21Rα and CD360. In some embodiments, the polypeptide has one, two, three, or all four of the following residues relative to X2 (SEQ ID NO: 933):

[0264] (a) X2 residue 4 is I or W;

[0265] (b) X2 residue 7 is V, D, E, or I;

[0266] (c) X2 residue 10 is K or R; and / or

[0267] (d) X2 residue 15 is A, I, L or M.

[0268] In another embodiment, the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO:937):

[0269] Residue 1: P, N, D, E, R, K, and H;

[0270] residue 2: G, F, Y, E, R, and K;

[0271] residue 3: V, I, M, L, F, T, and Q;

[0272] Residue 4: Y;

[0273] Residue 5: A, V, I, L and E;

[0274] residue 6: F;

[0275] residue 7: M and L;

[0276] residue 8: Q and E;

[0277] residue 9: D;

[0278] Residue 10: A, I, M, and L;

[0279] residue 11: A, V, I, and L;

[0280] residue 12: D, E, and K;

[0281] residue 13: A, V, I, L, and T;

[0282] residue 14: C, A, V, S, T, and E;

[0283] Residue 15: T, N, and K;

[0284] Residue 16: A, I, L, Y, W, S, Q, E, R, K and H;

[0285] Residue 17: A, V, and I;

[0286] Residue 18: A, L, Y, Q, E, R, and K;

[0287] Residue 19: T, D, E, R, and K; and

[0288] Residue 20: Y, T, N, E, and H.

[0289] In some embodiments, the polypeptide has one, two, or all three of the following residues relative to X3 (SEQ ID NO:937):

[0290] (a) X3 residue 2 is R or K;

[0291] (b) X3 residue 12 is D or E; and / or

[0292] (c) X3 residue 16 is H or Y / W.

[0293] In other embodiments, the polypeptide has one, two, three, four, or all five of the following residues relative to X3 (SEQ ID NO:937):

[0294] (a) X3 residue 1 is K or R;

[0295] (b) X3 residue 5 is I or V;

[0296] (c) X3 residue 11 is I or L;

[0297] (d) X3 residue 13 is I or V; and / or

[0298] (e) X3 residue 19 is R or K.

[0299] In another embodiment, (i) relative to X3 (SEQ ID NO: 937):

[0300] (a) All three of the following conditions hold: residue 4 is Y, residue 6 is F, and / or residue 9 is D;

[0301] (b) X3 residue 2 is R or K;

[0302] (c) X3 residue 12 is D or E;

[0303] (d) X3 residue 16 is H, Y, or W;

[0304] (e) X3 residue 1 is K or R;

[0305] (f) X3 residue 5 is I or V;

[0306] (g) X3 residue 11 is I or L;

[0307] (h) X3 residue 13 is I or V; and

[0308] (i) X3 residue 19 is R or K; and

[0309] (ii) relative to X2 (SEQ ID NO: 933):

[0310] (a) X2 residue 11 is K, E, or R;

[0311] (b) X2 residue 14 is R, E, or K;

[0312] (c) X2 residue 18 is V, R, H, or K; and

[0313] (d) X2 residue 21 is R or K;

[0314] (e) X2 residue 4 is I or W;

[0315] (f) X2 residue 7 is V, D, E, or I;

[0316] (g) X2 residue 10 is K or R; and

[0317] (h) X2 residue 15 is A, I, L or M.

[0318] In another embodiment, the antagonist polypeptide further comprises an X4 domain, wherein the X4 domain comprises a helical structure, wherein the X4 domain does not allow the polypeptide to simultaneously bind to the IL-21 receptor (IL-21Rα, CD360) and the γc, CD132 receptor, and wherein X2, X3, and X4 can be in any order in the polypeptide. In this embodiment, the X4 domain can be any helical domain suitable for the intended purpose, as long as it does not allow the polypeptide to simultaneously bind to the IL-21 receptor (IL-21Rα, CD360) and the γc, CD132 receptor.

[0319] In another embodiment, X4 comprises a 19-residue peptide. In one such embodiment, the X4 peptide has an amino acid selected from the following amino acid residues at each position:

[0320] Residue 1: R and K;

[0321] Residue 2: I, W, S, E, R and K;

[0322] residue 3: V;

[0323] residue 4: C, A, and M;

[0324] residue 5: A, L, S, E, R, and K;

[0325] residue 6: M, N, Q, D, E, R, and K;

[0326] residue 7: A, L, and F;

[0327] residue 8: A, V, and D;

[0328] residue 9: G, W, E, and K;

[0329] residue 10: A, E, and K;

[0330] residue 11: C and A;

[0331] Residue 12: G, I, E and K;

[0332] Residue 13: V, L, Q, D, E, and R;

[0333] Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E and K;

[0334] residue 15: I, L, and F;

[0335] residue 16: R, K, and H;

[0336] residue 17: S, R, and K;

[0337] Residue 18: L, F, Y, W, and E; and

[0338] Residue 19: L, F, Y and E.

[0339] In one embodiment of any of the Il-21 binding polypeptides disclosed herein, 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 comprises an amino acid selected from the group consisting of the following residues relative to X1 (SEQ ID NO: 936):

[0340] Residue 1: V, I, and L;

[0341] Residue 2: A, I, S, N, R and K;

[0342] residue 3: D and E;

[0343] Residue 4: V and I;

[0344] residue 5: M and F;

[0345] residue 6: K;

[0346] Residue 7: E;

[0347] residue 8: A and V;

[0348] Residue 9: A and R;

[0349] Residue 10: A, V, I, L, T, Q, E, and K;

[0350] Residue 11: C, V, I, M, F, Y, Q, R, and K;

[0351] residue 12: A;

[0352] Residue 13: R and K;

[0353] Residue 14: K;

[0354] Residue 15: E; and

[0355] Residue 16: A, I, M, L, F, Y, S, and N.

[0356] In one embodiment, the polypeptide does not have a cysteine ​​residue at position 11 relative to X1 (SEQ ID NO: 936).

[0357] The X2, X3, X4 (when present), and X1 (when present) domains can be present in any order in the polypeptides of any embodiment herein. The inventors have provided extensive teachings of "shuffled" versions of polypeptides; see Table 2 and the columns listing the domain order of the X1, X2, X3, and X4 domains. In one embodiment, the polypeptide comprises, 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, in domain order, X4-X3-X2, X3-X2, X1-X3-X2, X2-X1-X3, X3-X2-X1, X3-X1-X2, X2-X4-X3, X3-X2-X4, X3-X4-X2, X4-X2-X3, X1-X2-X3-X4, X1-X4-X3-X2, X2-X1-X4-X3, X2-X3-X4-X1, X3-X2-X1-X4, X3-X4-X1-X2, X4-X1-X2-X3, X3-X2, or X4-X3-X2-X1.

[0358] The polypeptides of the present disclosure may comprise an amino acid linker between any of the domains. In some embodiments, there is a linker between each of domains X2, X3, X4 (when present), and X1 (when present). In other embodiments, the polypeptide may not comprise a linker between domains, or may comprise a linker between some but not all domains. The linker may be of any length or amino acid composition. In various non-limiting embodiments, suitable linkers include, but are not limited to, GS, GGS, GGGGG (SEQ ID NO: 939), GSGGG (SEQ ID NO: 940), GGGGGG (SEQ ID NO: 941), GGSGGG (SEQ ID NO: 942), GGSGGSGGGSGGSGSG (SEQ ID NO: 943), GSGGSGGGSGGSGSG (SEQ ID NO: 944), GSGSGGGSGGSCKKISGGSGGGS GGGGS (SEQ ID NO: 945), and (GGGGX) n (SEQ ID NO: 946), wherein X is Q, E or S, and n is 2-5.

[0359] Depending on the intended use, the polypeptide can be fused with one or more additional domains. 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 with a polypeptide of the present disclosure. In various non-limiting embodiments, one or more functional domains include a cell targeting domain (including but not limited to an antibody, an antibody fragment, a domain that extends protein half-life (such as albumin, albumin-binding protein, antibody Fc fragment), a protein, antigen, ligand, peptide, etc. that binds to a biomarker) or a detectable domain (including but not limited to a fluorescent protein, a luminescent protein, a protein tag, etc.). In various embodiments, 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 NO: 599-606. The polypeptide and one or more functional domains can be directly adjacent to each other in the fusion protein, or can be connected by a polypeptide linker suitable for the intended purpose. One or more functional domains may be present at the N-terminus, the C-terminus, or at a position between the X2, X3, X4, or X1 (when present) domains.

[0360] In other embodiments, 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, 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, 716-717. 17-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931. In one embodiment, the polypeptide or fusion protein comprises an amino acid sequence 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 NO: 627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900 and 902-907. In this embodiment, the polypeptide has antagonistic 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 has agonist activity.

[0361] In another aspect, the present disclosure provides a conditionally active IL-21 receptor binding protein comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present simultaneously in the same fusion protein, wherein the first polypeptide component and the second polypeptide component collectively comprise domains X2, X3, and optionally X4, as defined in any embodiment or combination of embodiments of the polypeptide of the present disclosure, wherein:

[0362] (i) the first polypeptide component comprises at least one of X2, X3 and X4 (when present), but does not comprise each of X2, X3 and X4 (when present); and

[0363] (ii) the second polypeptide component comprises each of X2, X3 and X4 that are not present in the first polypeptide component;

[0364] The first polypeptide component and the second polypeptide component alone are not active receptor binding proteins, and the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein capable of binding to an IL-21 receptor (IL-21Rα, CD360).

[0365] In one embodiment:

[0366] (i) the first polypeptide component comprises at least one of X2, X3 and X4, but does not comprise each of X2, X3 and X4; and

[0367] (ii) the second polypeptide component comprises each of X2, X3 and X4 that is not present in the first polypeptide component;

[0368] The first polypeptide component and the second polypeptide component alone are not active receptor binding proteins, and the first polypeptide component 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 capable of binding to the γc, CD132 receptor.

[0369] In another embodiment,

[0370] (i) the first polypeptide component comprises at least one of X1, X2, X3 and X4, but does not comprise each of X1, X2, X3 and X4; and

[0371] (ii) the second polypeptide component comprises each of X1, X2, X3 and X4 that are not present in the first polypeptide component;

[0372] The first polypeptide component and the second polypeptide component alone are not active receptor binding proteins, and the first polypeptide component 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 capable of binding to the γc, CD132 receptor.

[0373] The present disclosure surprisingly presents a conditionally active receptor IL-21 agonist comprising the separate first polypeptide and the second polypeptide, wherein the separate first polypeptide and the second polypeptide are not receptor agonists, but can form an active agonist of IL-21 by binding to the IL-21 receptor (IL-21Rα, CD360) and γc, CD132 receptors rather than covalently interacting. Therefore, the conditionally active receptor agonist of the present disclosure can be used for any purpose for which the polypeptide agonist of the present disclosure can be used.

[0374] In another embodiment,

[0375] (i) the first polypeptide component comprises X2 but does not comprise X3; and

[0376] (ii) the second polypeptide component comprises X3;

[0377] The first polypeptide component and the second polypeptide component alone are not active receptor binding proteins, and the first polypeptide component 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) but not simultaneously binding to the γc, CD132 receptors.

[0378] This embodiment provides conditionally active receptor antagonists that can be used for any of the purposes for which the polypeptide antagonists disclosed herein can be used. Split IL-21 antagonists are designed to achieve targeted delivery of IL-21 antagonists with minimal off-target activity. In another embodiment, 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.

[0379] The polypeptide is typically cleaved at a site that does not interfere with protein function (e.g., a linker portion in embodiments with a linker). Furthermore, just as the X1 (when present), X2, X3, and X4 (when present) domains in a non-split polypeptide can be looped together in any order, the split protein can contain any combination of domains.

[0380] Thus, X1 (when present), X2, X3, and X4 (when present) can be in any order in the first polypeptide and the second polypeptide; in a non-limiting embodiment:

[0381] (i) the first polypeptide comprises X1, and the second polypeptide comprises X2, X3, and X4;

[0382] (ii) the first polypeptide comprises X2, and the second polypeptide comprises X1, X3, and X4;

[0383] (iii) the first polypeptide comprises X3, and the second polypeptide comprises X1, X2, and X4;

[0384] (iv) the first polypeptide comprises X4, and the second polypeptide comprises X1, X2, and X3;

[0385] (v) the first polypeptide comprises X1 and X2, and the second polypeptide comprises X3 and X4;

[0386] (vi) the first polypeptide comprises X1 and X3, and the second polypeptide comprises X2 and X4;

[0387] (vii) the first polypeptide comprises X1 and X4, and the second polypeptide comprises X2 and X3;

[0388] (viii) the first polypeptide comprises X2 and X3, and the second polypeptide comprises X1 and X4;

[0389] (ix) the first polypeptide comprises X2 and X4, and the second polypeptide comprises X1 and X3;

[0390] (x) the first polypeptide comprises X3 and X4, and the second polypeptide comprises X1 and X2;

[0391] (xi) the first polypeptide comprises X1, X2, and X3, and the second polypeptide comprises X4;

[0392] (xii) the first polypeptide comprises X1, X2, and X4, and the second polypeptide comprises X3;

[0393] (xiii) the first polypeptide comprises X1, X3, and X4, and the second polypeptide comprises X2; or

[0394] (xiv) The first polypeptide comprises X2, X3 and X4, and the second polypeptide comprises X1.

[0395] When the first polypeptide and / or the second polypeptide comprises more than one domain of X1, X2, X3 and X4, in some embodiments, these domains can be separated by amino acid linkers of any suitable length or amino acid composition. There are no requirements for linkers; in one embodiment, no linker is present between any of the domains. In other embodiments, an amino acid linker may be present between 0, 1 or 2 junctions between domains X1, X2, X3 and X4 in the first polypeptide and / or the second polypeptide. The amino acid linker can be any length deemed suitable for the intended use and can, for example, comprise any linker embodiment disclosed herein. In some embodiments, the linker is at the N-terminus or C-terminus and is referred to as a linker despite not connecting the two domains together.

[0396] In one embodiment, X1 (when present), X2, X3, and X4 are each as described in any embodiment or combination of embodiments of the polypeptides of the present disclosure. In another embodiment, the first polypeptide and the second polypeptide collectively comprise: (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: SEQ ID NO: NO: 1-598, 627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 68 1-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746-747, 749-798, 80 0-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; or (b) 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 NO: NO: 627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675 , 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 7 42-743, 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.

[0397] In another embodiment, the first polypeptide and the second polypeptide comprise:

[0398] (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;

[0399] (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;

[0400] (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;

[0401] (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;

[0402] (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;

[0403] (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;

[0404] (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;

[0405] (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;

[0406] (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.

[0407] In another embodiment, the first polypeptide and the second polypeptide comprise:

[0408] (a) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 609;

[0409] (b) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 612;

[0410] (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;

[0411] (d) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 613; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 614;

[0412] (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;

[0413] (f) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 617; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 618;

[0414] (g) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 619; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 620;

[0415] (h) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 621; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 622; or

[0416] (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.

[0417] In yet another embodiment, the first polypeptide and the second polypeptide comprise:

[0418] (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;

[0419] (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;

[0420] (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;

[0421] (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;

[0422] (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;

[0423] (f) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 617; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 618;

[0424] (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;

[0425] (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

[0426] (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.

[0427] In yet another embodiment, the first polypeptide and the second polypeptide comprise:

[0428] (a) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 609;

[0429] (b) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 612;

[0430] (c) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 610; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 611;

[0431] (d) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 613; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 614;

[0432] (e) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 615; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 616;

[0433] (f) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 617; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 618;

[0434] (g) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 619; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 620;

[0435] (h) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 621; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 622; or

[0436] (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.

[0437] In one embodiment, one or both of the first polypeptide and the second polypeptide are fused to a targeting domain, thereby allowing targeted delivery. In a non-limiting embodiment, the targeting domain may comprise an antibody or nanobody ( Figure 2C In this example, the first polypeptide and the second polypeptide were fused to anti-HER2-DARPin or anti-EGFR-DARPin (Table 4, SEQ ID NOs: 613-624) to demonstrate that the first polypeptide and / or the second polypeptide can be fused to such targeting domains for use and can be reconstructed ( Figure 2D ).

[0438] In another embodiment, the present disclosure provides polypeptides comprising the amino acid sequence of any of the first or second polypeptides described herein. These polypeptides can be used, for example, in the conditionally active receptor agonists of the present disclosure. In one embodiment, the polypeptide 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: 607-624.

[0439] In another aspect, the present disclosure provides a non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4, wherein:

[0440] (a) 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 of LAEIMKEVAECARKEA (SEQ ID NO: 936);

[0441] (b) X2 is 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 DTRILKVSLKKIVRA (SEQ ID NO: 933);

[0442] (c) X3 is 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and

[0443] (d) X4 is 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935);

[0444] wherein X1, X2, X3 and X4 can be in any order in the polypeptide;

[0445] wherein an amino acid linker may be present between any of the domains; and

[0446] The polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0447] The polypeptides of this aspect of the disclosure can be used, for example, to treat cancer and / or modulate immune responses.

[0448] In one embodiment, X1 residues 1-16 are selected from:

[0449] Residue 1: V, I, and L;

[0450] Residue 2: A, I, S, N, R and K;

[0451] residue 3: D and E;

[0452] Residue 4: V and I;

[0453] residue 5: M and F;

[0454] residue 6: K;

[0455] Residue 7: E;

[0456] residue 8: A and V;

[0457] Residue 9: A and R;

[0458] Residue 10: A, V, I, L, T, Q, E, and K;

[0459] Residue 11: C, V, I, M, F, Y, Q, R, and K;

[0460] residue 12: A;

[0461] Residue 13: R and K;

[0462] Residue 14: K;

[0463] Residue 15: E; and

[0464] Residue 16: A, I, M, L, F, Y, S, and N.

[0465] In another embodiment, X1 residue 16 is not A. In one embodiment, X2 residues 1-16 are selected from:

[0466] Residue 1: G, A, T, Q, D, R, K, and H;

[0467] residue 2: G, A, S, T, and N;

[0468] residue 3: A, M, Y, N, Q, E, and R;

[0469] Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E and H;

[0470] residue 5: M, L, Q, and K;

[0471] residue 6: V, I, L, Y, R, and K;

[0472] Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D and E;

[0473] residue 8: F, S, T, and E;

[0474] residue 9: L;

[0475] residue 10: K;

[0476] residue 11: K;

[0477] Residue 12: I;

[0478] Residue 13: V and I;

[0479] Residue 14: R; and

[0480] Residue 15: A, I, M, and L.

[0481] In various embodiments, 1, 2, 3, or all 4 of the following conditions are met: (a) X2 residue 3 is not R;

[0482] (b) X2 residue 4 is not 1;

[0483] (c) X2 residue 7 is not V; and / or

[0484] (d) Residue 8 of X2 is not S.

[0485] In another embodiment, X3 residues 1-20 are selected from:

[0486] Residue 1: P, N, R, and K;

[0487] residue 2: F, Y, and R;

[0488] residue 3: V, I, M, L, F, T, and Q;

[0489] Residue 4: E;

[0490] Residue 5: I;

[0491] Residue 6: R;

[0492] residue 7: M;

[0493] residue 8: V, L, F, Y, T, and R;

[0494] residue 9: G, A, and Q;

[0495] residue 10: I, M, and L;

[0496] residue 11: I and L;

[0497] residue 12: D;

[0498] Residue 13: I;

[0499] residue 14: C, A, V, S, and T;

[0500] residue 15: D and E;

[0501] Residue 16: H;

[0502] Residue 17: V;

[0503] Residue 18: K;

[0504] Residue 19: R; and

[0505] Residue 20: T, N, and E.

[0506] In various embodiments, 1, 2, 3, 4, 5, 6, or all 7 of the following conditions are met: (a) X3 residue 2 is not R;

[0507] (b) X3 residue 8 is not R;

[0508] (c) X3 residue 9 is not Q;

[0509] (d) X3 residue 10 is not L;

[0510] (e) X3 residue 11 is not 1;

[0511] (f) X3 residue 14 is not V; and / or

[0512] (g) X3 residue 15 is not D.

[0513] In one embodiment, X4 residues 1-19 are selected from:

[0514] Residue 1: K;

[0515] Residue 2: E;

[0516] residue 3: V;

[0517] residue 4: M;

[0518] Residue 5: E;

[0519] Residue 6: R;

[0520] Residue 7: A;

[0521] Residue 8: R and K;

[0522] residue 9: V, M, S, and T;

[0523] Residue 10: A;

[0524] residue 11: A;

[0525] Residue 12: Q;

[0526] residue 13: K;

[0527] Residue 14: C, G, A, V, I, M, L, S, T, Q, D and K;

[0528] residue 15: L;

[0529] residue 16: G;

[0530] Residue 17: R and K;

[0531] Residue 18: F, Y, and W; and

[0532] Residue 19: L, F and Y.

[0533] In various embodiments, one, two, or all three of the following conditions are true:

[0534] (a) X4 residue 8 is not K;

[0535] (b) X4 residue 9 is not S; and / or

[0536] (c) X4 residue 19 is not L.

[0537] In one embodiment, the polypeptide is an antagonist of the IL-21 receptor. In another embodiment, the X1 residue 11 is C. This embodiment helps increase the binding affinity to human common gamma chain (CD132) as well as mouse CD360 and mouse CD132.

[0538] In various embodiments, 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. In other embodiments, there are amino acid linkers between the domains.

[0539] 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.

[0540] DEELAEIMKEVAECARKEAEKIDNTDEDTRILKVSLKKIVRAANVIVRM REREADSKRFEIRMRQLIDIVDHVKREFASEDLKEVMERAKSAAQKILGRFL(SEQ ID NO: 938)

[0541] In this embodiment, the residues at the non-helical positions may be selected from the group consisting of:

[0542] Residue 01: V, I, M, F, Y, and D;

[0543] Residue 02: A, V, I, M, L, F, Y, W, Q, D, E, R, and H; Residue 03: E;

[0544] residue 04: D and E;

[0545] residue 21: A, T, and E;

[0546] Residue 22: R and K;

[0547] residue 23: I, M, L, F, and Y;

[0548] Residue 24: P, G, A, V, I, S, T, N, Q, D, R, and H; Residue 25: G, A, V, N, Q, and K;

[0549] residue 26: T, N, R, and K;

[0550] residue 27: G, A, S, T, N, D, R, K, and H;

[0551] residue 28: P, E, and H;

[0552] Residue 44: A and S;

[0553] Residue 45: N and K;

[0554] residue 46: G, A, V, S, T, N, R, K, and H;

[0555] Residue 47: V and I;

[0556] Residue 48: V, I, Y, Q, R, and K;

[0557] residue 49: L, R, and K;

[0558] residue 50: G, M, Y, S, Q, and H;

[0559] Residue 51: R;

[0560] residue 52: G, M, L, F, Y, W, N, D, E, and H;

[0561] residue 53: G, R, and K;

[0562] Residue 54: G, A, V, I, L, N, E and K;

[0563] Residue 55: A, V, I, M, L, F, Y, W, T, Q and H;

[0564] residue 56: D;

[0565] residue 57: P, A, V, Y, W, S, D, E, and H;

[0566] residue 78: F;

[0567] residue 79: A, Y, S, and K;

[0568] residue 80: S;

[0569] Residue 81: E;

[0570] residue 82: D; and

[0571] Residue 83: L and F.

[0572] In another embodiment, the polypeptide of this aspect of the disclosure 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 NO: 1-598, 627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 716-717. 17-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898 and 900-931.

[0573] In one embodiment, the polypeptide further comprises one or more functional domains. The polypeptide can 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 fluorescent protein, photoprotein, etc.). In one embodiment, the one or more functional domains are fused to the translation of the polypeptide.

[0574] In another embodiment of this aspect, the present disclosure provides a non-naturally occurring conditionally active IL-21 receptor binding protein comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in a fusion protein, wherein the first polypeptide component and the second polypeptide component together comprise domains X1, X2, X3, and optionally X4, as defined in any one of the preceding claims, wherein:

[0575] (i) the first polypeptide component comprises at least one of X1, X2, X3 and X4, but does not comprise each of X1, X2, X3 and X4; and

[0576] (ii) the second polypeptide component comprises each of X1, X2, X3 and X4 that are not present in the first polypeptide component;

[0577] wherein the first polypeptide component and the second polypeptide component alone are not active receptor binding proteins, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein.

[0578] In various embodiments, the polypeptide can be selected from the group consisting of:

[0579] (i) a polypeptide comprising X1 and excluding X2, X3 and X4;

[0580] (ii) a polypeptide comprising X2 and excluding X1, X3 and X4;

[0581] (iii) a polypeptide comprising X3 and excluding X1, X2 and X4;

[0582] (iv) a polypeptide comprising X4 and excluding X1, X2 and X3;

[0583] (v) a polypeptide comprising X1 and X2 but excluding X3 and X4;

[0584] (vi) a polypeptide comprising X1 and X3 but excluding X2 and X4;

[0585] (vii) a polypeptide comprising X1 and X4 but excluding X2 and X3;

[0586] (viii) a polypeptide comprising X2 and X3 but not X1 and X4;

[0587] (ix) a polypeptide comprising X2 and X4 but excluding X1 and X3;

[0588] (x) a polypeptide comprising X3 and X4 but excluding X1 and X2;

[0589] (xi) a polypeptide comprising X1, X2, and X3 but not X4;

[0590] (xii) a polypeptide comprising X1, X2, and X4 but not X3;

[0591] (xiii) a polypeptide comprising X1, X3, and X4 but not X2; and

[0592] (xiv) A polypeptide comprising X2, X3 and X4 and excluding X1.

[0593] In other embodiments, X1, X2, X3 and X4 respectively comprise amino acid sequences corresponding to 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, 749-750, 760-761, 761-762 85%, 90%, 95%, 98% or 100% identical amino acid sequence to any of 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.

[0594] In another aspect, the present disclosure provides nucleic acids, including isolated nucleic acids, encoding polypeptides, fusion proteins, and first and second polypeptides of the present disclosure. The isolated nucleic acid sequence may comprise RNA or DNA. Such isolated nucleic acid sequences may comprise additional sequences for promoting expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be clear to those skilled in the art which nucleic acid sequence will encode the polypeptides of the present invention.

[0595] On the other hand, the present disclosure provides expression vectors, which include nucleic acids of any aspect of the present disclosure that are operably linked to suitable control sequences. "Expression vectors" include vectors in which nucleic acid coding regions or genes are operably linked to any control sequences that can achieve expression of gene products. "Control sequences" that are operably linked to a nucleic acid sequence of the present invention are nucleic acid sequences that can affect the expression of nucleic acid molecules. A control sequence need not be adjacent to a nucleic acid sequence, as long as the control sequence functions to guide the expression of the nucleic acid sequence. Thus, for example, an intermediate untranslated but transcribed sequence can exist between a promoter sequence and a nucleic acid sequence, and the promoter sequence can still be considered to be "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, plasmids and viral-based expression vectors. The control sequence for driving the expression of the disclosed nucleic acid sequence in a mammalian system can be constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, EF) or inducible (driven by any of many inducible promoters, including but not limited to tetracycline, ecdysone, steroid responsiveness). The expression vector must be replicable in the host organism as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector may include a plasmid, a virus-based vector (including but not limited to a retroviral vector or an oncolytic virus) or any other suitable expression vector. In some embodiments, an expression vector may be applied in the method of the present disclosure to express a polypeptide in vivo to achieve therapeutic benefits. In non-limiting embodiments, the expression vector may be used to transfect or transduce a cell therapy target (including but not limited to CAR-T cells or tumor cells) to achieve the methods of treatment disclosed herein.

[0596] On the other hand, the present disclosure provides a host cell comprising an expression vector, a polypeptide, a fusion protein, a first polypeptide and / or a second polypeptide, a polypeptide component, a conditionally active agonist and antagonist and / or a nucleic acid and an expression vector disclosed herein, wherein the host cell can be a prokaryotic cell or a eukaryotic cell. Cells can be transiently or stably engineered to incorporate the expression vector of the present invention using techniques including, but not limited to, bacterial transformation, calcium phosphate coprecipitation, electroporation, or liposome-mediated, DEAE dextran-mediated, polycation-mediated, or viral-mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd edition (RI Freshney. 1987. Liss, Inc. New York, NY)). The method for producing a polypeptide according to the present invention is another part of the present invention. The method comprises the following steps: (a) cultivating a host according to this aspect of the present invention under conditions conducive to polypeptide expression, and (b) optionally, reclaiming the expressed polypeptide. The expressed polypeptides can be recovered from cell-free extracts, but preferably they are recovered from the culture medium.

[0597] On the other hand, the present disclosure provides a pharmaceutical composition comprising a polypeptide, a fusion protein, a first polypeptide, a second polypeptide, a recombinant nucleic acid, an expression vector and / or a recombinant host cell and a pharmaceutically acceptable carrier according to any embodiment or combination of embodiments herein. The pharmaceutical composition of the present disclosure can be used, for example, in the method of the present disclosure 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 active agents, including but not limited to checkpoint inhibitors or cancer vaccines. The pharmaceutical composition may also include (a) a lyoprotectant; (b) a surfactant; (c) an extender; (d) a tension regulator; (e) a stabilizer; (f) a preservative and / or (g) a buffer.

[0598] In some embodiments, the buffer in the pharmaceutical composition is Tris buffer, histidine buffer, phosphate buffer, citrate buffer or acetate buffer. The pharmaceutical composition can also include a lyophilization protectant, such as sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, phenol, meta-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid and various mixtures thereof. In other embodiments, the pharmaceutical composition includes an extender, such as glycine. In yet other embodiments, 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 a tonicity modifier, such as a compound that renders the formulation substantially isotonic or isotonic with human blood. Exemplary tonicity modifiers 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, when combined with the protein of interest, substantially prevents or reduces the chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0599] The polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector and / or recombinant host cell of any embodiment or combination of embodiments herein can be the only active agent in the pharmaceutical composition, or the composition can also contain one or more other active agents suitable for the intended use.

[0600] On the other hand, the present disclosure provides a method for treating cancer, comprising administering to a subject with cancer a polypeptide agonist or fusion protein of an embodiment of an IL-21 agonist herein, a conditionally active receptor agonist, a recombinant nucleic acid, an expression vector, a recombinant host cell, and / or a pharmaceutical composition in an amount effective to treat the tumor. In various embodiments, the cancer is selected from the group consisting of colon cancer, melanoma, renal cell 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 tumor type selected by a diagnostic test, such as microsatellite instability, tumor mutation load, PD-L1 expression level, or immune score determination (as developed by the Cancer Immunotherapy Consortium).

[0601] In another aspect, the present disclosure provides a method for regulating an immune response in a subject, comprising a polypeptide antagonist 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 of an embodiment of an IL-21 antagonist disclosed herein. In one embodiment, the immune response is an anti-cancer immune response.

[0602] As used herein, "treat" or "treating" means to accomplish one or more of the following: (a) reducing the severity of a condition; (b) limiting or preventing the development of symptoms characteristic of the condition or conditions being treated; (c) inhibiting the worsening of symptoms characteristic of the condition or conditions being treated; (d) limiting or preventing the recurrence of the condition or conditions in a patient who previously had the condition or conditions; and (e) limiting or preventing the recurrence of symptoms in a patient who previously had symptoms of the condition or conditions.

[0603] The subject can be any subject suffering from the relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cows, etc.

[0604] Example

[0605] Overview

[0606] Here, we created ultrastable de novo IL-21 mimetic proteins that recapitulate its interaction with the receptor and the biology of native IL-21 in humans and mice, making them very suitable for therapeutic use. The designed IL-21 mimetic proteins include agonists, antagonists, immune cytokines, or split agonists / antagonists, as detailed in the Examples and listed in Tables 1-6. Binding of the two receptors was measured in two settings: (i) binding to hIL-21R, only hIL-21R, and (ii) binding to hγ c Receptor binding, hγ c With hIL-21R. For hγ c Receptor binding, binding assays require hIL-21R because hγ c The affinity of hγ is very weak and therefore reasonable hγ binding is possible only in the presence of hIL-21R. c Combined measurements.

[0607] For (i) hIL-21R combination, in yeast surface display based on flow cytometry or biolayer interferometry (BLI), measure, as mentioned in the method part.For yeast surface display method, the cell of the IL-21 analogies that uses the hIL-21R of 1-1000nM SA-PE mark to express and be combined with hIL-21R together with anti-Myc FITC antibody mark.When testing with 1000nM hIL-21R, the protein display PE+FITC+ colony that is combined with hIL-21R is equal to or more than 0.5% of single cell gate (singlet-gated) colony.For BLI method, hIL-21R is loaded into and analyzes the association of the IL-21 analogies that test and provide with 100pM-1000nM scope.In addition, alternatively, the IL-21 analogies with polyhistidine tag can be loaded into and analyzes the association of the IL-21 analogies that is combined with 100-1000nM hIL-21R. In either case, the protein bound to hIL-21R showed a shift of at least 0.1 nm during association.

[0608] The yeast surface display data that provided show the result of the hIL-21R that uses 100nM PE mark.The BLI data that provided show the hIL-21R loading analysis method of the use aforementioned scope that calculates for suitable Kd and the protein loading analysis of the band polyhistidine tag of the use 100nM hIL-21R of conjunction with mensuration.Under yeast surface display and BLI situation, identical with hIL-21R conjunction with mensuration by test and the mouse cross reaction that is combined with mIL-21R, and demonstrate the result similar to people's receptor.

[0609] For (ii) hγ cReceptor binding was measured using flow cytometry-based yeast surface display or biolayer interferometry (BLI) as described in the Methods section. For the yeast surface display method, 1-1000 nM unlabeled hIL-21R and 1-1000 nM SA-PE-labeled hγ c The receptor was labeled with anti-Myc FITC antibody and expressed in the presence of hIL-21R and hγ c When treated with 1000nM hIL-21R and 1000nM hγ c In the presence of hIL-21R, hγ c Receptor-bound proteins showed a PE+FITC+ population equal to or greater than 0.5% of the single cell gated population. c The receptors were loaded onto the assay tip and tested for association with IL-21 mimetics provided in the range of 100 pM to 1000 nM in the presence of hIL-21R at a concentration 1.5 times that of the IL-21 mimetic. c The receptor and hIL-21R bound protein showed a shift of at least 0.1 nm during association.

[0610] The yeast surface display data presented show that 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 presented show the appropriate Kd calculated for hγ using the aforementioned ranges. c Receptor loading assay. In the case of yeast surface display and BLI, the receptor loading assay was tested with mγ c Receptor and mIL-21R bind to mouse cross-reactive hγ c The receptor binding assay was identical and showed similar results to the human receptor.

[0611] The agonist mimetics of IL-21 are designed to have two interfaces, one for IL-21R and the other for CD360. These designed agonists can bind to both receptors simultaneously so that the two receptor subunits heterodimerize. In addition, these designs have cross-reactions with human and mouse receptors. Antagonist IL-21 mimetics are only designed to retain the binding ability to IL-21R, but do not retain the binding ability to γ c The binding ability of the agonist allows it to occupy the same space as the agonist and still interfere with γ cThe association of heterodimerization with receptor subunits.Similarly, these designs retain human and mouse cross-reactivity.The immune cytokine of IL-21 mimics (IL-21 mimics of antibody fusion) is an example of molecular fusion for targeted cytokine delivery.Split IL-21 mimics is designed to achieve the targeted delivery of IL-21 mimics with minimal off-target cytokine activity.

[0612] Design initial hits and optimization

[0613] Based on the structure of the human IL-21 (hIL-21) / human IL-21R (hIL-21R) complex, we generated a scaffold that mimics the helical bundle structure of hIL-21 with improved secondary structure packing for enhanced stability. We optimized the helices by adjusting the length of the helices in the helical bundle scaffold, minimizing unstructured regions, and eliminating the non-ideal long loops in native hIL-21. Wild-type hIL-21 consists of four helices, of which helix A (the first helix from the N-terminus) and helix C (the third helix from the N-terminus) form the interface with hIL-21R, while helix D (the fourth helix from the N-terminus) forms the interface with hγ c The upper two helices B (the second helix from the N-terminus) and C were not long enough to form ideal intramolecular interactions and intermolecular contacts with the receptor chain. To optimize helices B and C, we extended them to replace most of the unstructured regions, including two long unstructured regions between the helix pairs (helix A / helix B and helix C / helix D), and adjusted the other two helices (helices A and D) to achieve improved packing. To design the interface of the de novo scaffold, we grafted the interface residues of hIL-21 into the structurally matching positions of the scaffold and used Rosetta Stone. TM The remaining residues were designed in the context of hIL-21R using Rosetta TM The designed proteins were evaluated and filtered using scoring indicators, and their folding trajectories were evaluated and filtered using FastForward Folding ( Figure 1A ).

[0614] Using yeast surface display as a screening platform, we identified five hits that could bind to hIL-21R; however, no hits showed binding to hγ c To further develop our best hit 21d26 (Table 5, SEQ ID NO: 627) and hγ c We used directed evolution and showed that it binds to hγ cAmong these mutants (Table 1, SEQ ID NOs: 1-8), we isolated variant 21JC15 with four mutations (W15C, W49R, Q73H, and H99G) on 21d26 (W15C is residue 15 in the X1 domain (SEQ ID NO: 936), W49R is residue 21 in the X2 domain (SEQ ID NO: 933), Q73H is residue 16 in the X3 domain (agonist form) (SEQ ID NO: 934), and H99G is residue 16 in the X4 domain (SEQ ID NO: 935)). This variant not only exhibited binding to hIL-21R and hIL-21R / hγ c Binding to mIL-21R and mIL-21R / mγ c In addition, we used site saturation mutagenesis (SSM) to mutate the interface residues and individually evaluate their effects on affinity ( Figure 5 ; Table 1, SEQ ID NO: 9-36).

[0615] agonists

[0616] Through this analysis and combining these mutations, we isolated the optimized mutant 21h10 (Table 1, SEQ ID NO: 24; Figure 1B 、 1C ), which exhibited significantly higher affinity than the parent 21JC15 (Table 1, SEQ ID NO: 8) while retaining cross-reactivity between human and mouse receptors ( Figures 1G-1J ), as well as other variants 21m1 to 21m6 and 21h1 to 21h9 (Table 1, SEQ ID NOs: 9-23). ​​In particular, 21h10 showed the best cross-reactivity and, interestingly, exhibited only 44.9% sequence identity to hIL-21 and only 23.5% sequence identity to mIL-21 as determined by BLASTP analysis. 21h10 was efficiently expressed in E. coli and showed monodispersity in size exclusion chromatography ( Figure 1D ). In addition, circular dichroism analysis confirmed the presence of helical secondary structure in the protein ( Figure 1E ) and demonstrated excellent thermal stability ( Figure 1F ).

[0617] Agonist diversification was achieved by redesigning non-interface residues and some interface residues using ProteinMPNN to allow for possible substitutions at these positions (Table 1, SEQ ID NOs: 37-163). In addition, agonists were further validated based on 21h10 (Table 1, SEQ ID NO: 24) by using SSM data (Table 1, SEQ ID NOs: 164-263; Figure 6 ) and ProteinMPNN (Table 1, SEQ ID NO: 264-432) weakened the affinity of the two interfaces and generated affinity variants. These affinity variants may achieve changes in the orientation and half-life of the receptor complex, which may affect EC50, Emax, signal transduction pathway deviation or further downstream differentiation phenotypes of natural IL-21 signal transduction. This confirms that the agonist IL-21 mimetic is stable, allowing interface engineering to achieve various affinities on the two interfaces. Natural-like high-affinity variants can be used to reproduce the full activity of natural IL-21 biology and its full human and mouse cross-reactivity, such as using these variants as substitutes for natural IL-21. Variants with lower affinity may have lower potency than natural IL-21 or have different functions from natural IL-21. However, it is useful when the IL-21 mimetic construct fused with an antibody or other targeting domain needs to be biodistributed by the target binding affinity of the fused targeting domain rather than the affinity between the fused IL-21 mimetic and the IL-21 receptor. Additionally, variants with lower affinity may have higher EC50 or lower Emax in cell signaling, which may be desirable features for use.

[0618] SEQ ID NOs: 1-8 in Table 1 are agonist mimetics derived from 21d26 (Table 5, SEQ ID NO: 627) with a combination of mutations (W15C, W49R, Q73H, and H99G), which were applied with all four mutations to generate 21JC15 (Table 1, SEQ ID NO: 8). SEQ ID NOs: 9-24 in Table 1 are agonist mimetics optimized from 21JC15 (Table 1, SEQ ID NO: 8) with forward mutations found in site-saturation mutagenesis (SSM) data. SEQ ID NOs: 25-36 in Table 1 are agonist mimetics from 21JC15 (Table 1, SEQ ID NO: 8) with a combination of mutations applied to 21h10 (Table 1, SEQ ID NO: 24). SEQ ID NOs: 37-163 in Table 1 are agonist mimetics derived from 21h10 (Table 1, SEQ ID NO: 24) that have been applied to proteins that do not contribute to IL-21R (CD360) or γ c(CD132) receptor binding interface residues. SEQ ID NO: 164-432 in Table 1 are agonist affinity variant mimetics derived from 21h10 (Table 1, SEQ ID NO: 24), which have been applied to the IL-21R (CD360) or γ-1 receptors by site saturation mutagenesis (SSM) data or ProteinMPNN. c Mutation of some of the residues in the (CD132) receptor binding interface resulted in attenuation of the interface binding affinity.

[0619] In Table 1, Kd1 is the Kd for hIL-21R, which indicates the binding affinity for hIL-21R, and Kd2 is the Kd for the IL-21 receptor complex (hIL-21R and hγ c receptor), which indicates the Kd for hγ c The binding affinity of hIL-21R and hIL-21R was measured by BLI. c The percentage of PE+FITC+ population relative to the single cell gated population under the presence of hIL-21R is shown, which indicates the presence of hγ c The binding of the receptors was measured by flow cytometry. Data using only the aforementioned concentrations are presented in Table 5, rather than all data within the 1-1000 nM range, because the concentrations used for the presented data achieve the best measurement of affinity compared to lower or higher concentrations within this range. A rise to a 1000 nM concentration is optimal for identifying binding agent hits, while a reduction to 1 nM is optimal for finding affinity-optimized variants in simulated species. Mouse receptor binding assays work identically to human receptor binding assays, and the results are similar to those of human receptors.

[0620] Retopology / shuffling and dividing cytokines

[0621] The designed agonists can be shuffled using their helical domains (X1, X2, X3 and X4) but still maintain their ability to heterodimerize the receptor subunits. They retain most of their binding affinity at both interfaces, which is required to act as agonists ( Figure 2A). It was demonstrated that X2-X3-X4-X1, X3-X4-X1-X2 and X4-X1-X2-X3 (Table 2) are engineered to maintain their given molecular structures while retaining interfaces for IL-21Rα (CD360) and γc (CD132) receptors to achieve their agonistic functions. When the helical domains were shuffled and reconnected, the polypeptide linker was a linker designed using Rosetta and ProteinMPNN computational design, or one of the (GS)n or (GGS)n linkers (n=2-10). The helical domain shuffling design binds to the IL-21 receptor, and this means that the helical domain has ultrastability and can be reorganized into any order outside the three suggested orders, retaining its interface with the IL-21 receptor, with new connections (retopology) between the helices.

[0622] The sequences of the helical domain shuffling constructs are listed in Table 2. SEQ ID NOs: 433-445 in Table 2 are agonist mimetics having helical domains shuffled in the order X2-X3-X4-X1. Similarly, SEQ ID NOs: 480-569 in Table 2 are agonist mimetics having helical domains shuffled in the order X2-X3-X4-X1 but with mutations in X1. SEQ ID NOs: 446-457 in Table 2 are agonist mimetics having helical domains shuffled in the order X3-X4-X1-X2. SEQ ID NOs: 458-479 in Table 2 are agonist mimetics having helical domains shuffled in the order X4-X1-X2-X3. TM Either ProteinMPNN, or one of the (GS)n or (GGS)n linkers (n=2-10) generated the linkers between the helical domains for reconnection.

[0623] This molecular stability is associated with the molecule's engineering potential and can be extended to split cytokine mimics to achieve conditional cytokines. IL-21 mimics are four-helix bundle proteins with three different ways of dividing the helices into two linear groups (segment X and segment Y), namely H1 / H234, H12 / H34, and H123 / H4 ( Figure 2B ; Table 4), and this can even be achieved through the reorganization of the helical domain to achieve diversification (Table 2). The purpose of these split IL-21 mimics for targeted delivery is to fuse each split fragment X and Y with the targeting domain, such as to guide the split cytokine mimics to the desired cells or tissues of interest ( Figure 2CHere, we present examples of split cytokines fused to anti-HER2-DARPin or anti-EGFR-DARPin (Table 4, SEQ ID NOs: 613-624) to demonstrate that these IL-21 mimetics can be fused to such targeting domains for use and can be reconstituted in vitro using biolayer interferometry, as described in the Methods section of the Examples ( Figure 2D ; Table 4). This confirms that the split IL-21 mimetic fragment can be fused to any available targeting domain.

[0624] The sequences of the split fragments of the IL-21 mimetic 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 NO: 24), namely X1 (Table 4, SEQ ID NO: 607), X1-X2 (Table 4, SEQ ID NO: 608), X3-X4 (Table 4, SEQ ID NO: 609), X4 (Table 4, SEQ ID NO: 610), X1-X2-X3 (Table 4, SEQ ID NO: 611), and X2-X3-X4 (Table 4, SEQ ID NO: 612). SEQ ID NOs: 613-618 in Table 4 are split fragments (Table 4, SEQ ID NOs: 607-612) fused to anti-HER2-DARPin (Table 4, SEQ ID NO: 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 a (GS)n or (GGS)n linker, with n = 2-10.

[0625] In Table 2, Kd1 is the Kd for hIL-21R, which indicates the binding affinity for hIL-21R, divided into kon1 and kdis1, all measured by BLI. kon1 is the association rate for hIL-21R, and kdis1 is the dissociation rate for hIL-21R. In Table 4, Kd2 is the Kd for the IL-21 receptor complex (hIL-21R and hγ c receptors), which indicates the Kd for hγ measured by BLI c Receptor binding affinity.

[0626] Antibody fusions

[0627] For targeting specific cells or tissues of interest, we have shown various antibody-fused IL-21 mimetic constructs (Table 3). IL-21 mimetics are fused to antibody heavy chains. For cis-action, such as targeting effector cells (such as CD8+ T cells), there are anti-human PD-1 targeting antibodies (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 antibodies (RED8) fusions (Table 3, SEQ ID NOs: 594-598) and anti-mouse CD8 targeting antibodies (G10.1) fusions (Table 3, SEQ ID NOs: 589-593). For trans-actions, such as targeting tumor tissue, there are 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 be constructed as a homodimer or heterodimer using knob / hole mutations in the antibody heavy chain ( Figure 3A ) and showed the expected size in 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 ). In addition, this construct can bind to two targets simultaneously ( Figure 3D In addition to targeting antibodies, we also prepared Fc fusions to achieve the purpose of half-life extension (Table 3, SEQ ID NOs: 570-571). This demonstrates that agonistic IL-21 mimetics can be fused to any available antibody or other targeting molecule to redirect their biodistribution.

[0628] SEQ ID NOs: 570-571 in Table 3 are Fc fragment fusion constructs in which the agonist mimetic 21AG-M-p3C1 (Table 1, SEQ ID NO: 161) is fused to a human Fc fragment (Table 3, SEQ ID NO: 599) or a murine Fc fragment (Table 3, SEQ ID NO: 600), respectively. The linker between the IL-21 mimetic and the Fc fragment is either a (GS)n or (GGS)n linker, with n = 2-10. SEQ ID NOs: 572-576, 601 in Table 3 are components used to prepare fusion constructs of the IL-21 mimetic and pembrolizumab. Two identical copies of the pembrolizumab light chain (Table 3, SEQ ID NO: 572) are paired with two identical copies or two different copies of the pembrolizumab heavy chain (Table 3, SEQ ID NOs: 573-576, 601). SEQ ID NOs: 577-581, 602 in Table 3 are components used to prepare fusion constructs of IL-21 mimetics with atezolizumab. Two identical copies of the atezolizumab light chain (Table 3, SEQ ID NO: 577) are paired with two identical copies or two different copies of the atezolizumab heavy chain (Table 3, SEQ ID NOs: 578-581, 602). SEQ ID NOs: 582-583, 603 in Table 3 are components used to prepare fusion constructs of IL-21 mimetics with TA99. Two identical copies of the TA99 light chain (Table 3, SEQ ID NO: 582) are paired with two identical copies or two different copies of the TA99 heavy chain (Table 3, SEQ ID NOs: 583, 603). SEQ ID NOs: 584-588, 604 in Table 3 are components used to prepare fusion constructs of IL-21 mimetics with anti-murine PD-1 monoclonal antibodies. Two identical copies of the anti-murine PD-1 monoclonal antibody light chain (Table 3, SEQ ID NO: 584) were paired with two identical copies or two different copies of the anti-murine PD-1 monoclonal antibody heavy chain (Table 3, SEQ ID NOs: 585-588, 604). SEQ ID NOs: 589-593, 605 in Table 3 are components used to prepare fusion constructs of an IL-21 mimetic with the anti-murine CD8 monoclonal antibody G10.1. Two identical copies of the G10.1 light chain (Table 3, SEQ ID NO: 589) were paired with two identical copies or two different copies of the G10.1 heavy chain (Table 3, SEQ ID NOs: 590-593, 605). SEQ ID NOs: 594-598, 606 in Table 3 are components used to prepare fusion constructs of an IL-21 mimetic with the anti-human CD8 monoclonal antibody RED8.Two identical copies of the RED8 light chain (Table 3, SEQ ID NO: 594) were paired with either two identical copies or two different copies of the RED8 heavy chain (Table 3, SEQ ID NOs: 595-598, 606).

[0629] In Table 3, Kd is measured for hIL-21R and antibody target (human PD-L1 in the case of atezolizumab fusion). The hIL-21R binding and hPD-L1 binding of atezolizumab-IL-21 mimic fusion constructs were tested by BLI. For hIL-21R binding assays, atezolizumab-21h10 fusion constructs (Table 3, SEQ ID NOs: 577, 578) were tested by immobilizing hIL-21R on SA tips and associating and dissociating in 0.313-20 nM fusion constructs. Figure 3C For hPD-L1 binding assays, atezolizumab-21h10 fusion constructs (Table 3, SEQ ID NOs: 577, 578) were tested by immobilizing hPD-L1 on SA tips and associating and dissociating at 0.027-20 nM of the fusion construct ( Figure 3C ). Due to its ability to bind to both hIL-21R and hPD-L1 simultaneously, in the first experiment, hPD-L1 was immobilized to the assay tip and conjugated with an atezolizumab-21h10 fusion construct (Table 3, SEQ ID NOs: 577, 578) and then with hIL-21R; in the second experiment, hIL-21R was immobilized to the assay tip and conjugated with an atezolizumab-21h10 fusion construct (Table 3, SEQ ID NOs: 577, 578) and then with hPD-L1.

[0630] antagonists

[0631] Antagonistic IL-21 mimetics bind only to IL-21R but not to γ c Binding Design. IL-21R binding was assessed by flow cytometry using 100-1000 nM human or mouse IL-21R labeled with streptavidin R-phycoerythrin conjugate (SAPE) and / or by biolayer interferometry using Avi-tagged human or mouse IL-21R loaded on streptavidin (SA) tips.

[0632] By flow cytometry, use 10-1000nM unlabeled people or mouse IL-21R and 10-1000nM with people or mouse Gc of streptavidin R-phycoerythrin conjugate (SAPE) mark, and / or by biolayer interferometry, use the people or mouse Gc with Avi tag that is loaded on streptavidin (SA) tip, assessed Gc combination.With IL-21R:IL-21 analogies=1.5:1 provide soluble unlabeled people or mouse IL-21R for test.

[0633] The generation of antagonists by design also means that IL-21 mimetics have excellent molecular stability, because the knockout of γ c Interface without destroying the overall molecular integrity or another interface (IL-21R interface) that is usually not retained by native IL-21. Based on the source or design method, there are four types of mimetics that have been experimentally tested - (i) initial antagonist hits (Table 5, SEQ ID NO: 627-631, Figure 4A ) and mimetics derived from the agonist 21h10 (Table 1, SEQ ID NO: 24) ( Figure 4B ), using (ii) Rosetta TM (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) ProteinMPNN (Table 5, 798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898 and 900-907). The resulting antagonists can be combined with IL-21R and can partially or completely not bind to γ c Binding to form an IL-21 receptor complex demonstrates that the IL-21 mimetic is stable and allows for interface mutations that achieve antagonistic properties.

[0634] SEQ ID NOs: 627-631 in Table 5 are five antagonist hits found during the initial screening 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 NO: 24) that were identified by RosettaTM In γ c - Mutations on interface residues. SEQ ID NOs: 688-751 in Table 5 (designated 21AT-Sp###) are mimetics derived from 21h10 (Table 1, SEQ ID NO: 24) based on site saturation mutagenesis (SSM) data in the γ c - Mutations on interface residues. SEQ ID NOs: 752-907 in Table 5 (named 21AT-Mp###) are mimetics derived from 21h10 (Table 1, SEQ ID NO: 24), which were synthesized by ProteinMPNN in γ c - Mutations on interface residues.

[0635] In Table 5, Q1 is the percentage of the PE+FITC+ population relative to the single cell gated population at 100 nM PE-labeled hIL-21R, which represents the binding to hIL-21R as measured by flow cytometry. Q2 is the percentage of the PE+FITC+ population relative to the single cell gated population at 100 nM PE-labeled hIL-21R as measured by flow cytometry. c The percentage of PE+FITC+ population relative to the single cell gated population under the presence of hIL-21R is shown, which indicates the presence of hγ c The binding of the receptors was measured by flow cytometry. Data using only the aforementioned concentrations are presented in Table 5, rather than all data within the 1-1000 nM range, because the concentrations used for the presented data achieve the best measurement of affinity compared to lower or higher concentrations within this range. A rise to a 1000 nM concentration is optimal for identifying binding agent hits, while a reduction to 1 nM is optimal for finding affinity-optimized variants in simulated species. Mouse receptor binding assays work identically to human receptor binding assays, and the results are similar to those of human receptors.

[0636] other

[0637] In the use of protein-based therapeutics, people often explore genetic fusion or chemical conjugation of functional domains for the purpose of extending the half-life of proteins or other applications. We designed and tested albumin binding domain fusion constructs with the goal of extending half-life (Table 6, SEQ ID NO: 908-909). We also designed a number of Cys and / or Lys repositioning variants (Table 6, SEQ ID NO: 910-931) from 21h10 (Table 1, SEQ ID NO: 24) or 21AG-M-p3C1 (Table 1, SEQ ID NO: 161). These variants can be chemically conjugated to PEG by having a chemical conjugation site within the IL-21 mimetic to achieve half-life extension or chemically conjugated to other functional domains to obtain conditional activation. 21h10 (Table 1, SEQ ID NO: 24) with Cys15 can be chemically conjugated to PEG of various sizes to achieve an increase in molecular size. This confirms that IL-21 mimetics can be fused to other functional domains as well as antibodies or the aforementioned targeting domains through genetic fusion or chemical conjugation to achieve their half-life extension or other functional benefits.

[0638] SEQ ID NOs: 908-909 in Table 6 are albumin-binding peptide fusion constructs designed for half-life extension, in which 21AG-M-p3C1 (Table 1, SEQ ID NO: 161) is fused to the albumin-binding peptide G148ABD3 (Table 6, SEQ ID NO: 932). Because the G148ABD3 domain binds to albumin in vivo, it increases the overall molecular weight, thereby extending the half-life of the construct. SEQ ID NOs: 910-931 in Table 6 are agonist mimetics derived from 21h10 (Table 1, SEQ ID NO: 24) or 21AG-M-p3C1 (Table 1, SEQ ID NO: 161), which have Lys (K) or Cys (C) added or removed from their non-interface residues (Table 6, SEQ ID NOs: 910-925) or interface residues (Table 6, SEQ ID NOs: 926-931) for chemical conjugation of functional domains, including but not limited to PEG, peptides and small molecules, for various purposes such as half-life extension and conditional activation.

[0639] method

[0640] Computational design of de novo IL-21 mimetics

[0641] The crystal structure of hIL-21 with hIL-21R (PDB: 3TGX) was used to design mimetics of native IL-21. PyRosetta with PDBInfoLabel metadata was used. TMGeneration of IL-21-like scaffolds. During scaffold generation and interface residue design, the following residues from human IL-21 were designated as fixed: 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. TM FastDesign designed with non-fixed residues and Rosetta with the 'beta_nov16' scoring function TM FastRelax Relax. Use Rosetta TM Designs were filtered using the following scoring metrics: packstat > 0.6, per-residue score < -2.3, and sspred > 0.8. Designs were filtered using fastfold to filter based on fold probability.

[0642] Yeast surface display and fluorescence-activated cell sorting

[0643] Based on the codon frequency table of Saccharomyces cerevisiae, the amino acid sequence of the designed protein was reverse transcribed into a DNA sequence, and pETcon3 was attached to the 5' and 3' ends. TM Vector homologous sequence. Using competent yeast cells EBY100, the designed DNA gene block was cloned into the linearized pETcon3 by yeast homologous recombination. TMThe vector was linearized by 100 times overdigestion of NdeI and XhoI (New England Biolabs) and then purified by gel extraction (Qiagen). Yeast transformation was verified by colony sequencing; the verified colonies were grown in SDCAA medium (2.0% glucose, 0.67% yeast nitrogen base, 0.5% casamino acids, 0.54% sodium dihydrogen phosphate, 0.86% sodium dihydrogen phosphate) and induced in SGCAA medium (2% galactose, 0.67% yeast nitrogen base, 0.5% casamino acids, 0.54% Na2HPO4, 0.86% NaH2PO4). The designed protein induced by SGCAA was expressed and presented on the yeast surface by Aga2p membrane protein. The induced cells were resuspended in running buffer (phosphate buffered saline (PBS) + 1% w / v bovine serum albumin, pH 7.2-7.4) to perform all flow cytometry analyses. The induced cell of having designed protein from the teeth outwards uses two kinds of fluorophores (FITC and PE) mark.The anti-c-mycMab mark myc tag that use is puted together with FITC represents the degree of protein expression.For the IL-21R mark, use people or mouse IL-21R (R&D Systems 991-R2, R&D Systems 596-MR) with people Fc label.For the receptor complex mark, use people IL-21R (R&D Systems 9249-R2) with His label and people γ with Fc label c (Acro Biosystems ILG-H5256), or mouse IL-21R with a His tag (Sino Biological 51184-M08H) and mouse γ with an Fc tag c (R&D Systems 784-MR). Target receptors fused to the Fc domain were labeled with the biotinylated ZZ domain of protein A and streptavidin-PE (SA-PE), which binds to the biotin of protein A. PE indicates the level of receptor binding. For binding hits, their signal is proportional to the expression level of the protein. Yeast were analyzed by flow cytometry (BD AccuriC6, Thermo Fisher Attune NxT) or sorted by fluorescence activated cell sorting (FACS) cell sorter (Sony SH800).

[0644] Directed evolution and design optimization

[0645] Use random mutagenesis library test kit (Agilent 200550), amplify 21d26 DNA, and use BL21 (DE3) (New England Biolabs) that library is carried out partial sequencing.Sequencing display, when using fallibility PCR amplification DNA, about 2-4 sudden change is applied to design.Next, use electroporation technology that library is converted into yeast, its diversity is approximately 3.4E7.Use FACS to sort for direct evolution through labeled receptor subunit with different combinations and concentration to yeast library, have carried out above-mentioned mark.

[0646] To analyze and enhance the interfacial affinity of the 21JC15 interface, we used site-saturation mutagenesis (SSM) to analyze the two interfaces of the protein in detail. The protein sequence of interest was split into two halves with corresponding single mutations and synthesized as two fragments for assembly (Integrated DNA Technologies). By PCR, the protein was cloned using pETcon3 TM Four different receptor conditions ((1) labeled human IL-21R, (2) unlabeled human IL-21R and labeled human γ c , (3) labeled mouse IL-21R, and (4) unlabeled mouse IL-21R and labeled mouse γ c ) sorting saturates mutants with favorable mutations and depletes mutants with unfavorable mutations. TM (Illumina), using Zymoprep TM DNA from the sorted SSM libraries was prepared by qPCR (Zymo Research), gel extraction (Qiagen). The sorted SSM libraries revealed several markers that could improve binding to each receptor (human IL-21R, human γ c , mouse IL-21R and mouse γ c ) binding affinity mutations.

[0647] By combining positive mutations, we constructed a combinatorial library incorporating the mutations by assembling eight primer pools using PCR (Integrated DNA Technologies), incorporating mutations with degenerate codons, and then sorting them for either human or mouse receptors. This yielded 16 candidates with significantly improved affinity for the receptor, 10 human receptor-optimized candidates (21h1-21h10) and 6 mouse receptor-optimized candidates (21m1-21m6). The Kd of the 16 candidates for each receptor was measured by biolayer interferometry (ForteBio, see methods).

[0648] Expression of cytokine mimics and mitotic cytokine mimics (E. coli)

[0649] The DNA fragment encoding the designed protein was cloned (Integrated DNA Technology) into a pET-29b(+) plasmid with an N-terminal polyhistidine tag. The cloned plasmid was transformed into competent BL21 cells (New England Biolabs), and the culture was grown in Terrific Broth II and induced with 1 mM isopropyl b-D-thiogalactopyranoside (IPTG). The culture was grown in baffled flasks in a shaker at 225 rpm at 37°C until they reached an OD of approximately A600 =0.8 for induction. The culture was induced at 18°C ​​for 14 hours. The harvested culture was ultrasonically treated (Qsonica TM The lysed culture was ultracentrifuged at 18,000 g for 30 minutes and purified using immobilized metal affinity chromatography (IMAC). The lysed culture was purified by HPLC size exclusion chromatography using Superdex TM 75 10 / 300GL column (GE Healthcare) in Akta TM (GE Healthcare AKTA TM Pure) and separate the eluted protein.

[0650] Immunocytokine (Antibody-Cytokine Fusion) Protein Expression (Mammalian Expression)

[0651] Antibody-cytokine fusion constructs were synthesized in the CMVR plasmid (Genscript). The cytokine mimetic was fused to the C-terminus of the antibody heavy chain using a linker. The fusion construct was expressed using HEK293 cells and purified by protein A capture (Genscript). SDS-PAGE and SEC-HPLC were used for purification.

[0652] Biolayer interferometry

[0653] Binding data were collected using OctetRED96 and OctetR8 (Sartorius). Octet binding buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) + 1% w / v bovine serum albumin, pH 7.2-7.4) was used for all biolayer interferometry analyses. To measure binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems AVI9249) or biotinylated mIL-21R (Acro Biosystems ILR-M82E3) were immobilized on a streptavidin-coated biosensor (SAForteBio) at 5 μg / ml in octet binding buffer until a 0.8-1.0 nm offset was loaded, and the ligand was serially diluted 2-fold starting at 1 uM, associated for 300 seconds and dissociated for 300 seconds. Similarly, to measure binding affinity to hIL-21Ra / hγ c or mIL-21Ra / mγ c The binding affinity of biotinylated hγ c (Acro Biosystems ILG-H85E8) or biotinylated mγ c (Acro Biosystems ILA-M82E3) were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg / ml in octet binding buffer and tested 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 association and 250 seconds of dissociation. For loading of polyhistidine-tagged IL-21 mimetics, anti-penta-his (His1K) tips were used for immobilization and loading to a 1.0-1.5 nm offset. For ligands, hIL-21 (R&D Systems 8879-IL), mIL-21 (R&D Systems 594-ML), or IL-21 mimetics were used (see Methods). Data were processed using ForteBio Data Analysis Software Version 9.0.0.10, and parameters are reported with standard errors.

[0654] Circular dichroism and thermal stability determination

[0655] Far-UV circular dichroism measurements were performed using a spectropolarimeter (JASCO J-1500 spectropolarimeter). The protein was dissolved in PBS (pH 7.4) at 0.5 mg / mL in a 1 mm pathlength cuvette. Wavelength scans were performed over the wavelength range of 195 nm to 260 nm. Thermal melting experiments were performed by heating the sample from 25°C to 95°C and cooling it back to 25°C while observing the absorption at 222 nm.

[0656] References

[0657] 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).

[0658] 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).

[0659] 3. Petrella, TMet al. Interleukin-21 has activity in patients with metastatic melanoma:aphase II study. J. Clin. Oncol. 30, 3396-3401 (2012).

[0660] 4.Petrella,T.M.et al.Final efficacy results of NCIC CTG IND.202:Arandomized phase II study of recombinant interleukin-21(rIL21)in patientswith recurrent or metastatic melanoma(MM).J.Clin.Oncol.(2013)doi:10.1200 / jco.2013.31.15_suppl.9032.

[0661] 5.Coquet,J.M.,Skak,K.,Davis,I.D.,Smyth,M.J.&Godfrey,D.I.IL-21Modulates Activation of NKT Cells in Patients with Stage IV MalignantMelanoma.Clin Transl Immunology 2,e6(2013).

[0662] 6.Bhatia,S.et al.Recombinant interleukin-21plus sorafenib formetastatic renal cell carcinoma:a phase 1 / 2study.J Immunother Cancer 2,2(2014).

[0663] 7.Hamming,O.J.et al.Crystal structure of interleukin-21receptor(IL-21R)bound to IL-21reveals that sugar chain interacting with WSXWS motif isintegral part of IL-21R.J.Biol.Chem.287,9454-9460(2012).

[0664] 8.Silva,D.-A.et al.De novo design of potent and selective mimics ofIL-2and IL-15.Nature565,186-191(2019).

[0665] 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).

[0666] 10. Abhiraman, G. C. et al. A structural blueprint for interleukin-21 signal modulation. Cell Rep. 42, 112657 (2023).

[0667] 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).

[0668] 12. Dauparas, J. et al. Robust deep learning-based protein sequence design using ProteinMPNN. Science 378, 49 - 56 (2022).

[0669] 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 - 5,40 (2023).

[0670] Table 1. Agonists

[0671] *Kd1 = Kd(hIL2IR)

[0672] *Kd2 = Kd(hIL21R / hGc)

[0673] *Q_{3}=Q_{2}(1 μM_hIL21R / 1 μM_hGc)

[0674] Note: In the translation of item , "5,40" should be "540" in the original text. It seems to be a typo in the original, and this translation is adjusted accordingly. Also, in the translation of item , the subscript format is used as it is more common in scientific notation representation.

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696] Table 2. Retopology / shuffling

[0697] *kon1=kon(hIL21R)

[0698] *kdis1=kdis(hIL21R)

[0699] *Kd1=Kd(hIL21R)

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713] Table 3. Antibody fusions

[0714] *LC: Antibody light chain, HC: Antibody heavy chain

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

[0724]

[0725]

[0726]

[0727]

[0728]

[0729] Table 4. Split

[0730] *Kd2-Kd(hIL21R / hGc)

[0731]

[0732]

[0733]

[0734]

[0735]

[0736] Table 5. Antagonist and other designs

[0737] *Q1=100nM_hIL21R

[0738] *Q2=1uM_hIL21R / 200nM_hGc

[0739]

[0740]

[0741]

[0742]

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751] Table 6. Others

[0752]

[0753]

Claims

1. A polypeptide comprising domains X2, X3 and optionally X4, wherein: (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO:933); (b) X3 is 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 (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934) or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO:937), wherein one, two or all three of the following conditions are true relative to SEQ ID NO:937: residue 4 is Y, residue 6 is F, and / or residue 9 is D; and (c) X4 is optional, and when present, comprises a helical structure; wherein X2, X3 and X4, when present, can be in any order in said polypeptide; wherein an amino acid linker may be present between any of the domains; and The polypeptide is capable of binding to IL-21 receptor (IL-21Rα, CD360).

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

3. The polypeptide according to claim 1 or 2, wherein the polypeptide has an amino acid selected from the following residues relative to X2 (SEQ ID NO: 933): Residue 1: G, A, S, T, Q, D, E, R, K, and H; residue 2: G, A, L, S, T, N, and Q; residue 3: A, M, Y, W, N, Q, E, R, and K; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E and H; residue 5: M, L, Q, E, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E and R; residue 8: A, L, F, S, T, E, and H; residue 9: A, V, I, and L; residue 10: E, R, and K; residue 11: V, E, K, R, and H; residue 12: A, I, and L; Residue 13: V and I; residue 14: S, E, K, and R; Residue 15: A, I, M, L and E; residue 16: C, A, and S; Residue 17: N and R; residue 18: V, S, N, E, K, R, and H; residue 19: A, V, I, and L; Residue 20: V and I; Residue 21: W, S, E, K, and R; and Residue 22: M and D.

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

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

6. The polypeptide according to any one of claims 2 to 5, wherein the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO: 934): Residue 1: P, N, D, E, R, K, and H; residue 2: G, F, Y, E, R, and K; residue 3: V, I, M, L, F, T, and Q; residue 4: A, I, L, S, T, E, R, and K; Residue 5: A, V, I, L and E; residue 6: A, V, L, E, R, and K; residue 7: M and L; residue 8: V, L, F, Y, T, N, R, and K; residue 9: G, A, V, Q, and E; residue 10: A, I, M, and L; residue 11: A, V, I, and L; residue 12: D, E, and K; residue 13: A, V, I, L, and T; residue 14: C, A, V, S, T, and E; Residue 15: A, D, E, and R; Residue 16: A, I, L, Y, W, S, Q, E, R, K and H; Residue 17: A, V, and I; Residue 18: A, L, Y, Q, E, R, and K; Residue 19: T, D, E, R, and K; and Residue 20: Y, T, N, E and H.

7. The polypeptide according to any one of claims 2 to 6, wherein the polypeptide comprises 1, 2, 3 or all 4 of the following residues relative to X3 (SEQ ID NO: 934): (a) X3 residue 2 is R or K; (b) X3 residue 6 is R or K; (c) X3 residue 8 is R or K; (d) X3 residue 12 is D or E; and / or (e) X3 residue 16 is H or Y or W.

8. The polypeptide of any one of claims 2-7, wherein the polypeptide comprises 1, 2, 3, 4, 5 or all 6 of the following residues relative to X3 (SEQ ID NO: 934): (a) X3 residue 1 is K or R; (b) X3 residue 5 is I or V; (c) X3 residue 9 is Q or G; (d) X3 residue 11 is I or L. (e) X3 residue 13 is I or V; and / or (f) X3 residue 19 is R or K.

9. The polypeptide according to any one of claims 2 to 8, wherein the polypeptide has an amino acid selected from the following residues relative to X4 (SEQ ID NO: 935): Residue 1: R and K; Residue 2: I, W, S, E, R and K; Residue 3: A, V, I, and L; residue 4: C, A, and M; residue 5: A, L, S, E, R, and K; residue 6: M, N, Q, D, E, R, and K; residue 7: A, L, and F; residue 8: Y, S, E, R, and K; residue 9: V, M, S, and T; residue 10: A, E, and K; Residue 11: C and A; Residue 12: Q and R; Residue 13: A and K; Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E and K; residue 15: I, L, and F; residue 16: G, F, and Y; Residue 17: S, R, and K; Residue 18: L, F, Y, W, and E; and Residue 19: L, F, Y and E.

10. The polypeptide according to any one of claims 2 to 9, wherein the polypeptide comprises one or two of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 9 is S or T; and / or (b) X4 residue 16 is G or Y.

11. The polypeptide according to any one of claims 2 to 10, wherein the polypeptide comprises 1, 2, 3, 4 or all 5 of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 8 is K or R; (b) X4 residue 12 is Q; (c) X4 residue 13 is K; (d) X4 residue 15 is L or I; and / or (e) X4 residue 19 is L or F / Y.

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

13. The polypeptide according to claim 1, wherein: (a) X2 is 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO:933); and (b) X3 is 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 KRFYIFMQDLIDIVTHVKRE (SEQ ID NO:937), wherein one, two or all three of the following conditions are true relative to SEQ ID NO:937: residue 4 is Y, residue 6 is F, and / or residue 9 is D; wherein X2 and X3 can be in any order in the polypeptide; wherein an amino acid linker can be present between the domains; and The polypeptide can bind to IL-21 receptor (IL-21Rα, CD360) but cannot bind to γc and CD132 receptors at the same time.

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

15. The polypeptide of claim 13, wherein relative to SEQ ID NO: 937, all three of the following conditions are true: residue 4 is Y, residue 6 is F, and / or residue 9 is D.

16. The polypeptide according to any one of claims 13 to 15, wherein the polypeptide has an amino acid selected from the following residues relative to X2 (SEQ ID NO: 933): Residue 1: G, A, S, T, Q, D, E, R, K, and H; residue 2: G, A, L, S, T, N, and Q; residue 3: A, M, Y, W, N, Q, E, R, and K; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E and H; residue 5: M, L, Q, E, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E and R; residue 8: A, L, F, S, T, E, and H; residue 9: A, V, I, and L; residue 10: E, R, and K; residue 11: V, E, K, R, and H; residue 12: A, I, and L; Residue 13: V and I; residue 14: S, E, K, and R; Residue 15: A, I, M, L and E; residue 16: C, A, and S; Residue 17: N and R; residue 18: V, S, N, E, K, R, and H; residue 19: A, V, I, and L; Residue 20: V and I; Residue 21: W, S, E, K, and R; and Residue 22: M and D.

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

18. The polypeptide of any one of claims 13-17, which has 1, 2, 3 or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 4 is I or W; (b) X2 residue 7 is V, D, or E or I; (c) X2 residue 10 is K or R; and / or (d) X2 residue 15 is A, I, L or M.

19. The polypeptide according to any one of claims 13 to 18, wherein the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO: 937): Residue 1: P, N, D, E, R, K, and H; residue 2: G, F, Y, E, and R and K; residue 3: V, I, M, L, F, T, and Q; Residue 4: Y; Residue 5: A, V, I, L and E; Residue 6: F; residue 7: M and L; Residue 8: Q and E; residue 9: D; 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.

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

21. The polypeptide of any one of claims 13-20, wherein the polypeptide has 1, 2, 3, 4 or all 5 of the following residues relative to X3 (SEQ ID NO: 937): (a) X3 residue 1 is K or R; (b) X3 residue 5 is I or V; (c) X3 residue 11 is I or L; (d) X3 residue 13 is I or V; and / or (e) X3 residue 19 is R or K.

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

23. The polypeptide according to any one of claims 13-22, further comprising an X4 domain, wherein the X4 domain does not allow the polypeptide to bind to the IL-21 receptor (IL-21Rα, CD360) and γc, CD132 receptor simultaneously, and wherein X2, X3 and X4 can be in any order in the polypeptide.

24. The polypeptide of claim 23, wherein X4 comprises a 19 residue peptide, and wherein the peptide has an amino acid at each position selected from the group consisting of: Residue 1: R and K; Residue 2: I, W, S, E, R and K; Residue 3: V; residue 4: C, A, and M; residue 5: A, L, S, E, R, and K; residue 6: M, N, Q, D, E, R, and K; residue 7: A, L, and F; residue 8: A, V, and D; residue 9: G, W, E, and K; residue 10: A, E, and K; Residue 11: C and A; Residue 12: G, I, E and K; Residue 13: V, L, Q, D, E, and R; Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E and K; residue 15: I, L, and F; residue 16: R, K, and H; Residue 17: S, R, and K; Residue 18: L, F, Y, W, and E; and Residue 19: L, F, Y and E.

25. The polypeptide of any one of claims 1-24, further comprising an X1 domain, wherein the X1 domain comprises a helical structure, and wherein X1, X2, X3 and X4, when present, can be in any order in the polypeptide.

26. The polypeptide of claim 25, wherein 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 LAEIMK EVAECARKEA (SEQ ID NO: 936).

27. The polypeptide of claim 26, wherein the polypeptide has an amino acid selected from the following residues relative to X1 (SEQ ID NO: 936): Residue 1: V, I, and L; Residue 2: A, I, S, N, R and K; residue 3: D and E; Residue 4: V and I; Residue 5: M and F; Residue 6: K; Residue 7: E; Residue 8: A and V; Residue 9: A and R; Residue 10: A, V, I, L, T, Q, E, and K; residue 11: C, V, I, M, F, Y, Q, R, and K; Residue 12: A; Residue 13: R and K; Residue 14: K; Residue 15: E; and Residue 16: A, I, M, L, F, Y, S, and N.

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

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

30. The polypeptide according to any one of claims 1 to 15, wherein the polypeptide comprises, in domain order, X4-X3-X2, X3-X2, X1-X3-X2, X2-X1-X3, X3-X2-X1, X3-X1-X2, X2-X4-X3, X3-X2-X4, X3-X4-X2, X4-X2-X3, X1-X2-X3-X4, X1-X4-X3-X2, X2-X1-X4-X3, X2-X3-X4-X1, X3-X2-X1-X4, X3-X4-X1-X2, X4-X1-X2-X3, X3-X2 or X4-X3-X2-X1.

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

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

33. A fusion protein according to 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.

34. The polypeptide or fusion protein according to any one of claims 1 to 33, comprising: (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 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, 716-717, 7-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; or (b) 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 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.

35. A conditionally active IL-21 receptor binding protein comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in the same fusion protein at the same time, wherein the first polypeptide component and the second polypeptide component together comprise the domains X2, X3 and optionally X4 as defined in any one of claims 1 to 34, wherein: (i) the first polypeptide component comprises at least one of X2, X3 and X4 (when present), but does not comprise each of X2, X3 and X4 (when present); and (ii) the second polypeptide component comprises each of X2, X3 and X4 (when present) that are not present in the first polypeptide component; The first polypeptide component and the second polypeptide component alone are not active receptor binding proteins, and the first polypeptide component and the second polypeptide component interact to form an active IL-21 receptor binding protein capable of binding to an IL-21 receptor (IL-21Rα, CD360).

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

37. The conditionally active IL-21 receptor binding protein according to claim 35, comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in the same fusion protein at the same time, wherein the first polypeptide component and the second polypeptide component collectively comprise the domains X1, X2, X3 and X4 defined in any one of claims 1 to 34; wherein: (i) the first polypeptide component comprises at least one of X1, X2, X3 and X4, but does not comprise each of X1, X2, X3 and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3 and X4 that are not present in the first polypeptide component; The first polypeptide component interacts with the second polypeptide to form an active IL-21 receptor binding protein capable of binding to IL-21 receptors (IL-21Rα, CD360) and to γc, CD132 receptors.

38. The conditionally active IL-21 receptor binding protein of any one of claims 35-37, wherein the first polypeptide and the second polypeptide together comprise: (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 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, 716-717, 7-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; or (b) 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 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.

39. The conditionally active IL-21 receptor binding protein of any one of claims 35-38, wherein the first polypeptide and the second polypeptide comprise: (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; (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.

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

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

42. The polypeptide of claim 41, wherein X1 residues 1-16 are selected from the group consisting of: Residue 1: V, I, and L; Residue 2: A, I, S, N, R and K; residue 3: D and E; Residue 4: V and I; Residue 5: M and F; Residue 6: K; Residue 7: E; Residue 8: A and V; Residue 9: A and R; Residue 10: A, V, I, L, T, Q, E, and K; residue 11: C, V, I, M, F, Y, Q, R, and K; Residue 12: A; Residue 13: R and K; Residue 14: K; Residue 15: E; and Residue 16: A, I, M, L, F, Y, S, and N.

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

44. The polypeptide of any one of claims 41-43, wherein X2 residues 1-15 are selected from the group consisting of: Residue 1: G, A, T, Q, D, R, K and H; residue 2: G, A, S, T, and N; Residue 3: A, M, Y, N, Q, E, and R; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E and H; residue 5: M, L, Q, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D and E; residue 8: F, S, T, and E; Residue 9: L; Residue 10: K; Residue 11: K; Residue 12: I; Residue 13: V and I; Residue 14: R; and Residue 15: A, I, M and L.

45. The polypeptide of any one of claims 41-44, wherein one, two, three or all four of the following conditions are met: (a) X2 residue 3 is not R; (b) X2 residue 4 is not 1; (c) X2 residue 7 is not V; and / or (d) X2 residue 8 is not S.

46. ​​The polypeptide of any one of claims 41-45, wherein X3 residues 1-20 are selected from: Residue 1: P, N, R, and K; Residue 2: F, Y, and R; residue 3: V, I, M, L, F, T, and Q; Residue 4: E; Residue 5: I; Residue 6: R; residue 7: M; residue 8: V, L, F, Y, T, and R; residue 9: G, A, and Q; 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.

47. The polypeptide of any one of claims 41-46, wherein one, two, three, four, five, six or all seven of the following conditions are met: (a) X3 residue 2 is not R; (b) X3 residue 8 is not R; (c) X3 residue 9 is not Q; (d) X3 residue 10 is not L; (e) X3 residue 11 is not 1; (f) X3 residue 14 is not V; and / or (g) X3 residue 15 is not D.

48. The polypeptide of any one of claims 41-47, wherein X4 residues 1-19 are selected from: Residue 81: K; Residue 2: E; Residue 3: V; residue 4: M; Residue 5: E; Residue 6: R; Residue 7: A; Residue 8: R and K; residue 9: V, M, S, and T; Residue 10: A; Residue 11: A; Residue 12: Q; Residue 13: K; Residue 14: C, G, A, V, I, M, L, S, T, Q, D and K; Residue 15: L; Residue 16: G; Residue 17: R and K; Residue 18: F, Y, and W; and Residue 19: L, F and Y.

49. The polypeptide of any one of claims 41-48, wherein one, two, three, four, five, six or all seven of the following conditions are met: (a) X4 residue 8 is not K; (b) X4 residue 9 is not S; and / or (c) X4 residue 19 is not L.

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

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

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

53. A polypeptide according to any one of claims 41-52, 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.

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

55. A polypeptide according to any one of claims 41-54, 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.

56. The polypeptide of claim 55, wherein the residues at the non-helical positions are selected from the group consisting of: Residue 01: V, I, M, F, Y, and D; Residue 02: A, V, I, M, L, F, Y, W, Q, D, E, R and H; Residue 03: E; Residue 04: D and E; residue 21: A, T, and E; Residue 22: R and K; residue 23: I, M, L, F, and Y; Residue 24: P, G, A, V, I, S, T, N, Q, D, R and H; residue 25: G, A, V, N, Q, and K; residue 26: T, N, R, and K; residue 27: G, A, S, T, N, D, R, K, and H; residue 28: P, E, and H; Residue 44: A and S; Residue 45: N and K; residue 46: G, A, V, S, T, N, R, K, and H; Residue 47: V and I; Residue 48: V, I, Y, Q, R, and K; residue 49: L, R, and K; residue 50: G, M, Y, S, Q, and H; Residue 51: R; residue 52: G, M, L, F, Y, W, N, D, E, and H; residue 53: G, R, and K; Residue 54: G, A, V, I, L, N, E and K; Residue 55: A, V, I, M, L, F, Y, W, T, Q and H; residue 56: D; residue 57: P, A, V, Y, W, S, D, E, and H; Residue 78: F; residue 79: A, Y, S, and K; Residue 80: S; Residue 81: E; residue 82: D; and Residue 83: L and F.

57. A polypeptide according to any one of claims 41-56, 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 NO: 1-598, 627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 716-717, 718-720, 721-723, 724-725, 726-727 17-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898 and 900-931.

58. The polypeptide of any one of claims 41-57, further comprising one or more functional domains.

59. The polypeptide of claim 58, wherein the one or more functional domains are translationally fused to the polypeptide.

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

61. The conditionally active IL-21 receptor binding protein of claim 60, wherein X1, X2, X3 and X4 each comprise an amino acid sequence according to any one of claims 52-70.

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 the polypeptide, the first polypeptide or the second polypeptide according to any one of the preceding claims.

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 according to any one of the preceding claims.

66. A pharmaceutical composition comprising the polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector and / or recombinant host cell according to any one of the preceding claims and a pharmaceutically acceptable carrier.

67. A method for treating cancer, comprising administering to a subject suffering from cancer an amount effective to treat the tumor of the polypeptide, conditionally active IL-21 receptor agonist, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector, recombinant host cell and / or pharmaceutical composition according to any one of the preceding claims.

68. The method of claim 67, wherein the cancer is selected from the group consisting of colon cancer, melanoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, gastric cancer, urothelial carcinoma, Hodgkin lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor type selected by a diagnostic test such as microsatellite instability, tumor mutational burden, PD-L1 expression level, or an immune score assay (as developed by the Cancer Immunotherapy Consortium).

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

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

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