CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of US Provisional Patent Application No. 63/244,551, filed September 15, 2021, and US Provisional Patent Application No. 63/247,179, filed September 22, 2021; each of which is incorporated by reference herein in its entirety for any purpose.

FIELD

[002] The present invention is related to, inter alia, de novo IL-2RP binding polypeptides.

BACKGROUND

[003] A de novo protein immunotherapeutic, Neoleukin-2/15 (also known as Neo-2/15), has recently been described. Neo-2/15 is a de novo protein that mimics the function of both human interleukin-2 (hIL-2) and human interleukin- 15 (hIL-15). To accomplish its biological function, Neo-2/15 induces the hetero-dimerization of two IL-2 cell membrane receptors, the IL-2 receptor beta (IL-2RP) and the IL-2 receptor common gamma IL-2Ry _c . The IL-2 heterodimeric receptor is also known as IL-2RPYc. The hetero-dimerization of IL-2RPYc caused by hIL-2 (and also Neo-2/15), initiates a signaling cascade that is responsible for stimulating the activation and proliferation of several types of immune cells (such as T-cells, among others). Unlike IL-2, Neo-2/15 signals independently of CD25 and unlike IL-15, it also signals independently of CD-215. Neo-2/15 is a highly thermostable protein that demonstrates potent IL-2 like signaling on both human and mouse cells. Neo-2/15 has been shown to have anticancer therapeutic activity in several murine models. Conversely, for other diseases, inhibiting the signaling cascade induced by dimerization of IL-2Rpy _c is desirable, and several studies have sought to exploit such a paradigm to combat inflammation-related disease using IL-2R antibodies. These therapeutic regimens have met limited success. The identification of de novo proteins that can specifically and potently block IL-2 and/or IL- 15 signaling involved in inflammation and autoimmune disease has the potential to translate into successful clinical candidates that can be used for a multitude of diseases, including those of autoimmune nature. The present disclosure addresses this and other needs. DRAWINGS

[004] Figure 1A-1B: Figure 1A demonstrates the binding of Neo-2/15, P3 and P4 to IL- 2Ry _c as measured by biolayer interferometry. Figure IB demonstrates the pSTAT5 signaling elicited by increasing concentrations of PEGylated Neo-2/15v (black triangles), P3 (filled in circles), P4 (open circles) in human Pan T cells.

[005] Figures 2A-2C demonstrates human IL-2 pSTAT5 signaling inhibition by P3 (filled-in circles) and P4 (open circles) at different concentrations of human IL-2. Y-axis represents percentage of cells stained positive for pSTAT5 signaling.

[006] Figures 3A-3C show biolayer interferometry (OCTET) binding assays of S4 (3 A), P5 (3B), and P6 (3C) against immobilized hIL-2Rp.

[007] Figures 4A-4C show biolayer interferometry (OCTET) binding assays of S4 (4A), P5 (4B), P6 (4C), and Neo-2/15 (4A-4C) in complex with hIL-2Rp, against immobilized hIL-2RYc.

[008] Figures 5A-5C show the inhibition of binding of hIL-2 to hIL-2RPYc by S4 (5 A), P5 (5B), and P6 (5C).

[009] Figures 6A-6D show percentage IL-2R pSTAT5 signaling inhibition on all T cells (6A), CD4+ T cells (6B), CD8+ T cells (6C) and Regulatory T cells (6D) by P5, P6, S4, and an anti-IL-2 IgG. Y-axis represents percent reduction of pSTAT5 signaling, normalized to maximal pSTAT5 signaling.

[0010] Figures 7A-7B show the pSTAT5 signaling ability of P6 (7 A) and P5 (7B) on all T cells, CD4+ T Cells, CD8+ T Cells and Regulatory T cells. Y-axis represents percentage of cells stained positive for pSTAT5 signaling.

[0011] Figures 8A-8D show the pSTAT5 signaling ability of S4 on Regulatory T cells (8A), CD4+CD25- T cells (8B), CD4+CD25+ T cells (8C) and CD8+ T cells (8D). Y-axis represents percentage of cells stained positive for pSTAT5 signaling.

[0012] Figures 9A-9B show the pSTAT5 signaling ability of S4 on PBMC cells (9A) and NK cells (9B). Y-axis represents percentage of cells stained positive for pSTAT5 signaling.

[0013] Figures 10A-10D show percentage IL-15R pSTAT5 signaling inhibition on all T cells (10A), CD4+ T cells (10B), CD8+ T cells (10C) and Regulatory T cells (10D) by S4 and an anti-IL-15 IgG. Y-axis represents percent reduction of pSTAT5 signaling, normalized to maximal pSTAT5 signaling. [0014] Figures 11 A-l 1C show circular dichroism (CD) of S4 (A), P5 (B), and P6 (C) at pH 8. Far UV wavelength spectra is shown at 20°C, after heating to about 98°C and after cooling the heated sample to 20°C.

[0015] Figures 12A-12C show temperature -induced unfolding curves at pH 8 for S4 (12A), P5 (12B), and P6 (12C) obtained from 20 to 98 °C by monitoring the CD signal at 222 nm.

[0016] Figures 13A-13D show biolayer interferometry (OCTET) binding assays of C1(13A), C9 (13B), CIO (13C) and C7 (13D) against immobilized hIL-2Rp.

[0017] Figures 14A-14D show biolayer interferometry (OCTET) binding assays of C1(14A), C9 (14B), CIO (14C) and C7 (14D), in complex with hIL-2Rp, against immobilized hIL-2RYc.

[0018] Figures 15A-15D show the binding inhibition of hIL-2 to hIL-2RPYc by Cl (15 A), C9 (15B), CIO (15C) and C7 (15D).

[0019] Figures 16A-6C show percentage IL-2R pSTAT5 signaling inhibition on all T cells (16A), CD4+ T cells (16B), and CD8+ T cells (16C) by S4, P5, P6, Cl, C9, CIO, C7, and an anti-IL-2 IgG. Y-axis represents percent reduction of pSTAT5 signaling, normalized to maximal pSTAT5 signaling.

[0020] Figures 17A-17C show percentage IL-15R pSTAT5 signaling inhibition on all T cells (17A), CD4+ T cells (17B), and CD8+ T cells (17C) by S4, C7, C8, C7 and C8 fusion proteins, and an anti-IL-15 IgG. Y-axis represents percent reduction of pSTAT5 signaling, normalized to maximal pSTAT5 signaling.

[0021] Figures 18A-18C show percentage IL-2R and IL-15R pSTAT5 signaling inhibition on all T cells (18A), CD4+ T cells (18B), and CD8+ T cells (18C) by C7 and C8, and an anti-IL-15 and IL-2 IgG control. Y-axis represents percentage of cells stained positive for pSTAT5 signaling.

[0022] Figures 19A-19D show aggregation levels and temperature -induced unfolding curves at pH 8 (19A), pH 6 (19B), pH 4 (19C), and pH 2 (19D) for S4 obtained from 20 to 98 °C by monitoring the CD signal at 222 nm.

[0023] Figures 20A-20D show aggregation levels and temperature -induced unfolding curves at pH 8 (20 A), pH 6 (20B), pH 4 (20C), and pH 2 (20D) for Cl obtained from 20 to 98 °C by monitoring the CD signal at 222 nm. [0024] Figures 21A-21D show aggregation levels and temperature -induced unfolding curves at pH 8 (21A), pH 6 (21B), pH 4 (21C), and pH 2 (21D) for C9 obtained from 20 to 98 °C by monitoring the CD signal at 222 nm.

[0025] Figures 22A-22D show aggregation levels and temperature -induced unfolding curves at pH 8 (22 A), pH 6 (22B), pH 4 (22C), and pH 2 (22D) for CIO obtained from 20 to 98 °C by monitoring the CD signal at 222 nm.

[0026] Figures 23 A-23D show aggregation levels and temperature -induced unfolding curves at pH 8 (23 A), pH 6 (23B), pH 4 (23C), and pH 2 (23D) for C7 obtained from 20 to 98 °C by monitoring the CD signal at 222 nm.

[0027] Figures 24A-B - show body weight (A) and survival (B) of NSG mice in a murine model of GVHD. Mice were treated with a control (PBS - filled in circles), C7i fusion construct at 20 mg/kg (filled in squares) and 2 mg/kg (filled in triangles).

[0028] Figure 25 - shows that human PBMCs stimulated with increasing concentrations of IL2 and fixed anti-CD3 induces robust proliferation (triangles) as measured by CellTiter Gio whereas increasing concentrations of C7i results in dose dependent inhibition of IL2 induced proliferation (diamonds) and C7i alone does not produce proliferation (circles ).

[0029] Figure 26 - shows human PBMCs stimulated with increasing concentrations of IL2 and fixed anti-CD3 induces robust IFN gamma production (triangles) as measured by AlphaLisa whereas increasing concentrations of C7i results in dose dependent inhibition of IL2 induced IFN gamma production (diamonds) and C7i alone does not produce IFN gamma (circles).

SUMMARY

[0030] The present inventors have identified novel methods to modulate the activity of IL- 2. In some aspects, the methods are effective at reducing (i.e. inhibiting) one or more activities of IL-2 or IL-15. In some aspects, the methods are effective at blocking one or more activities of IL-2 or IL-15. In particular, the present inventors have created polypeptides that bind to IL-2RP but have no binding site for IL-2Ra and have reduced binding affinity (including fully ablated binding) to IL-2Ry _c (as compared to IL-2 or Neo- 2/15). In some aspects, the polypeptides of the present invention have substantially the same or increased binding affinity to IL-2RP as compared to Neo-2/15. In some particularly preferred aspects, the polypeptides of the present invention have increased binding affinity to IL-2RP as compared to IL-2. [0031] In some aspects, polypeptides of the present invention act to limit (i.e., inhibit) or prevent IL-2 from binding to and co-localizing IL-2RP with IL-2Ry _c thereby limiting (i.e., inhibiting) or blocking the ability of IL-2 to signal through IL-2R. In such a manner, exemplary polypeptides of the present invention antagonize the activity of IL-2. Accordingly, in some aspects, the polypeptides of the present invention act as antagonists of the biological function of IL-2. Polypeptides that act as antagonists of the biological function of IL-2 by competing for the IL-2 receptor can also be referred to as IL-2R antagonists.

[0032] The IL- 15 receptor shares two signaling subunits with the IL-2 receptor, namely IL-2RP and IL-2Ry _c . In some aspects, exemplary polypeptides of the present invention act to limit (i.e., inhibit) or prevent IL-15 from binding to the shared IL-2Rpyc. Accordingly, in some aspects, the polypeptides of the present invention act as antagonists of the biological function of IL-15. Polypeptides that act as antagonists of the biological function of IL-15 by competing for the IL-15 receptor can also be referred to as IL-15R antagonists. [0033] Exemplary polypeptides of the present invention inhibit the binding of IL-2 to the IL-2 receptor and/or signaling via the IL-2 receptor in select IL-2RP positive cell types. In some embodiments, IL-2RP positive cell types are IL-2Ry _c positive but are either IL-2Ra positive or IL-2Ra negative. In some embodiments, polypeptides of the present invention inhibit the binding of IL-2 to the IL-2 receptor and/or signaling via the IL-2 receptor to a greater extent in cells that are IL-2Ra negative as compared to cells that are IL-2Ra positive. In some embodiments, polypeptides of the present invention inhibit the binding of IL-2 to the IL-2 receptor and/or signaling via the IL-2 receptor by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in IL-2RP positive cells that are IL-2Ra negative. In some embodiments, polypeptides of the present invention inhibit the binding of IL-2 to the IL-2 receptor and/or signaling via the IL-2 receptor by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in IL-2RP positive cells that are IL-2Ra negative and by not more than 50%, not more than 30% or by not more than 20% in IL- 2RP positive cells that are IL-2Ra positive.

[0034] In some embodiments, polypeptides of the present invention have limited ability or no ability themselves to induce the heterodimerization or dimerization of IL-2Rpyc and, as such, have a reduced ability (including to negligible and/or undetectable levels) to simulate STAT5 phosphorylation as compared to IL-2. In some embodiments, polypeptides of the present invention stimulate STAT5 phosphorylation at a level that is at least 50% less than the level that IL-2 stimulates STAT5 phosphorylation in the same type of cell. In some embodiments, polypeptides of the present invention stimulate STAT5 phosphorylation at a level that is at least 60% less, at least 70% less, at least 80% less, at least 85% less, at least 90% less, or at least 95% less than the level that IL-2 stimulates STAT5 phosphorylation in the same type of cell.

[0035] In some embodiments, polypeptides of the present invention stimulate STAT5 phosphorylation at negligible levels, including undetectable levels. In some embodiments, polypeptides of the present invention have a reduced ability (including to negligible and/or undetectable levels) to simulate STAT5 phosphorylation as compared to Neo-2/15.

[0036] Exemplary polypeptides of the present invention inhibit the ability of IL-2 and/or IL-15 to stimulate STAT5 phosphorylation in IL-2RP positive cell types. In some embodiments, polypeptides of the present invention inhibit the ability of IL-2 to stimulate STAT5 phosphorylation in IL-2RP positive cell types by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. In some embodiments, polypeptides of the present invention inhibit the ability of IL-2 to stimulate STAT5 phosphorylation by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in IL-2RP positive cells that are IL-2Ra negative. In some embodiments, polypeptides of the present invention inhibit the ability of IL-2 to stimulate STAT5 phosphorylation by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in IL-2RP positive cells that are IL-2Ra negative and by not more than 50%, not more than 40%, not more than 30% or by not more than 20% in IL-2RP positive cells that are IL-2Ra positive.

[0037] Polypeptides of the present invention were created using Neo-2/15 as a parental scaffold and, as such, possess the advantages of de novo proteins. One such advantage is increased thermostability as compared to many native proteins and derivatives thereof. [0038] Also provided herein are pharmaceutical compositions comprising polypeptides of the present invention and pharmaceutically acceptable carriers; as well as methods of using such polypeptides and pharmaceutical compositions.

[0039] Methods of antagonizing IL-2R and/or IL-15R are provided herein. Such methods comprise administering to a subject at least one polypeptide of the present invention. Also provided herein are methods of inhibiting IL-2 and/or IL-15 activity in a subject comprising administering to a subject at least one polypeptide of the present invention. Methods of treating diseases associated with IL-2 and/or IL- 15 activity in a subject are also provided. The subject can be a mammalian subject. In some embodiments, the subject is a non-human primate or a human.

[0040] Methods of making the polypeptides of the present invention are further described by using nucleic acids encoding the polypeptides, expression vectors comprising the nucleic acids, and recombinant host cells.

[0041] IL-2, IL- 15 and the IL-2 receptor can be from a mammalian source. In any of the embodiments described herein, IL-2, IL- 15 and the IL-2 receptor can be human IL-2, human IL- 15 and/or the human IL-2 receptor. In some embodiments, the IL-2 receptor is the mouse IL-2 receptor. In some aspects, the IL-2 receptor is the human IL-2 receptor.

DESCRIPTION

[0042] As used herein, “IL-2” refers to native wild-type IL-2 or recombinant IL-2. “Human IL-2” or “hIL-2” refers to native wild-type human IL-2 or recombinant IL-2 (rhIL2, or simply rIL-2, or hIL-2). The amino acid sequence of native human wild-type IL- 2 is found in the Genbank under accession locator NP 000577.2 and is as set forth in SEQ ID NO:48. (SEQ ID NO:48 - MYRMQLLSCIALSLALVTNS

APTSSSTKKTQLQLEHLLLDLQMILNGINN

YKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHL RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT).

The amino acid sequence of mature native human wild-type IL-2 lacks the N-terminal 20 amino acid signal peptide. An exemplary recombinant form of IL-2 does not have the N terminal alanine of wild-type IL-2 and serine is substituted for cysteine at amino acid position 125.

[0043] As used herein, “Neo-2/15” refers to the de novo IL-2 mimic Neo-2/15 described in Silva et al. The amino acid sequence of Neo-2/15 is as set forth in SEQ ID NO:57. (SEQ ID NO:57 -

PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQ KD EAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS).

[0044] “Numbered in accordance with Neo-2/15” or “according to the numbering of Neo- 2/15” means identifying an amino acid with reference to the position at which that amino acid occurs in the sequence of Neo-2/15, for example L17 refers to the seventeenth amino acid, leucine, that occurs in SEQ ID NO: 57.

[0045] “Affinity” or “binding affinity” refers to the strength of the sum total of non- covalent interactions between a binding site of a molecule and its binding partner. The affinity of a molecule for its partner can generally be represented by the dissociation constant (KD).

[0046] “Reduced binding” refers to a decrease in affinity for the respective interaction. The term also includes reduction of the affinity to zero (or below the detection limit of the analytic method), i.e., complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[0047] As used herein, the natural amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; 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 (Vai; V). As used herein “any amino acid” typically refers to the 20 natural amino acids. The skilled practitioner will appreciate, however, that one or more, (e.g., from 1 to 10, 1 to 5, 1 to 3, or 1 or 2) unnatural amino acids can be used in place of a natural amino acid. As used herein, the term “unnatural amino acid” refers to an amino acid other than the 20 amino acids that occur naturally in protein. Unnatural amino acids are known in the art. :

[0048] The “IL-2 receptor common gamma” or “IL-2RYc” or “ IL-2RG” refers to the IL-2 gamma receptor and is a member of the type I cytokine receptor family that is a cytokine receptor subunit to the receptor complexes for at least six different interleukin receptors including, but not limited to, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. The “hIL-2 receptor common gamma” or “hIL-2RYc” or “ hIL-2RG” refers to the human IL-2 gamma receptor. A nucleic acid sequence of the human IL-2 gamma receptor is found in Genbank under accession locator NM 000206. An amino acid sequence of the human IL-2 gamma receptor is found in Genbank under accession locator NP 000197 and is set forth in SEQ ID NO:49:

MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPL PE VQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGC QLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQL ELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRS RFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWL ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGG ALGEGPGASPCNQHSPYWAPPCYTLKPET.

[0049] “IL-2RP receptor” or “IL-2R receptor beta” or “IL-2RB receptor” refers to the IL-2 beta receptor. “hIL-2Rp receptor” or “hIL-2R receptor beta” or “hIL-2RB receptor” refers to the human IL-2 beta receptor. An amino acid sequence of the human IL-2 beta receptor is found in Genbank under accession locator NP 001333152.1 and is as set forth in SEQ ID NO:50

MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDT SCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREG VRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHY

FERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEF T TWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLK K VLKCNTPDP SKFF S QL S SEHGGD VQKWL S SPFP S S SF SPGGL APEISPLE VLER DKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYS EEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGG SGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAG PREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV.

[0050] “IL2RBG” or “IL-2RpYc” refers to the IL-2Rp and IL-2RYc heterodimer.

“hIL2RBG” or “hIL-2RpYc” refers to the hIL-2Rp and hIL-2RYc heterodimer. IL-2RpYc is also known as the intermediate affinity IL-2 Receptor.

[0051] The terms "polypeptide”, “protein” and “peptide” are used interchangeably to refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).

[0052] An “agonist” is a compound that interacts with a target to cause or promote an increase in the activation of the target.

[0053] A “partial agonist” is a compound that interacts with the same target as an agonist (and in a similar fashion/ structural-mechanism) but does not produce as great a magnitude of a biochemical and/or physiological effect as the agonist at a given concentration, even by increasing the dosage of the partial agonist.

[0054] An “IL-2 antagonist” or “IL-2R antagonist” as used herein is a compound that opposes one or more actions of IL-2 or one or more activities of IL-2. The term antagonist refers to both full antagonists and partial antagonists. For example, a “partial antagonist” is an antagonist that does not-fully interrupt the biochemical effect of IL-2, but that is sufficient to interrupt selected targeted cellular and/or physiological activities promoted by IL-2. An antagonist of IL-2 might, under certain biological scenarios, have ability to induce IL-2-like signaling on its own (i.e., pSTAT5 signaling). In some embodiments, the ability to induce IL-2-like signaling will be at a lower level than the signaling induced by IL-2.

[0055] “Operably linked” is intended to mean that the nucleotide sequence of is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). “Regulatory sequences” include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). The expression constructs of the invention can be introduced into host cells to thereby produce the polypeptides of the present invention.

[0056] The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell but are still included within the scope of the term as used herein.

[0057] Polypeptides and polynucleotides can be provided in “isolated” form. This means that they are separated from one or more components with which they occur in nature or during production. In some aspects, an isolated polypeptide is at least 50% w/w pure of other components present during its production and/or purification but does not exclude the possibility that it is combined with an excess of pharmaceutically acceptable carrier or other vehicle intended to facilitate its use.

[0058] As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun, or electroporation.

[0059] As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions.

Polypeptides

[0060] The polypeptides of the present invention are modeled on Neo-2/15 but contain amino acid substitutions that interfere with binding to the IL-2 common gamma receptor. At the same time, the polypeptides retain binding to IL-2RP either by retaining the amino acids of Neo-2/15 at the positions responsible for binding to IL-2RP or by substituting select amino acids with ones that retain the polypeptides’ ability to IL-2RP or even increase affinity for IL-2Rp. In some embodiments, when bound to IL-2RP in the same site where IL2 does, the exemplary polypeptides of the present invention inhibit IL-2’s ability to induce IL-2R beta and common gamma heterodimerization and signaling via IL-2R. In some embodiments, polypeptides of the present invention have reduced ability, including no detectable ability, to induce signaling via IL-2R as compared to IL-2 in cells that express IL-2Rp. In some embodiments, polypeptides of the present invention inhibit IL-2’s binding to and/or signaling via the intermediate affinity IL-2 receptor complex (i.e., the heterodimer of the IL-2RP and the common gamma chain) to a greater degree than they inhibit IL-2’s binding to and/or signaling via the high affinity IL-2 receptor complex (heterotrimer of the IL-2R beta, gamma, and alpha chains). In some embodiments, polypeptides of the present invention inhibit IL-15’ s binding to and/or signaling via the intermediate affinity IL-2 receptor complex (i.e., the heterodimer of the IL-2RP and the common gamma chain) to a greater degree than they inhibit IL-15’ s binding to and/or signaling via the high affinity IL- 15 receptor complex (heterotrimer of the IL-2R beta, IL-2R gamma and IL-15R alpha chains). In some embodiments, exemplary polypeptides of the present invention selectively inhibit binding of IL-2 to IL-2R, i.e., they are able to inhibit IL-2 binding to a greater degree in certain cell types than in others. Accordingly, in some particularly preferred embodiments, the polypeptides of the present invention are able to selectively modulate the activity of IL-2 and its ability to bind to and signal via IL-2R. In some aspects, polypeptides of the present invention are able to inhibit IL-2’s binding to and/or signaling via IL-2R to a greater degree in cell types that don’t express or transiently express CD25 as compared to cells that constitutively express CD25. In some aspects, polypeptides of the present invention are able to inhibit IL-2’s binding to and/or signaling via IL-2R to a greater degree in cells that don’t express CD25 or express only low to medium levels of CD25 than in those cells that express high levels of CD25. In some aspects, polypeptides of the present invention are able to inhibit IL-2’s binding to and/or signaling via IL-2R to a greater degree in CD4+CD25- cells and CD8+CD25- cells as compared to T regulatory cells. In some aspects, polypeptides of the present invention only minimally inhibit or don’t inhibit IL-2’s binding to and/or signaling via IL-2R in T regulatory cells. Because of the ability of polypeptides of the present invention to inhibit IL-2’s ability to activate and induce proliferation of CD8+ and CD4+ T cells that are involved in inflammation, autoimmunity, organ graft rejection, GVHD and other disease, they are well suited to treat diseases associated with dysfunction of CD4+CD25- T cells, and CD8+CD25- T cells. In some embodiments, they can do so while having little or no inhibitory effect on IL-2’s binding to and/or signaling via IL-2R in regulatory T cells. Accordingly, in some aspects, polypeptides of the present invention can be used to attenuate (e.g., inhibit or ablate) IL-2R signaling in certain cell types and not others. In some embodiments, IL-2 signaling will be attenuated (e.g., inhibited or ablated) in NK cells. In some embodiments, IL-2 signaling may be attenuated (e.g., inhibited or ablated) in CD8+CD25- T cells. In some embodiments, IL-2 signaling may be attenuated (e.g., inhibited or ablated) in CD4+CD25- T cells.

[0061] In some embodiments, polypeptides of the present invention bind IL-2Ry _c with an affinity that is at least 5 fold, 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 1000 fold, or at least 10,000 fold or more lower than IL-2 when tested in the same assay under the same conditions. In some embodiments, exemplary polypeptides do not detectably bind to IL-2Ry _c . The binding affinity of subject polypeptides for IL-2Ry _c can be measured using any suitable method known in the art. Suitable methods for measuring IL-2Ry _c binding, include, but are not limited to, isothermal titration calorimetry binding assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET) and surface plasmon resonance assays (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009)); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays). A preferred method for determining affinity is biolayer interferometry, for example, as described in the examples. In a particularly preferred method, binding to IL-2Ry _c is measured by determining binding to IL-2Ry _c in the presence of IL-2RB as shown in Example 6. In some particularly preferred embodiments, IL-2Ry _c is hIL-2Ry _c . [0062] In some aspects, in order to create a strong antagonist to IL-2R, it is desirable to have increased binding affinity to IL-2RP as compared to IL-2. In some aspects, polypeptides of the present invention bind IL-2RP with an affinity that is at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 500 fold, or at least 1000 fold or more fold stronger that that of IL-2 in the same assay under the same conditions. In some aspects, polypeptides of the present invention bind IL-2RP with substantially the same affinity as Neo-2/15. In some aspects, polypeptides of the present invention bind IL-2RP with an affinity that is at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 500 fold, or at least 1000 fold or more fold stronger that that of Neo-2/15 in the same assay under the same conditions. In some embodiments, a polypeptide of the present invention binds IL-2RP with a KD of 20 nM or lower, a KD of 10 nM or lower, a a KD of 5 nM or lower, or a KD of 1 nM or lower. Binding can be assessed by any suitable method known to those in the art. A preferred method for determining affinity is biolayer interferometry, for example, as described in the examples. In a particularly preferred method, binding is measured as shown in Example 6. In some particularly preferred embodiments, IL-2RP is hIL-2Rp.

STRUCTURAL AND SEQUENCE CHARACTERISTICS OF EXEMPLARY POLYPEPTIDES OF THE PRESENT INVENTION

[0063] The present inventors have identified a combination of mutations that can be made to the Neo-2/15 polypeptide to result in a polypeptide that retains binding to hIL-2Rp but has significantly reduced binding to, or no detectable binding to, hIL-2RYc.

[0064] Not only have the present inventions discovered combinations of mutations that are effective at significantly reducing binding to IL-2Ry _c , resulting in polypeptides with reduced ability to induce IL-2R signaling, they have shown that by increasing the binding to IL-2RP while at the same time decreasing and/or abolishing binding to IL-2Ry _c , they can create polypeptides with improved ability to competitively inhibit the binding of IL2 to its receptor thereby creating IL-2R antagonists. Methods of testing polypeptides of the present invention for signaling activity and/or ability to competitively inhibit IL-2 binding to IL-2R are known in the art and are described herein. One particular method of determining the ability of a polypeptide to competitively inhibit IL-2 binding to IL-2R is shown in Example 6. [0065] Accordingly, provided herein are, inter alia, polypeptides that (i) bind to IL-2RP, (ii) are designed to have no binding site for IL-2Ra and (iii) have reduced binding to, or no detectable binding to, IL-2Ry _c . In some embodiments, IL-2RP is hIL-2Rp.

[0066] In some aspects, reduction of binding is as compared to IL-2. In some aspects, reduction of binding is as compared to Neo-2/15. In some aspects, the polypeptides have increased binding affinity to IL-2Rp. In some aspects, increased binding affinity to IL-2RP is as compared to Neo-2/15. In some aspects, increased binding affinity to IL-2RP is as compared to IL-2. In some embodiments, IL-2RP is hIL-2Rp.

[0067] The polypeptides of the present invention were created using Neo-2/15 as a parental scaffold and, as such, are also de novo proteins, and are therefore non-naturally occurring proteins. In some embodiments, exemplary polypeptides of the present invention comprise at least 4 domains. In some embodiments, the domains are helical domains. IL-2 comprises 4 domains, and binding sites for IL-2Ra, IL-2RP, and IL-2Ry _c . Neo-2/15 comprises 4 domains, including binding sites to IL-2RP and IL-2Ry _c , but has no binding site for IL-2Ra.

[0068] In exemplary embodiments of the present invention, polypeptides of the present invention comprise 4 domains. The four domains are referred to herein as DI, D2, D3, and D4. In exemplary embodiments, domains DI and D3, as in Neo-2/15, interact with IL-2R via binding to IL-2Rp. Whereas in Neo-2/15, D4 is primarily responsible for interacting with IL-2R via binding to IL-2Ry _c , in the exemplary polypeptides of the present invention, a selection of amino acid residues in D4 involved in binding to IL-2Ry _c have been mutated in order to reduce binding affinity to IL-2Ry _c . The D2 domain has little interaction with the IL-2R and for this reason can have a great deal of variability in its amino acid composition on the surface.

[0069] The inventors have identified positions in Neo-2/15 that may be mutated in order to interfere with the interaction between the polypeptide and hIL-2Ry _c and create an IL-2 and/or IL- 15 antagonist. In particular, amino acid substitutions at a selection of positions 13, 17, 91, 92, 95, 96 and 99 (numbered according to SEQ ID NO: 57) may be introduced to interfere with the interactions between the polypeptide and IL-2Ry _c and create an IL-2 and/or IL-15 antagonist. In some aspects, substitutions that result in a change in amino acid charge, polarity, and/or steric hindrance may disrupt interactions (e.g., hydrophobic or ionic interactions or hydrogen bonding) between the polypeptide and hIL-2Ry _c . In exemplary embodiments, certain substitutions at positions 17, 91, 92, 95, 96 and 99 (numbered according to SEQ ID NO: 57) may create a strong IL-2 antagonist, however, the present invention also provides polypeptides with a subset of the noted mutations, including a subset of the noted mutations in combination with other mutations, for example, substitution at position 13 (numbered according to SEQ ID NO:57). In some such embodiments, polypeptides with substitutions at a subset of the noted positions may possess weaker antagonistic properties compared to a polypeptide with substitutions at all of the noted positions. Typically, at least three or at least four amino acid substitutions are selected from positions 13, 17, 91, 92, 95, 96 and 99 (numbered according to SEQ ID NO: 57) in order to substantially interfere with the interactions between the polypeptide and IL- 2Ry _c . Substitutions at positions 21 or 89, although present in some of the exemplary polypeptides, are not directly involved in binding to hIL-2RYc.

[0070] The present inventors have also demonstrated that it is possible to introduce mutations in the polypeptide at positions that interact with IL-2RP and still retain binding to IL-2RP, or even increase binding to IL-2RP, including both human and mouse IL-2Rp. For example, in order to design a polypeptide with stronger binding to human and/or mouse IL- 2RP as compared to Neo-2/15, the present inventors introduced mutations at one or more of positions 2, 3, 7, 8, 11, 14, 18, 22, 29, 30, 33, 46, 37, and 54 (numbered according to SEQ ID NO: 57). The inventors have also demonstrated that it is possible to introduce mutations at positions not involved in binding to IL-2RP and retain the polypeptide’s desired activity. Further, the inventors have demonstrated that it is possible to introduce cysteines into the polypeptide sequence and create disulfide bonds that may improve the polypeptide’s stability, as measured by, for example, increased resistance to protease degradation.

[0071] The polypeptides optionally comprise linkers between the domains. In some embodiments, a linker is composed of amino acids. Such linkers, including amino acid linkers, function to connect the four domains. They are typically not directly involved in binding and, for those reasons, there is great variability permitted in the length of the linker and the identity of the amino acids. In various embodiments, the linkers can be of any length. In some aspects, the linkers are from 1 to 100 amino acids in length, such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 2-10 or 1-5 amino acids in length. The skilled practitioner can use the teachings in the art (See, for example, Silva et al., Nature, 2019 Jan;565(7738): 186-191; T.W. Linsky et al., Science 10.1126/science.abe0075 (2020)) in combination with the teachings of the present specification to construct linkers for connecting the domains while maintaining the desirable properties of the polypeptides. In addition to variability in the length and identity of the amino acids, there is also a great deal of variability permitted in the ordering of the domains, DI, D2, D3, and D4. In various embodiments, the domains can be linked via amino acid linkers in varying order and still be properly folded and presented for binding to IL-2Rp. As noted, the order of domains in Neo-2/15 and in some of the exemplary polypeptides of the present invention is D1-D3-D2- D4. The skilled artisan will understand, however, that the domains can be re-ordered and still result in polypeptides having the desired activities. Exemplary alternative ordering of domains includes, for example, D4-D1-D3-D2, D2-D4-D1-D3, and D3-D2-D4-D1.

[0072] Included are embodiments wherein the order of the domains is DI, D3, D2 and D4, wherein there is a first linker between domains DI and D3, a second linker between domains D3 and D2, and a third linker between D2 and D4. In some aspects, the first linker is 10 amino acids in length, the second linker is 2 amino acids in length, and the third linker is 3 amino acids in length. An exemplary sequence for the first linker is VKTNSPPAEE (SEQ ID NO:51). An exemplary sequence for the second linker is DQ and an exemplary sequence for the third linker is TAS (SEQ ID NO:52).

[0073] Exemplary polypeptides of the present invention bind IL-2RP and comprise the domains DI, D2, D3, and D4 wherein:

DI comprises the amino acid sequence:

KIQX XsAEXnAL X14DAX17X18ILX21I (SEQ ID NO: 1);

D2 comprises an amino acid sequence at least 8 amino acids in length;

D3 comprises the amino acid sequence:

X33LEDYAFN FELILX46EIAR LFESG (SEQ ID NO:2); and

D4 comprises the amino acid sequence:

EDEQEEMANX89I X91X92ILX95X96WIX99S (SEQ ID NO:3) wherein DI, D2, D3 and D4 may be in any order in the polypeptide; amino acid linkers may be present between any of the domains, X7 is leucine, or glutamic acid; Xs is any amino acid; Xu is histidine or tyrosine; X14 is tyrosine or leucine; X17 is arginine, lysine, glutamic acid, aspartic acid, glutamine or asparagine; Xis is methionine, tyrosine, glutamine, asparagine, isoleucine, or leucine; X21 is any amino acid other than proline; X33 is cysteine, tyrosine, lysine, glutamic acid, or aspartic acid; X46 is glutamic acid or aspartic acid; Xs9 is alanine, arginine or lysine; X91 is arginine, lysine, aspartic acid, glutamic acid, glutamine or asparagine; X92 is arginine, lysine, aspartic acid, glutamic acid, , glutamine, asparagine, tyrosine, or phenylalanine; X95 is threonine, serine, glutamic acid, alanine, or aspartic acid; X96 is lysine, arginine, aspartic acid, glutamic acid, glutamine or asparagine; and X99 is arginine, lysine, aspartic acid, glutamic acid, glutamine or asparagine; and wherein the polypeptide contains a total of no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, no more than four, no more than three, no more than two, no more than one, or zero substitutions at amino acid positions not designated as X. Included in the present invention are embodiments wherein such polypeptides comprise an intramolecular bond. Included in the present invention are embodiments wherein such polypeptides do not comprise an intramolecular bond. Included in the present invention are embodiments wherein X17 is arginine, lysine, glutamic acid, or aspartic acid; X91 is arginine, lysine, aspartic acid, or glutamic acid; X92 is arginine, lysine, aspartic acid, or glutamic acid; X95 is threonine, serine, glutamic acid, or aspartic acid; X96 is lysine, arginine, aspartic acid, or glutamic acid; and X99 is arginine, lysine, aspartic acid, or glutamic acid. In some embodiments, at least one or at least two of the following is true: X91 is glutamic acid, aspartic acid, glutamine or asparagine; X92 is glutamic acid, aspartic acid, glutamine, asparagine, tyrosine or phenylalanine; X99 is glutamic acid, aspartic acid, glutamine or asparagine; Xrzis arginine, lysine, glutamine or asparagine; and Xse is arginine, lysine, glutamine or asparagine. In some embodiments, at least one of the following is true: X7 is not leucine, X11 is not histidine, X14 is not tyrosine, Xis is not methionine, or X46 is not glutamic acid. In some embodiments, at least 2, at least 3, at least 4, or all 5 of the following is true: X7 is not leucine, Xu is not histidine, X14 is not tyrosine, Xis is not methionine, or X46 is not glutamic acid. In some embodiments, X7 is glutamic acid. In some embodiments, Xn is tyrosine. In some embodiments, Xu is leucine. In some embodiments, X17 is glutamic acid or aspartic acid. In some embodiments, X17 is glutamic acid. In some embodiments, Xis is tyrosine or isoleucine. In some embodiments, Xis is tyrosine. In some embodiments, X7 is leucine, Xu is histidine, X14 is tyrosine, Xis is methionine, and X46 is glutamic acid. In some embodiments, X21 is alanine, asparagine, aspartic acid, arginine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, X21 is asparagine or lysine. In some embodiments, X21 is lysine. In some embodiments, Xs is histidine, tyrosine, phenylalanine, or alanine. In some embodiments, Xs is tyrosine or phenylalanine. In some embodiments, Xs is phenylalanine. In some embodiments, if the isoleucine at position 19 of SEQ ID NO: 1 is substituted, it is substituted with phenylalanine. In some embodiments, if the glutamic acid (E) at position 7 of SEQ ID NO: 1 is substituted, it is substituted with alanine. In some embodiments, if the leucine at position 10 of SEQ ID NO: 1 is substituted, it is substituted with arginine or lysine. In some embodiments, position 10 of SEQ ID NO:2 is not substituted. In some embodiments, X33 is cysteine, tyrosine, glutamic acid or aspartic acid. In some embodiments, X33 is glutamic acid or aspartic acid. In some embodiments, X33 is glutamic acid. In some embodiments, if the tyrosine at position 5 of SEQ ID NO:2 is substituted, it is substituted with histidine. In some embodiments, the alanine at position 6 of SEQ ID NO:2 is substituted with cysteine and the polypeptide comprises a second cysteine, wherein the cysteine at position 6 of SEQ ID NO:2 and the second cysteine form a disulfide bond. In some embodiments, the second cysteine is located within 40 amino acids of the C- terminus of D3. In some embodiments, X46 is aspartic acid. In some embodiments, if the serine at position 22 of SEQ ID NO:2 is substituted, it is substituted with glycine. In some embodiments, one or more of position 5 of SEQ ID NO:2, position 6 of SEQ ID NO:2, position 22 of SEQ ID NO:2, position 7 of SEQ ID NO:1, position 10 of SEQ ID NO: 1, or position 19 of SEQ ID NO: 1 is substituted. In some embodiments, Xs9 is lysine or arginine. In some embodiments, X91 is lysine or arginine. In some embodiments, X92 is lysine or arginine. In some embodiments, X95 is threonine, glutamic acid, or aspartic acid. In some embodiments, X95 is threonine or glutamic acid. In some embodiments, X95 is glutamic acid. In some embodiments, X96 is glutamic acid or aspartic acid. In some embodiments, X99 is lysine or arginine. In some embodiments, X17 is glutamic acid; X91 is arginine; X92 is lysine; X96 is glutamic acid; and X99 is arginine. All of above embodiments can be used in combination, unless the context clearly dictates otherwise. In some embodiments, a polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 38:

KIQX7X8AX10X11AL X14DAX17X18ILX21X22 VKTNSPX29X30X31E X33LEDX37X38FN FELILX46EIARLFEX54G DQK X59EAEKAKRMKEWMKRIX75TTAS EDEQEEMANX89I X91X92ILX95X96WIX99S (SEQ ID NO: 38). wherein: X7, Xs, Xu, X14, X17, Xis, X21, X33, X46, Xs9, X91, X92, X95, X96, and X91 are as described in any of the embodiments above; X10 is glutamic acid, aspartic acid, or alanine; X22 is isoleucine or phenylalanine; X29 is proline or leucine; X30 is alanine or valine; X31 is glutamic acid, aspartic acid, lysine, or arginine; X37 is tyrosine or histidine; X38 is alanine or cysteine; X54 is serine or glycine; X59 is glutamic acid, aspartic acid, lysine, or arginine; and X75 is any amino acid. In some embodiments, X31 is glutamic acid or lysine. In some embodiments, X31 is lysine. In some embodiments, X59 is aspartic acid or lysine. In some embodiments, X59 is lysine. In some embodiments, X75 is lysine. In some embodiments, X75 and X38 are cysteine, and form a disulfide bond.

[0074] As used herein, a “position” in a SEQ ID NO refers to the sequential position in the amino acid sequence identified by the SEQ ID NO, including any X residues. For example, position 12 of SEQ ID NO: 1, which has the sequence KIQX7X8AEX11AL X14DAX17X18ILX21I, is aspartic acid (underlined). The X residues are numbered according to the sequential numbering of Neo2/15, for consistency between sequences.

[0075] Exemplary polypeptides of the present invention bind IL-2RP and comprise the domains DI, D2, D3, and D4 wherein:

(a) DI comprises the amino acid sequence: KIQEFAEYAL LDAEX18ILKX22 (SEQ ID NO: 5)

(b) D2 comprises an amino acid sequence at least 8 amino acids in length;

(c) D3 comprises the amino acid sequence:

ELEDYX38FN FELILDEIAR LFESG (SEQ ID NO: 6); and

(d) D4 comprises the amino acid sequence: EDEQEEMANX89I RKILX95EWIRS (SEQ ID NO: 7)

[0076] wherein: DI, D2, D3 and D4 may be in any order in the polypeptide; amino acid linkers may be present between any of the domains; Xis is tyrosine or isoleucine; X22 is isoleucine or phenylalanine; X38 is any amino acid; Xs9 is arginine or lysine; and X95 is threonine, serine, glutamic acid, alanine, or aspartic acid; and wherein the polypeptide contains a total of no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, no more than four, no more than three, no more than two, no more than one, or zero substitutions at amino acid positions not designated as X; provided that at least 2, at least 3, at least 4, or all 5 of the following are true: (i) if there is a substitution of the glutamic acid at position 14 of SEQ ID NO:5, the substitution is to aspartic acid, lysine, arginine, asparagine or glutamine; (ii) if there is a substitution of the arginine at position 12 of SEQ ID NO:7, the substitution is to lysine, aspartic acid, glutamic acid, asparagine or glutamine; (iii) if there is a substitution of the lysine at position 13 of SEQ ID NO:7, the substitution is to arginine, aspartic acid, glutamic acid, asparagine, glutamine, tyrosine or phenylalanine; (iv) if there is a substitution of the glutamic acid at position 17 of SEQ ID NO:7, the substitution is to lysine, arginine, aspartic acid, asparagine, or glutamine; and (v) if there is a substitution of the arginine at position 20 of SEQ ID NO:7, the substitution is to lysine, aspartic acid, glutamic acid, asparagine, or glutamine. In some embodiments at least 2, at least 3, at least 4, or all 5 of the following are true: (i) if there is a substitution of the glutamic acid at position 14 of SEQ ID NO:5, the substitution is to aspartic acid; (ii) if there is a substitution of the arginine at position 12 of SEQ ID NO:7, the substitution is to lysine; (iii) if there is a substitution of the lysine at position 13 of SEQ ID NO:7, the substitution is to arginine; (iv) if there is a substitution of the glutamic acid at position 17 of SEQ ID NO:7, the substitution is to aspartic acid; and (v) if there is a substitution of the arginine at position 20 SEQ ID NO:7, the substitution is to lysine. In some embodiments, at least one, at least two or at least three of positions 14 of SEQ ID NO:5, 12 of SEQ ID NO:7, 13 of SEQ ID NO:7, 17 of SEQ ID NO:7 and 20 of SEQ ID NO:7 is not substituted. In some embodiments, Xis is tyrosine. In some embodiments, X22 is isoleucine. In some embodiments, X38 is alanine or cysteine. In some embodiments, X38 is alanine. In some embodiments, X38 is cysteine and the polypeptide comprises a second cysteine, wherein the cysteine at X38 and the second cysteine form a disulfide bond. In some embodiments, the second cysteine is located within 40 amino acids of the C-terminus of D3. In some embodiments, at least one, at least two, at least three or all four of the following is true: the amino acid at position 4 of SEQ ID NO:5 is not substituted, the amino acid at position 8 of SEQ ID NO:5 is not substituted, the amino acid at position 11 of SEQ ID NO:5 is not substituted and the amino acid at position 14 of SEQ ID NO:6 is not substituted, and any combination thereof. In some embodiments, at least one, at least two, or all three of the following is true: if there is a substitution at position 4 of SEQ ID NO:5, it is not to leucine; if there is a substitution at position 11 of SEQ ID NO:5, it is not to tyrosine, and if there is a substitution at position 14 of SEQ ID NO:6, it is not to glutamic acid, and any combination thereof. In some embodiments, position 10 of SEQ ID NO:6 is not substituted. All of above embodiments can be used in combination, unless the context clearly dictates otherwise.

[0077] Exemplary polypeptides of the present invention bind IL-2RP and comprise the domains DI, D2, D3, and D4 wherein:

(a) DI comprises the amino acid sequence: KIQEFAEYALLDAX17YILKI (SEQ ID NO: 8)

(b) D2 comprises an amino acid sequence at least 8 amino acids in length;

(c) D3 comprises the amino acid sequence:

ELEDYCFNFE LILDEIARLF ESG (SEQ ID NO: 9); and

(d) D4 comprises the amino acid sequence:

EDEQEEMANK IRKILX95EWIX99 S (SEQ ID NO:4) wherein DI, D2, D3 and D4 may be in any order in the polypeptide; amino acid linkers may be present between any of the domains; X17 and X99 are each independently selected from arginine, lysine, glutamic acid, aspartic acid, glutamine, or asparagine; and X95 is threonine, serine, glutamic acid, alanine, or aspartic acid; and wherein the polypeptide contains a total of no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, no more than four, no more than three, no more than two, no more than one, or zero substitutions at amino acid positions not designated as X. In some embodiments, X17 is selected from glutamic acid or aspartic acid and X99 is selected from arginine or lysine. In some embodiments, X17 is glutamic acid and X99 is arginine. In some embodiments, X95 is threonine, glutamic acid, or aspartic acid. In some embodiments, X95 is threonine or glutamic acid. In some embodiments, X95 is glutamic acid. In some embodiments, if there is a substitution of the lysine at position 10 of SEQ ID NO:4, it is a substitution to arginine or alanine. In some embodiments, at least 1, at least 2, or all 3 of the following are true: (i)if there is a substitution of the arginine at position 12 of SEQ ID NO:4, the substitution is to lysine, aspartic acid, glutamic acid, asparagine or glutamine; (ii) if there is a substitution of the lysine at position 13 of SEQ ID NO:4, the substitution is to arginine, aspartic acid, glutamic acid, asparagine, glutamine, tyrosine or phenylalanine; and (iii) if there is a substitution of the glutamic acid at position 17 of SEQ ID NO:4, the substitution is to lysine, arginine, aspartic acid, asparagine, or glutamine. In some embodiments, at least 1, at least 2, or at least 3 of the following are true: (i) if there is a substitution of the arginine at position 12 of SEQ ID NO:4, the substitution is to lysine; (ii) if there is a substitution of the lysine at position 13 of SEQ ID NO:4, the substitution is to arginine; and (iii) if there is a substitution of the glutamic acid at position 17 of SEQ ID NO:4, the substitution is to aspartic acid. In some embodiments, at least one, at least two, at least three or all four of the following is true: the amino acid at position 4 of SEQ ID NO:8 is not substituted, the amino acid at position 8 of SEQ ID NO:8 is not substituted, the amino acid at position 11 of SEQ ID NO:8 is not substituted and the amino acid at position 14 of SEQ ID NO:9 is not substituted, and any combination thereof. In some embodiments, at least one, at least two, or all three of the following is true: if there is a substitution at position 4 of SEQ ID NO:8, it is not to leucine; if there is a substitution at position 11 of SEQ ID NO:8, it is not to tyrosine, and if there is a substitution at position 14 of SEQ ID NO:9, it is not to glutamic acid, and any combination thereof. In some embodiments, the polypeptide comprises a second cysteine, wherein the cysteine at position 6 of SEQ ID NO:9 and the second cysteine form a disulfide bond. In some embodiments, the second cysteine is located within 40 amino acids of the C-terminus of D3. In some embodiments, position 10 of SEQ ID NO:9 is not substituted. In some embodiments, one, two, three, four, five or all six of the following is true: position 5 of SEQ ID NO:9 is substituted, position 6 of SEQ ID NO:9 is substituted, position 22 of SEQ ID NO:9 is substituted, position 7 of SEQ ID NO:8 is substituted, position 10 of SEQ ID NO:8 is substituted, or position 19 of SEQ ID NO:8 is substituted is substituted. In some embodiments, DI comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or 100% identical to the amino acid sequence KIQEFAEYALLDAEYILKI (SEQ ID NO: 10). In some embodiments, D3 comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or 100% identical to the amino acid sequence ELEDYAFNFELILDEIARLFESG (SEQ ID NO: 11) or ELEDYCFNFELILDEIARLFESG (SEQ ID NO: 9). In some embodiments, D4 comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or 100% identical to the amino acid sequence EDEQEEMANKIRKILEEWIRS (SEQ ID NO: 12). All of above embodiments can be used in combination, unless the context clearly dictates otherwise.

[0078] Exemplary polypeptides of the present invention bind IL-2RP and comprise the domains DI, D2, D3, and D4 wherein:

(a) DI comprises the amino acid sequence: KIQEFAEYALLDAX17YILKI (SEQ ID NO: 8)

(b) D2 comprises an amino acid sequence at least 8 amino acids in length;

(c) D3 comprises the amino acid sequence:

ELEDYCFNFE LILDEIARLF ESG (SEQ ID NO: 9); and

(d) D4 comprises the amino acid sequence:

EDEQEEMANX89 IX91X92ILX95X96WIX99 S (SEQ ID NO:3) wherein DI, D2, D3 and D4 may be in any order in the polypeptide; amino acid linkers may be present between any of the domains; X17, X91, X96, and X99 are each independently selected from arginine, lysine, glutamic acid, aspartic acid, glutamine, or asparagine; X92 is selected from arginine, lysine, glutamic acid, aspartic acid, glutamine, asparagine, tyrosine, or phenylalanine; X89 is lysine or arginine; and X95 is threonine, serine, glutamic acid, alanine, or aspartic acid; and wherein the polypeptide contains a total of no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, no more than four, no more than three, no more than two, no more than one, or zero substitutions at amino acid positions not designated as X. In some embodiments, X91 is lysine or arginine. In some embodiments, X92 is lysine or arginine. In some embodiments, X96 is glutamic acid or aspartic acid. In some embodiments, X99 is lysine or arginine. In some embodiments, X17 is glutamic acid or aspartic acid. In some embodiments, X17 is glutamic acid. In some embodiments, X95 is threonine, glutamic acid, or aspartic acid. In some embodiments, X95 is threonine or glutamic acid. In some embodiments, X95 is glutamic acid. In some embodiments, X17 is glutamic acid; X91 is arginine; X92 is lysine; X96 is glutamic acid; and X99 is arginine. In some embodiments, at least one, at least two, at least three or all four of the following is true: the amino acid at position 4 of SEQ ID NO:8 is not substituted, the amino acid at position 8 of SEQ ID NO:8 is not substituted, the amino acid at position 11 of SEQ ID NO:8 is not substituted and the amino acid at position 14 of SEQ ID NO:9 is not substituted, and any combination thereof. In some embodiments, at least one, at least two, or all three of the following is true: if there is a substitution at position 4 of SEQ ID NO:8, it is not to leucine; if there is a substitution at position 11 of SEQ ID NO:8, it is not to tyrosine, and if there is a substitution at position 14 of SEQ ID NO:9, it is not to glutamic acid, and any combination thereof. In some embodiments, the polypeptide comprises a second cysteine, wherein the cysteine at position 6 of SEQ ID NO: 9 and the second cysteine form a disulfide bond. In some embodiments, the second cysteine is located within 40 amino acids of the C-terminus of D3. In some embodiments, position 10 of SEQ ID NO:9 is not substituted. In some embodiments, one, two, three, four, five or all six of the following is true: position 5 of SEQ ID NO:9 is substituted, position 6 of SEQ ID NO:9 is substituted, position 22 of SEQ ID NO:9 is substituted, position 7 of SEQ ID NO:8 is substituted, position 10 of SEQ ID NO:8 is substituted, or position 19 of SEQ ID NO:8 is substituted. In some embodiments, DI comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or 100% identical to the amino acid sequence KIQEFAEYALLDAEYILKI (SEQ ID NO: 10). In some embodiments, D3 comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or 100% identical to the amino acid sequence ELEDYAFNFELILDEIARLFESG (SEQ ID NO: 11) or ELEDYCFNFELILDEIARLFESG (SEQ ID NO: 9). In some embodiments, D4 comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or 100% identical to the amino acid sequence EDEQEEMANKIRKILEEWIRS (SEQ ID NO: 12). All of above embodiments can be used in combination, unless the context clearly dictates otherwise.

[0079] Exemplary polypeptides of the present invention bind IL-2RP and comprise the domains DI, D2, D3, and D4 wherein:

(a) DI comprises an amino acid sequence at least 70% identical to the amino acid sequence:

KIQEFAEYAL LDAEYILKI (SEQ ID NO: 10) or

PNKKIQEFAE YALLDAEYIL KI (SEQ ID NO: 13)

(b) D2 comprises an amino acid sequence at least 8 amino acids in length; (c) D3 comprises an amino acid sequence at least 70% identical to the amino acid sequence:

ELEDYCFNFE LILDEIARLF ESG (SEQ ID NO: 9); and

(d) D4 comprises an amino acid sequence at least 70% identical to the amino acid sequence:

EDEQEEMANK IRKILEEWIR S (SEQ ID NO: 12) wherein DI, D2, D3 and D4 may be in any order in the polypeptide; and amino acid linkers may be present between any of the domains. In some embodiments, at least one, at least two, at least three or all four of the following is true: the amino acid at position 4 of SEQ ID NO: 10 is not substituted, the amino acid at position 8 of SEQ ID NO: 10 is not substituted, the amino acid at position 11 of SEQ ID NO: 10 is not substituted and the amino acid at position 14 of SEQ ID NO:9 is not substituted, and any combination thereof. In some embodiments, at least one, at least two, or all three of the following is true: if there is a substitution at position 4 of SEQ ID NO: 10, it is not to leucine; if there is a substitution at position 11 of SEQ ID NO: 10, it is not to tyrosine, and if there is a substitution at position 14 of SEQ ID NO:9, it is not to glutamic acid and any combination thereof. In some embodiments, DI comprises at amino acid sequence at least 78%, at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, at least one, at least two, at least three or all four of the following is true: the amino acid at position 7 of SEQ ID NO: 13 is not substituted, the amino acid at position 11 of SEQ ID NO: 13 is not substituted, the amino acid at position 14 of SEQ ID NO: 13 is not substituted and the amino acid at position 14 of SEQ ID NO:9 is not substituted, and any combination thereof. In some embodiments, at least one, at least two, or all three of the following is true: if there is a substitution at position 7 of SEQ ID NO: 13, it is not to leucine; if there is a substitution at position 14 of SEQ ID NO: 13, it is not to tyrosine, and if there is a substitution at position 14 of SEQ ID NO:9, it is not to glutamic acid, and any combination thereof. In some embodiments, DI comprises an amino acid sequence at least 77%, at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, D3 comprises an amino acid sequence at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, D4 comprises an amino acid sequence at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, at least 3, at least 4, at least 5, or all 6 of the following are true: (i) if there is a substitution of the glutamic acid at position 14 of SEQ ID NO: 10 or position 17 of SEQ ID NO: 13, the substitution is to aspartic acid, lysine, arginine, asparagine or glutamine; (ii) if there is a substitution of the glutamic acid at position 16 of SEQ ID NO: 12, it is a substitution to threonine, serine, alanine, or aspartic acid; (iii) if there is a substitution of the arginine at position 12 of SEQ ID NO: 12, the substitution is to lysine, aspartic acid, glutamic acid, asparagine or glutamine; (iv) if there is a substitution of the lysine at position 13 of SEQ ID NO: 12, the substitution is to arginine, aspartic acid, glutamic acid, asparagine, glutamine, tyrosine or phenylalanine; (v) if there is a substitution of the glutamic acid at position 17 of SEQ ID NO: 12, the substitution is to lysine, arginine, aspartic acid, asparagine, or glutamine; and (vi) if there is a substitution of the arginine at position 20 of SEQ ID NO: 12, the substitution is to lysine, aspartic acid, glutamic acid, asparagine, or glutamine, and any combination thereof. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5 or all 6 of the following are true: (i) if there is a substitution of the glutamic acid at position 14 of SEQ ID NO:10 or position 17 of SEQ ID NO: 13, the substitution is to aspartic acid; (ii) if there is a substitution of the glutamic acid at position 16 of SEQ ID NO: 12, it is a substitution to threonine, serine, alanine, or aspartic acid; (iii) if there is a substitution of the arginine at position 12 of SEQ ID NO: 12, the substitution is to lysine; (iv) if there is a substitution of the lysine at position 13 of SEQ ID NO: 12, the substitution is to arginine; (v) if there is a substitution of the glutamic acid at position 17 of SEQ ID NO: 12, the substitution is to aspartic acid; and (vi) if there is a substitution of the arginine at position 20 of SEQ ID NO: 12, the substitution is to lysine; and any combination thereof. In some embodiments, if there is a substitution of the lysine at position 10 of SEQ ID NO: 12, the substitution is to arginine. In some embodiments, the cysteine at position 6 of SEQ ID NO:9 is not substituted and the polypeptide comprises a second cysteine, wherein the cysteine at position 6 of SEQ ID NO:9 and the second cysteine form a disulfide bond. In some embodiments, the second cysteine is located within 40 amino acids of the C- terminus of D3. All of above embodiments can be used in combination, unless the context clearly dictates otherwise.

[0080] In any of the above embodiments, D2 can be at least 19 amino acids in length. In any of the above embodiments, D2 can comprise an amino acid sequence at least 84%, at least 89%, at least 94%, or 100% identical to the amino acid sequence KDEAEKAKRMKEWMKRIKT (SEQ ID NO: 46), or KKEAEKAKRMKEWMKRIKT (SEQ ID NO: 47). In any of the above embodiments, D2 can comprise an amino acid sequence at least 84%, at least 89%, at least 94%, or 100% identical to the amino acid sequence KKEAEKAKRMKEWMKRICT (SEQ ID NO: 14) or KDEAEKAKRMKEWMKRICT (SEQ ID NO: 15). In some embodiments, D2 comprises an amino acid sequence at least 84%, at least 89%, at least 94%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14, the cysteine at position 18 of SEQ ID NO: 14 is not substituted and the polypeptide comprises a second cysteine, wherein the cysteine at position 18 of SEQ ID NO: 14 and the second cysteine form a disulfide bond. In some embodiments, D2 comprises an amino acid sequence at least 84%, at least 89%, at least 94%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15, the cysteine at position 18 of SEQ ID NO: 15 is not substituted and the polypeptide comprises a second cysteine, wherein the cysteine at position 18 of SEQ ID NO: 15 and the second cysteine form a disulfide bond. In some embodiments, the second cysteine is present in D3. In some embodiments, position 13 of SEQ ID NO: 14 and SEQ ID NO: 15 is not substituted. All of above embodiments can be used in combination, unless the context clearly dictates otherwise.

[0081] In any of the above embodiments, DI, D3, and D4 can be each at least 19 amino acids in length. In any of the above embodiments, the amino acid linkers can be 1-100, 1- 90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, or 2-10 amino acids in length. In any of the above embodiments, the order of the four domains can be D1-D3-D2-D4. In some embodiments, DI is not:

KIQLXsAEHAL YDAX17MILX21I (SEQ ID NO: 16), KIQLYAEHAX13 YDAX17MILNI (SEQ ID NO: 17), PKKKIQLYAE HALYDAEMIL KF (SEQ ID NO: 18), or

PKEKIQLYAE HALYDAEMIL KF (SEQ ID NO: 19); wherein: X13 is arginine, lysine, or leucine; Xs is any amino acid; X17 is glutamic acid or aspartic acid; and X21 is an amino acid. In any of the above embodiments, the polypeptide can optionally comprise an intramolecular disulfide bond. In various embodiments, the disulfide bond can be formed, for example, between cysteine residues located in any of domains DI, D2, D3, and/or D4. [0082] Exemplary polypeptides of the present invention comprises an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 20-26 and 53-54: KIQEFAEYALLDAEYILKIVKTNSPPAEEELEDYAFNFELILDEIARLFESGDQKDE

AEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:20)

KIQEFAEYALLDAEYILKIVKTNSPPAEEELEDYCFNFELILDEIARLFESGDQKDE AEKAKRMKEWMKRICTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:21)

KIQEFAEYALLDAEYILKIVKTNSPPAKEELEDYAFNFELILDEIARLFESGDQKK EAEKAKRMKEWMKRIKTTASEDEQEEMANKIRKILEEWIRS (SEQ ID NO:22)

KIQEFAEYALLDAEIILKIVKTNSPPAEEELEDYAFNFELILDEIARLFESGDQKDE AEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:23)

KIQEFAEYALLDAEYILKFVKTNSPPAEEELEDYAFNFELILDEIARLFESGDQKD EAEI<AI<RMI<EWMI<RII<TTASEDEQEEMANRIRI<ILE EWIRS (SEQ ID NO:24)

KIQEFAEYALLDAEIILKFVKTNSPPAEEELEDYAFNFELILDEIARLFESGDQKDE AEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:25)

KIQEFAEYALLDAEYILKIVKTNSPPAKEELEDYCFNFELILDEIARLFESGDQKKE AEKAKRMKEWMKRICTTASEDEQEEMANKIRKILEEWIRS. (SEQ ID NO:26).

KIQLYAEHALYDAEMILKIVKTNSPPAEEELEDYAFNFELILEEIAR LFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRI RKILEEWIRS (SEQ ID NO:53)

KIQLFAEHALYDAEMILKIVKTNSPPAEEELEDYAFNFELILEEIAR LFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANKIRKILEEWIRS (SEQ ID NO: 54)

In some embodiments, at least four, at least five, at least six or all seven of positions 14, 86, 88, 89, 93, 96, and 92 and any combination thereof of any one of SEQ ID NOs: 20-26 or SEQ ID Nos: 53-54 are not substituted. In some embodiments, at least one, at least two, at least three or all four of positions 4, 8, 11, or 43 and any combination thereof of any one of SEQ ID NO: 20-26 or SEQ ID Nos: 53-54 are not substituted. In some embodiments, there is a cysteine at position 35 and position 72 of any one of SEQ ID NO: 20-26 and the two cysteines can form a disulfide bond. In some embodiments, there is no substitution at position 39 of any one of SEQ ID NO: 20-26 or SEQ ID Nos: 53-54. In some embodiments, there is no substitution at position 67 of any one of SEQ ID NO: 20-26 or SEQ ID Nos: 53- 54. In some embodiments, the polypeptide comprises at least two amino acids N-terminal and attached to the amino acid at position 1, wherein the two amino acids are lysine-lysine, asparagine-lysine, glutamine-lysine, arginine-lysine, lysine-glutamic acid, asparagineglutamic acid, glutamine-glutamic acid, arginine-glutamic acid, lysine-aspartic acid, asparagine-aspartic acid, glutamine-aspartic acid, or arginine-aspartic acid, wherein the position numbering is according to SEQ ID NO: 20. In some embodiments, the polypeptide comprises lysine-lysine, asparagine-lysine, or arginine-lysine N-terminal and attached to the amino acid at position 1, wherein the position numbering is according to SEQ ID NO: 20. All of above embodiments can be used in combination, unless the context clearly dictates otherwise.

[0083] Exemplary polypeptides of the present invention comprises an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 27-37, 39- 45, and 55-56:

PNKKIQEFAEYALLDAEYILKIVKTNSPPAEEELEDYAFNFELILDEIARLFESGDQ KDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:27)

PNKKIQEFAEYALLDAEYILKIVKTNSPPAEEELEDYCFNFELILDEIARLFESGDQ KDEAEKAKRMKEWMKRICTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:28)

PNKKIQEFAEYALLDAEYILKIVKTNSPPAKEELEDYAFNFELILDEIARLFESGD QKKEAEKAKRMKEWMKRIKTTASEDEQEEMANKIRKILEEWIRS (SEQ ID NO:29)

PNKKIQEFAEYALLDAEIILKIVKTNSPPAEEELEDYAFNFELILDEIARLFESGDQ

KDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO: 30) PNKKIQEFAEYALLDAEYILKFVKTNSPPAEEELEDYAFNFELILDEIARLFESGD

QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID

NO:31)

PNKKIQEFAEYALLDAEIILKFVKTNSPPAEEELEDYAFNFELILDEIARLFESGDQ

KDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:32)

PNKKIQEFAEYALLDAEYILKIVKTNSPPAKEELEDYCFNFELILDEIARLFESGDQ

KKEAEKAKRMKEWMKRICTTASEDEQEEMANKIRKILEEWIRS (SEQ ID NO:33)

PKKKIQEFAAYALLDAEYILKIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQ KDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:34)

PKKKIQEAAEYALLDAEQILKIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQ

KDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO: 35)

PKKKIQLYAEHALYDAEMILKIVKTNSPPAEEELEDYAFNFELILDEIARLFESGD QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:36)

PKKKIQLYAEHALYDAEMILKIVKTNSPPAEEELEDYAFNFELILDEIARLFESGD QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:37)

PKKKIQLYAEHALYDAEIILKIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQ KDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO: 39)

PNKKIQLYAEHALYDAEMILKIVKTNSPPAEEELEDYAFNFELILEEIARLFESGD QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:40)

PRKKIQLYAEHALYDAEMILKIVKTNSPPAEEELEDYAFNFELILEEIARLFESGD QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:41)

PKKKIQLYAEHALYDAELILKIVKTNSPPAEEELEDHAFNFELILEEIARLFEGGD QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:42) PKEKIQLYAEHALYDAEMILKIVKTNSPPAEEELEDYAFNFELILEEIARLFESGD

QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIR (SEQ ID NO:43)

PKKKIQLYAEHALYDAEMILKIVKTNSPLVEEELEDYAFNFELILEEIARLFESGD QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:44)

PNKKIQLYAEHALYDAEMILKFVKTNSPPAEEELEDYAFNFELILEEIARLFESGD QKDEAEKAKRMKEWMKRIKTTASEDEQEEMANRIRKILEEWIRS (SEQ ID NO:45)

PKKKIQLYAE HALYDAEMIL KIVKTNSPPA EEELED YAFN FELILEEIAR LFESGDQKDE AEI<AI<RMI<EW MKRIKTTASE DEQEEMANRI RKILEEWIRS (SEQ ID NO:55).

PKKKIQLF AE HALYDAEMIL KIVKTNSPPA EEELED YAFN FELILEEIAR LFESGDQKDE AEI<AI<RMI<EW MKRIKTTASE DEQEEMANKI RKILEEWIRS (SEQ ID NO:56)

[0084] In some embodiments, at least four, at least five, at least six or all seven of positions 17, 89, 91, 92, 96, 99, and 95 and any combination thereof of any one of SEQ ID NOs: 27-37, 39-45 or SEQ ID NOs: 55-56 are not substituted. In some embodiments, at least one, at least two, at least three, at least four or five of positions 2, 7, 11, 14 and 46 and any combination thereof of any one of SEQ ID NOs: 27-37, 39-45 or SEQ ID NOs: 55-56 are not substituted. In some embodiments, there is a cysteine at position 38 and position 75 of any one of SEQ ID NOs: 27-37, 39-45 or SEQ ID NOs: 55-56 and the two cysteines can form a disulfide bond. In some embodiments, there is no substitution at position 70 of any one of SEQ ID NOs: 27-37, 39-45 or SEQ ID NOs: 55-56. In some embodiments, there is no substitution at position 42 of any one of SEQ ID NOs: 27-37, 39-45 or SEQ ID NOs: 55-56. In some embodiments, the polypeptide comprises an intramolecular disulfide bond. All of above embodiments can be used in combination, unless the context clearly dictates otherwise.

[0085] Exemplary polypeptides of the present invention include a mutant Neo-2/15 polypeptide comprising at least five amino acid substitutions, wherein each of the at least five amino acid substitutions is at a position corresponding to position 17, 91, 92, 95, 96, or 99 of Neo-2/15 (SEQ ID NO:57), wherein the mutant Neo-2/15 polypeptide does not detectably bind IL-2Ry _c in the presence of IL-2RP or binds IL-2Ry _c in the presence of IL- 2RP with at least 5 fold, at least 10 fold, at least 100 fold, at least 1000 fold or at least 10,000 fold lower affinity than does IL-2. In some embodiments, the polypeptide comprises two additional substitutions to cysteine residues, wherein the cysteine residues form an intramolecular disulfide bond. In some embodiments, the polypeptide comprises at least one additional substitution at at least one position corresponding to position 7, 11, 14, or 18 of Neo-2/15, wherein the mutant Neo-2/15 has increased affinity for IL-2 receptor beta as compared to a Neo-2/15 polypeptide. In some embodiments, the polypeptide comprises at least two additional substitutions at at least two positions corresponding to positions 7, 8, 11, 14, or 18 of Neo-2/15, wherein the mutant Neo-2/15 has increased affinity for IL-2 receptor beta as compared to a Neo-2/15 polypeptide. In some embodiments, the polypeptide comprises one, two or three substitutions at one, two or three positions corresponding to position 2, 8 or 33 of Neo-2/15, wherein the mutant Neo-2/15 has increased affinity for IL-2 receptor beta as compared to a Neo-2/15 polypeptide. In some embodiments, the polypeptide comprises a substitution at a position corresponding to position 89 of Neo-2/15.

[0086] In some embodiments, the polypeptide is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or 95% identical to Neo-2/15 (SEQ ID NO: 57). In some embodiments, the polypeptide has 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to Neo-2/15 (SEQ ID NO: 57).

POLYPEPTIDE VARIABILITY

[0087] In order to retain the optimal binding and functional characteristics of the polypeptides, in some embodiments, it may be desirable that no amino acids are added or deleted within domains DI, D3, and D4 of the polypeptides of the present invention. The teachings provided herein can be used to determine the optimal sites for mutating amino acid residues and retaining their desired functional characteristics, i.e., diminished binding to IL-2RG and retained binding to IL-2Rp.

[0088] The term “identity”, as used herein in reference to polypeptide sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained (including gaps if necessary). Identity may be calculated, in various embodiments, using published techniques and widely available computer programs, such as the GCG program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured, for example, using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), using the default parameters. Unless indicated otherwise, percent identity is determined across the length of the reference sequence.

[0089] In some aspects, amino acid substitutions relative to the reference peptide domains may be conservative amino acid substitutions. As used herein, “conservative amino acid substitution” means a given amino acid can be replaced by an amino acid having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity is retained. Amino acids can be grouped according to similarities in the properties of their side chains (see, e.g., A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Gly (G), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Vai; Leu to He or to Vai; Lys to Arg, to Gin or to Glu; Met to Leu, to Tyr or to He; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Vai, to He or to Leu. [0090] In some aspects, a polypeptide of the present invention is multivalent (e.g. bivalent, trivalent, tetravalent), which means it comprises two or more IL-2RP binding units and as such, can bind two or more IL-2 receptors. In some such aspects, two or more amino acid sequences described herein are joined together via a linker to create a multivalent polypeptide.

[0091] As noted above, exemplary polypeptides of the present invention comprise 4 domains, DI, D2, D3, and D4. In some aspects, the 4 domains joined together are 85-300 amino acids in length, 85-200 amino acids in length, 85-120 amino acids in length, 90-300 amino acids in length, 90-200 amino acids in length, 90-120 amino acids in length, 95-300 amino acids in length, 95-200 amino acids in length, or 95-120 amino acids in length.

[0092] In a further aspect, the present disclosure provides antibodies that selectively bind to the polypeptides of the disclosure. The antibodies can be polyclonal, monoclonal antibodies, humanized antibodies, and fragments thereof, and can be made using techniques known to those of skill in the art. As used herein, “selectively bind” means preferential binding of the antibody to the protein of the disclosure, as opposed to one or more other biological molecules, structures, cells, tissues, etc., as is well understood by those of skill in the art.

Activity of the Exemplary Polypeptides of the Present Invention

[0093] Polypeptides of the present invention are designed to have differential activity as compared to IL-2 and/or Neo-2/15. Certain polypeptides of the present invention are IL-2 and/or IL-15 receptor antagonists (e.g., hIL-2 and hIL-15 receptor antagonists). In exemplary embodiments, polypeptides of the present invention inhibit IL-2 binding to the IL-2 receptor py _c heterodimer in vitro and/or in vivo. In exemplary embodiments, polypeptides of the present invention inhibit IL-2 signaling in vitro and/or in vivo. In some embodiments, the inhibition of IL-2 binding and/or signaling is selective. In some aspects, inhibition of IL-2 binding and/or signaling is more pronounced in CD25 negative cells or cells with low or medium levels of CD25 than in cells with high levels of CD25. In some aspects, IL-2 binding and/or signaling is inhibited in CD4 ⁺ T CD8 ⁺ T cells that are CD25- but to a lesser extent in CD25 ⁺ T regulatory cells. In some aspects, IL-2 binding and/or signaling are not inhibited or minimally inhibited in CD25 ⁺ T regulatory cells.

[0094] In exemplary embodiments, polypeptides of the present invention have a reduced ability to stimulate STAT5 phosphorylation as compared to Neo-2/15 and/or IL-2 in select cell types. In some aspects, stimulation of STAT5 phosphorylation by a polypeptide of the present invention is at a level that is at least 50% less, at least 75% less, at least 90% less, or at least 95% less than the level of STAT5 phosphorylation stimulated by IL-2 in that same cell type. In some aspects, stimulation of STAT5 phosphorylation by a polypeptide of the present invention is at level that is at least 50% less, at least 75% less, at least 90% less, or at least 95% less than the level of STAT5 phosphorylation stimulated by Neo-2/15 in that same cell type. In some aspects, polypeptides of the present invention do not detectably stimulate STAT5 phosphorylation in select IL-2RPYc positive cell types. In some embodiments, the cell is a T cell (for example, a CD8 ⁺ CD25‘ T cell, a CD4 ⁺ CD25‘ T cell, or a NK cell). In some embodiments, the cell is an effector T cell. STAT5 signaling can be measured by any method known in the art, including, for example, using a method shown in the examples. For example, STAT5 phosphorylation can be measured using antibodies specific for phosphorylated STAT5 and flow cytometry analysis. In some aspects, the polypeptides of the present invention inhibit or prevent IL- 15 from stimulating STAT5 phosphorylation in select cell types (such as a CD8+ T cell or NK cell).

[0095] In some aspects, polypeptides of the present invention block more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70% more than 80%, or more than 90% of IL-2 binding to IL-2R as determined using assays known in the art. In some embodiments, IL-2R is the intermediate affinity IL-2 receptor. In some aspects, polypeptides of the present invention block more than 20%, more than 30% more than 40%, more than 50%, block more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of IL-2 binding to IL-2R in CD4 ⁺ CD25‘ cells, and CD8 ⁺ CD25‘ cells but not in T regulatory cells.

[0096] Included herein are polypeptides that have (i) decreased (including negligible) ability to bind hIL-2Ry _c and hIL-2Rpy _c (ii) increased human IL-2 beta receptor (hIL-2RP) binding affinity as compared to IL-2 and/or Neo-2/15, and/or (iii) act as IL-2R and/or IL- 15R antagonists. In some aspects, such exemplary polypeptides act as competitive inhibitors of IL-2 and/or IL-15. Without intending to be bound by any particular theory, polypeptides of the invention may function by interfering with the binding of hIL-2 to the hIL-2 receptor and inhibiting the heterodimerization of the hIL-2R beta and common gamma receptors. Because IL- 15 signals via this pathway as well, exemplary polypeptides of the present invention may, in some embodiments, act as dual antagonists for both IL-2 and IL- 15. [0097] Exemplary polypeptides of the present invention possess improved characteristics of de novo proteins with respect to thermostability as compared to native proteins and variants thereof. Benefits of increased stability may include the elimination of requirement for cold chain storage and a tolerance of mutations, genetic fusions, and chemical modifications.

[0098] In some embodiments, the polypeptides have a pepsin digestion half-life of at least 200 minutes, at least 300 minutes, at least 400 minutes, at least 500 minutes, at least 800 minutes, at least 1000 minutes at pH 2. Pepsin digestion half-life at pH 2 can be measured using any suitable method known in the art. In a particularly preferred method, pepsin digestion half-life at pH 2 is measured as shown in the examples.

[0099] In some embodiments, the polypeptides have a melting temperature (Tm) at pH 8 of at least 60°C, at least 70°C, or at least 80°C. Melting temperatures at pH 8 can be measured using any suitable method known in the art. In a particularly preferred method, melting temperatures at pH 8 are measured as shown in the examples.

[00100] In some embodiments, the polypeptides have a melting temperature (Tm) at pH 2 of at least 60°C, at least 70°C, or at least 80°C. Melting temperatures at pH 2 can be measured using any suitable method known in the art. In a particularly preferred method, melting temperatures at pH 2 are measured as shown in the examples.

[00101] In some embodiments, the polypeptides maintain or recover at least 70%, 80%, or 90% of their folded structure after thermal stability testing, and/or maintain or recover at least 80% of their ellipticity spectrum after thermal stability testing, and/or maintain or recover at least 70% or 80% of their antagonistic activity after thermal stability testing. In some embodiments, thermal stability is measured by circular dichroism (CD) spectroscopy at 222 nM. In some embodiments, the thermal stability test comprises heating the polypeptide from 25°C to 95°C in a one hour time frame, and cooling the polypeptide to 25°C in a 5 minute time frame. In some aspects, ellipticity of the polypeptide at 222 nm is monitored. In some aspects, after cooling the polypeptides, the ability of the polypeptide to act as an antagonist of the biological function of IL-2 is tested.

Nucleic Acids, Expression Vectors and Host Cells.

[00102] In a further aspect, the present invention provides nucleic acids, including isolated nucleic acids, encoding polypeptides of the present invention. The nucleic acid sequence may comprise RNA or DNA. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the invention.

[00103] In another aspect, the present invention provides recombinant expression vectors comprising the nucleic acid of any aspect of the invention. In some aspects, the nucleic acid is operatively linked to a suitable control sequence. “Recombinant expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the invention are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors include but are not limited to, plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector (including but not limited to a retroviral vector or oncolytic virus), or any other suitable expression vector. In some embodiments, the expression vector can be administered in the methods of the disclosure to express the polypeptides in vivo for therapeutic benefit.

[00104] In a further aspect, the present disclosure provides host cells that comprise the nucleic acids and recombinant expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the invention, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2 ^nd Ed. (R.L Freshney. 1987. Liss, Inc. New York, NY)). A method of producing a polypeptide according to the invention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide. The expressed polypeptide can be recovered from the cell free extract, but preferably they are recovered from the culture medium.

Fusion Proteins and Conjugates

[00105] Exemplary polypeptides of the present invention can be prepared as fusion or chimeric proteins that include a polypeptide of the present invention and a heterologous polypeptide. In some embodiments, heterologous polypeptides can increase the circulating half-life of the resultant chimeric polypeptide in vivo, and may, therefore, further enhance the properties of the proteins of the present invention. In various embodiments, the polypeptide that increases the circulating half-life may be a serum albumin, such as human serum albumin, or the Fc region of an IgG subclass of antibodies. Exemplary Fc regions can include one or more mutations that inhibit complement fixation and/or Fc receptor binding or may be lytic, i.e., able to bind complement or to lyse cells via another mechanism, such as antibody-dependent complement lysis. In some embodiments, a Fc region is a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain produced by digestion of IgG with papain. The fusion proteins can include the entire Fc region, or a smaller portion that retains a desired activity, such as the ability to extend the circulating half-life of a chimeric polypeptide of which it is a part. In addition, full-length or fragmented Fc regions can be variants of the wild-type molecule. That is, they can contain mutations that may or may not affect the function of the polypeptides. For example, a Fc region may have effector function or may be modified as to have one or more activities associated with effector function reduced or completely eliminated. Effector function refers to certain biological activities attributable to the Fc region of an immunoglobulin, which may vary with the immunoglobulin isotype. Examples of effector function include, but are not limited to, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors, and B cell activation.

[00106] In some exemplary embodiments, the polypeptides of the present invention comprise an IgGl, IgG2, IgG3, or IgG4 Fc region. In some exemplary embodiments, the polypeptides of the present invention comprise a variant IgGl, IgG2, IgG3, or IgG4 Fc region. In some aspects, the variant Fc region lacks effector function. In some embodiments, amino acid mutations have been made to the Fc region in order to silence or reduce effector function. One example of amino acids that have been reported to eliminate complement binding and fixation as well as ADCC activity are the LALA-PG mutations (L234A, L235A, P329G).

[00107] In other embodiments, the polypeptides of the present invention may be linked to other types of stabilization compounds to promote an increased half-life in vivo.

Accordingly, polypeptides of the present invention can comprise one or more stabilizing agents.

[00108] In some aspects, a polypeptide of the present invention is linked to a targeting domain. The targeting domain can direct cellular localization of the polypeptide of the present invention. For example, in some aspects, it might be desirable to target the polypeptides of the present invention to inflammatory cells involved in autoimmune disease.

[00109] When a targeting domain is a polypeptide, the targeting domain can be any suitable polypeptide that bind to one or more targets of interest and can be attached or associated with a polypeptide of the present invention. In non-limiting embodiments, the targeting domain may include but is not limited to an scFv, a F(ab), a F(ab’)2, a B cell receptor (BCR), a DARPin, an affibody, a monobody, a nanobody, diabody, an antibody (including a monospecific or bispecific antibody); a cell-targeting oligopeptide including but not limited to RGD integrin-binding peptides, de novo designed binders, aptamers, a bicycle peptide, conotoxins, small molecules such as folic acid, and a virus that binds to the cell surface. The targeting domain may be covalently or non-covalently bound to the protein.

[00110] In another embodiment, the targeting domain, when present, is a translational fusion with the protein. In this embodiment, the protein and the targeting domain may directly about each other in the translational fusion or may be linked by a polypeptide linker suitable for an intended purpose. Exemplary such linkers include, but are not limited, to those disclosed in WO2016178905, WO2018153865 (in particular, at page 13), and WO 2018170179 (in particular, at paragraphs [0316]-[0317]). Methods of making fusion proteins and conjugates are known in the art and not discussed herein in detail.

Methods of Treatment

[00111] In certain embodiments, polypeptides described herein are useful for the treatment of one or more conditions wherein suppression of one or more IL-2 and/or IL- 15 dependent functions is desirable.

[00112] The present disclosure provides, inter alia, methods for modulating an immune response in a subject by administering to the subject a polypeptide of the present invention. [00113] As used herein, an "immune response" refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus. In some embodiments, the response is specific for a particular antigen (an "antigen-specific response"), such as a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor. In some embodiments, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response. In some embodiments of the compositions and methods described herein, an immune response being modulated is T-cell mediated. Methods of measuring an immune response are known in the art and include, for example, measuring pro- inflammatory cytokines such as IL-6, IL- 12 and TNF-alpha as well as co-stimulatory molecules, such as CD80, CD86, and chemokine receptor.

[00114] The polypeptides of the present invention can be used, for example, to treat diseases associated with IL- 15 and/or IL-2 activity. The polypeptides of the present invention can be used, for example, to treat a subject, e.g., a human subject, who is suffering from a disease associated with IL- 15 and/or IL-2 activity. In some embodiments, the disease associated with IL- 15 and/or IL-2 activity is an autoimmune disease. In some embodiments, the polypeptides of the present invention are used to treat an autoimmune disease in a subject. Autoimmune diseases include, but are not limited to the following: (1) a rheumatic disease such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome or Behcet's disease (2) type II diabetes (3) an autoimmune disease of the thyroid, such as Hashimoto's thyroiditis or Graves' Disease (4) an autoimmune disease of the central nervous system, such as multiple sclerosis, myasthenia gravis, or encephalomyelitis (5) a variety of phemphigus, such as phemphigus vulgaris, phemphigus vegetans, phemphigus foliaceus, Senear-Usher syndrome, or Brazilian phemphigus, (6) psoriasis, (7) inflammatory bowel disease (e.g., ulcerative colitis or Crohn's Disease) and (8) celiac disease. In some embodiments, treatment of the subject is via administration of a polypeptide of the present invention.

[00115] In some embodiments, the disease associated with IL-15 and/or IL-2 activity is an inflammatory disease and the polypeptides of the present invention are used to treat the inflammatory disease in a subject. In some embodiments, the disease associated with IL- 15 and/or IL-2 activity is uveitis and the polypeptides of the present invention are used to treat uveitis in a subject.

[00116] In some embodiments, a polypeptides of the present invention can be used, for example, to treat a subject, e.g., a human subject, who has received a transplant of biological materials, such as an organ, tissue, or cell transplant. For example, the polypeptides of the invention may be particularly suitable in promoting graft survival (allograft or xenograft) and/or in treating patients with graft versus host disease. In some embodiments, treatment of the subject is via administration of a polypeptide of the present invention.

[00117] The polypeptides of the present invention can also be used, for example, as research tools to study the differential effects of IL-2 and/or IL- 15 agonism and antagonism.

Pharmaceutical Compositions

[00118] The polypeptides of the present invention can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings. Administration can be systemic or local. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In one aspect, the IL-2 receptor agonists are administered parenterally. In yet another aspect, the IL-2 receptor agonists are administered intravenously or subcutaneously. In specific embodiments, it can be desirable to administer an IL-2 receptor agonist locally to the area in need of treatment. In one embodiment, administration can be by direct injection at the site. In one aspect, the polypeptide are delivered orally.

[00119] Pharmaceutical compositions can be formulated to improve the bioavailability of the polypeptides of the present invention upon administration of the composition to a patient. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of polypeptides of the present invention, the manner of administration, and the composition employed.

[00120] The polypeptides of the present invention may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use.

[00121] In order to treat disease, the polypeptides of the present invention are provided in a therapeutically effective amount. This refers to an amount of the polypeptide effective for treating the disease or having the desired effect. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Dosage regimens can be adjusted by clinicians to provide the optimum desired response. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the polypeptides can include a single treatment or, can include a series of treatments.

[00122] The following examples are provided to describe certain embodiments of the invention provided herein and are not to be construed to as limiting.

EXAMPLES

[00123] IL-2RP binding proteins were recombinantly expressed and purified from E. coli. Genes encoding the designed protein sequences were synthesized and cloned into pET- 28b(+) E. coli plasmid expression vectors (GenScript, N-terminal 6xHis tag and thrombin cleavage site). Plasmids were then transformed into chemically competent E. coli Lemo21 cells (NEB). Protein expression was performed using Terrific Broth and M salts, cultures were grown at 37°C until OD ⁶⁰⁰ reached approximately 0.8, then expression was induced with 1 mM of isopropyl P-D-thiogalactopyranoside (IPTG), and temperature was lowered to 18 °C. After expression for approximately 18 hours, cells were harvested and lysed with a Microfluidics Ml 10P microfluidizer at 18,000 psi, then the soluble fraction was clarified by centrifugation at 24,000 g for 20 minutes. The soluble fraction was purified by Immobilized Metal Affinity Chromatography (Qiagen) followed by FPLC size-exclusion chromatography (Superdex 75 10/300 GL, GE Healthcare). The purified proteins were characterized by Mass Spectrum (MS) verification of the molecular weight of the species in solution (Thermo Scientific), Size Exclusion - MultiAngle Laser Light Scattering (SECMALLS) in order to verify monomeric state and molecular weight (Agilent, Wyatt), SDS- PAGE, and endotoxin levels.

[00124] Protein 1 (Pl) comprises the same sequence as Neo-2/15 (SEQ ID NO: 57) except for the following mutations: L13R, L17E, Q95T, and F99R.

[00125] Protein 2 (P2) comprises the same sequence as Neo-2/15 (SEQ ID NO: 57) except for the following mutations: L17E, N21K, A89R, I91R, T92K, Q95E, S96E, and F99R. [00126] Protein 3 (P3) comprises the same sequence as Pl except for two additional mutations: H8Y, and K33E.

[00127] Protein 4 (P4) comprises the same sequence as P2 except for two additional mutations: H8Y, and K33E. P4 is also referred to herein as S4.

[00128] Protein 5 (P5) comprises the same sequence as Neo-2/15 (SEQ ID NO: 57) except for the following mutations: L17E, N21K, A89K, I91R, T92K, Q95E, S96E, and F99R. [00129] Protein 6 (P6) comprises the same sequence as P5 except for two additional mutations: Y8F and K33E.

[00130] Neo-2/15 used in the examples is as set forth in SEQ ID NO:57. As used herein PEGylated Neo-2/15v refers to a PEGylated variant of Neo-2/15 wherein the PEG molecule is attached to an introduced cysteine at position 62.

EXAMPLE 1 - Polypeptides Pl and P2 demonstrated little or no binding to the human IL-2 gamma receptor and significantly reduced pSTAT5 signaling as compared to Neo-2/15.

[00131] The affinity for hIL2 receptor gamma was calculated from binding and dissociation kinetics at different protein concentration. hIL2 receptor gamma was immobilized in the surface of anti-human IgG Fc biosensors (ForteBio). For this purpose, sensors were soaked in samples containing 20 nM of hIL2RG-fc chimera (AcroBiosystems) for 300 sec. Subsequently, sensors were dipped in five-fold serial dilutions (5000 nM - 1.6 nM) of Neo- 2/15, Pl or P2, and binding was measured for 600 sec. Finally, sensors were removed from the protein samples and dipped in buffer solutions to promote and measure dissociation (1500 sec). The buffer used to prepare all the samples and soak the sensors was HBS-EP+ from GE Healthcare, which contains 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05 % v/v surfactant P20, pH 7.4. Data were acquired at 30 °C using an Octet RED96e system (ForteBio) and processed using the instrument’s integrated software. Kinetics were fitted to a 1 : 1 binding model after subtracting a buffer signal baseline and the Kd values were calculated. Kd values were estimated from response vs protein concentration plots and are shown in Table 1 below.

[00132] The affinity for hIL2 receptor beta was calculated from binding and dissociation kinetics at different protein concentration. hIL2 receptor beta was immobilized in the surface of anti-human IgG Fc biosensors (ForteBio). For this purpose, sensors were soaked in samples containing 20 nM of hIL2RB-fc chimera (AcroBiosystems) for 300 sec. Subsequently, sensors were dipped in five-fold serial dilutions (1000 nM - 0.32 nM) of Neo-2/15, Pl or P2, and binding was measured for 600 sec. Finally, sensors were removed from the protein samples and dipped in buffer solutions to promote and measure dissociation (1500 sec). The buffer used to prepare all the samples and soak the sensors was HBS-EP+ from GE Healthcare, which contains 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05 % v/v surfactant P20, pH 7.4. Data were acquired at 30 °C using an Octet RED96e system (ForteBio) and processed using the instrument’s integrated software.

Kinetics were fitted to a 1 : 1 binding model after subtracting a buffer signal baseline and the Kd values were calculated. Kd values were estimated from response vs protein concentration plots and are shown in Table 1 below.

[00133] Approximately 2xl0 ⁵ YT-1 CTLL-2 cells were plated in each well of a 96-well plate and re-suspended in RPMI complete medium containing serial dilutions of Neo-2/15, Pl or P2. Cells were stimulated for 15 min at 37°C and immediately fixed by addition of formaldehyde to 1.5% and 10 min incubation at room temperature. Permeabilization of cells was achieved by resuspension in ice-cold 100% methanol for 30 min at 4°C. Fixed and permeabilized cells were washed twice with FACS buffer (phosphate-buffered saline [PBS] pH 7.2 containing 0.1% bovine serum albumin) and incubated with Alexa Fluor® 647- conjugated anti-STAT5 pY694 (BD Biosciences) diluted in FACS buffer for 2 hours at room temperature. Cells were then washed twice in FACS buffer and MFI was determined on a CytoFLEX flow cytometer (Beckman-Coulter). Dose-response curves were fitted to a logistic model and half-maximal effective concentration (ECso values) were calculated using GraphPad Prism data analysis software after subtraction of the mean fluorescence intensity (MFI) of unstimulated cells and normalization to the maximum signal intensity. Pl and P2 demonstrated significantly reduced pSTAT5 signaling as compared to Neo-2/15.

Table 1

[00134] A Neo-2/15 mutant with an alanine in position 95 was also made to evaluate the contribution of residue Q95 in the interaction with the IL-2 gamma receptor. Biolayer interferometry experiments demonstrated that the Neo-2/15 Q95A mutant had reduced binding for the IL-2 gamma receptor (data not shown).

[00135] These results indicate that the interaction with hIL2RG was successfully disrupted in Pl and P2, nevertheless, the affinity for hIL2RB needs to be improved in order to obtain an ideal hIL2 antagonist.

EXAMPLE 2 - Polypeptides P3 and P4 demonstrated increased binding to IL2 receptor beta and decreased binding to IL2 receptor gamma as compared to Neo-2/15 [00136] The affinity for hIL2 receptor beta was calculated from binding and dissociation kinetics at different protein concentration. hIL2 receptor beta was immobilized in the surface of anti-human IgG Fc biosensors (ForteBio). For this purpose, sensors were soaked in samples containing 20 nM of hIL2RB-fc chimera (Symansis) for 300 sec. Subsequently, sensors were dipped in two-fold serial dilutions (200 nM - 6.1 uM) of Neo-2/15, P3 or P4, and binding was measured for 600 sec. Finally, sensors were removed from the protein samples and dipped in buffer solutions to promote and measure dissociation (1500 sec). The buffer used to prepare all the samples and soak the sensors was HBS-EP+ from GE Healthcare, which contains 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05 % v/v surfactant P20, pH 7.4. Data were acquired at 30 °C using an Octet RED96e system (ForteBio) and processed using the instrument’s integrated software. Kinetics were fitted to a 1 : 1 binding model after subtracting a buffer signal baseline and the Kd values were calculated. Kd values were estimated from response vs protein concentration plots and are shown in Table 2 below. [00137] hIL2 receptor gamma binding kinetics were measured for Neo-2/15, P3 and P4. hIL2RG was immobilized in the surface of anti-human IgG Fc biosensors (ForteBio), for this purpose, sensors were soaked in samples containing 20 nM of hIL2RG-fc chimera (Symansis) for 300 sec. Subsequently, sensors were dipped in 200 nM of Neo215, P3 or P4 + 200 nM hIL2RB-fc chimera (Symansis) , and binding was measured for 600 sec. All samples were prepared using HBS-EP+ buffer from GE Healthcare, which contains 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05 % v/v surfactant P20, pH 7.4. Data were acquired at 30 °C using an Octet RED96e system (ForteBio) and processed using the instrument’s integrated software. See Figure 1A and Table 2.

[00138] The mutations made to Pl and P2 to arrive at P3 and P4 resulted in proteins with higher affinity for hIL2RB.

Table 2

EXAMPLE 3 - Polypeptides P3 and P4 demonstrated little to no pSTAT5 signaling on Human Pan T cells.

[00139] Human pan T cells were purified from PBMC and frozen for later use. Cells were thawed and rested overnight by culturing in X-VIVO 15 media (Lonza) without IL-2. The following day, the T cells were enumerated and plated at 50,000 cells per well in 96 well plates. P3 and P4 proteins were added to T cells starting at final concentration of 1 uM, titrating 8 points with 1 : 10 dilutions to 0.01 pM. As a positive signaling control, PEGylated Neo-2/15v was added to cells starting at a final concentration of 100 nM, titrating 8 points with 1 : 10 dilutions to 0.01 pM. Cultures were incubated at 37°C for 20 minutes before fixing cells in wells by adding formaldehyde to a final concentration of 1.5% by volume. Cells were fixed for 10 minutes at room temperature, centrifuged, and resuspended in 200 uL ice cold methanol to permeabilize the cells. Methanol was washed out twice with FACS buffer before adding anti-human pSTAT5 pY694 (BD Biosciences) diluted 1 :25 in FACS buffer. Cells were incubated with antibody for 1 hour before washing twice and resuspending in 150 uL FACS buffer. Cells were acquired on a Guava EasyCyte HT flow cytometer (Millipore) and analyzed with FlowJo software (FlowJo LLC) to determine the percentage of cells demonstrating phosphorylated STAT5. Results were graphed in Prism software (GraphPad). P3 and P4 demonstrated little to no pSTAT5 signaling (Figure IB).

EXAMPLE 4 - Polypeptides P3 and P4 competitively inhibited binding of Neo-2/15 to the human IL-2 gamma receptor

[00140] Binding to human or mouse IL2GB was measured for Neo-2/15 in the presence of different concentrations of P3 and P4. hIL2RG or mIL2RG was immobilized in the surface of anti-human IgG Fc biosensors (ForteBio), for this purpose, sensors were soaked in samples containing 20 nM of hIL2RG-fc chimera (Symansis) for 300 sec. Subsequently, sensors were dipped in in twofold serial dilutions of P3 or P4 (200 uM - 6.1 uM) + 20 nM of Neo215 + 50 nM hIL2RB-fc chimera (Symansis) , and binding was measured for 600 sec. All samples were prepared using HBS-EP+ buffer from GE Healthcare, which contains 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05 % v/v surfactant P20, pH 7.4. Data were acquired at 30 °C using an Octet RED96e system (ForteBio) and processed using the instrument’s integrated software. IC50 values were estimated from response vs antagonist concentration plots. The binding of Neo-2/15 to human IL-2 receptor gamma and beta was inhibited by both P3 and P4. The binding of Neo-2/15 to mouse IL-2 receptor gamma and beta was inhibited by both P3 and P4, but to a lesser degree. ICso values are shown in Table 3.

Table 3

EXAMPLE 5 - Polypeptides P3 and P4 inhibited human IL-2 pSTAT5 Signaling on Human Pan T cells.

[00141] Human pan T cells were purified from PBMC and frozen for later use. Cells were thawed and rested overnight by culturing in X-VIVO 15 media (Lonza) without human IL- 2. The following day, the T cells were enumerated and plated at 50,000 cells per well in 96 well plates. P3 and P4 were added to T cells starting at final concentration of 100 nM, titrating 8 points with 1 :5 dilutions to roughly 1 pM. Recombinant human IL-2 (R&D Systems) was then added to cultures at a final concentration of 10 nM. Cultures were incubated at 37 °C for 20 minutes before fixing cells in wells by adding formaldehyde to a final concentration of 1.5% by volume. Cells were fixed for 10 minutes, centrifuged and resuspended in 200 uL ice cold methanol to permeabilize the cells. Methanol was washed out twice with FACS buffer before adding anti-human pSTAT5 pY694 (BD Biosciences) diluted 1 :25 in FACS buffer. Cells were incubated with antibody for 1 hour before washing twice and resuspending in 150 ul FACS buffer Cells were acquired on a Guava EasyCyte HT flow cytometer (Millipore) and analyzed with FlowJo Software (FlowJo LLC) to determine the percentage of cells demonstrating phosphorylated STAT5. P3 and P4 proteins inhibited human IL-2 pSTAT5 at shown concentrations (Figures 2A-C) . ICso values are shown in Table 4

Table 4

EXAMPLE 6: Polypeptides P4, P5 and P6 demonstrated high binding affinity to hlL- 2RB, no detectable binding to hIL-2RG, and inhibited binding of hIL-2 to hIL-2RBG [00142] Binding of P4, P5, and P6 to human IL2 receptor beta (hIL2RB) was analyzed by biolayer interferometry. Biotinylated hIL2RB molecules were immobilized to Streptavidin sensors (SA, ForteBio) at 2 pg/mL in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk). After baseline measurement in the binding buffer alone, the binding kinetics were monitored by dipping the biosensors in wells containing two-fold dilutions of the corresponding protein (20 to 0.62 nM) for 500sec (association) and then dipping the sensors back into baseline wells for 500 sec (dissociation). Data were collected at 25 °C in an Octet RED96 (ForteBio) and processed using the instrument’s integrated software; kinetics were globally fit using a 1 : 1 binding to calculate the reported KD values. See figures 3 A-C

[00143] Binding of P4, P5, and P6 to human common gamma receptor (hIL2RG) was analyzed by biolayer interferometry. Biotinylated hIL2RG molecules were immobilized to Streptavidin sensors (SA, ForteBio) at 2 pg/mL in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk). After baseline measurement in the binding buffer alone, the binding kinetics were monitored by dipping the biosensors in wells containing 200 nM of protein and 200 nM of hIL2RB for 300 sec. Data were collected at 25 °C in an Octet RED96 (ForteBio) and processed using the instrument’s integrated software. See figures 4A-C

[00144] hIL2 binding inhibition assays were performed by biolayer interferometry. Biotinylated hIL2RG molecules were immobilized to Streptavidin sensors (SA, ForteBio) at 2 pg/mL in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk). After baseline measurement in the binding buffer alone, the binding kinetics were monitored by dipping the biosensors in wells containing 50 nM of hIL2, 200 nM of hIL2RB, and increasing concentrations of antagonist (0 to 200 nM). Data were collected at 25 °C in an Octet RED96 (ForteBio) and processed using the instrument’s integrated software. Binding kinetics were fit using a one-association model to calculate the signal values once the equilibrium is reached (Req). The plot of Req vs the antagonist concentration was fit using a one-site binding model to calculate the ICso. See figures 5A-C.

EXAMPLE 7: Polypeptides P4, P5 and P6 inhibit IL-2R signaling in T cells

[00145] Human pan T cells were purified from whole blood and rested overnight by culturing in X-VIVO 15 media (Lonza) without human IL-2. The following day, the T cells were enumerated and plated at 100,000 cells per well in 96 well plates. Anti-IL-2 IgG, P5, P6, and S4 were added to T cells starting at final concentration of 100 nM, titrating 8 points with 1 :10 dilutions to 0.01 pM and incubated at 37 °C for 30 minutes. Recombinant human IL-2 (R&D Systems) was then added to cultures at a final concentration of 1 nM. Cultures were incubated at 37 degrees Celsius for 30 minutes before fixing cells in wells by adding formaldehyde to a final concentration of 1.5% by volume. Cells were fixed for 10 minutes at room temperature, centrifuged, and resuspended in 200 uL ice cold methanol to permeabilize the cells. Methanol was washed out twice with FACS buffer before adding antibodies (BD Biosciences) for anti-human pSTAT5 pY694 diluted 1 :20, anti-human CD4 L200 diluted 1 :50, anti-human CD8a SKI diluted 1 :20, anti-human CD25 M-A251 diluted 1 :20, and anti-human CD127 HIL-7RM21 diluted 1 : 10 in FACS buffer. Cells were incubated with antibody for 1 hour before washing twice and resuspending in 100 ul FACS buffer. Cells were acquired on a Cytek Aurora flow cytometer (Cytek Biosciences) and analyzed with FlowJo Software (FlowJo LLC) to determine the percentage of cells demonstrating phosphorylated STAT5. Live cells were gated on FSC and SSC plots. CD4 ⁺ cells and CD8 ⁺ cells were gated on live cells, and regulatory T cells were defined as the CD25 ⁺ CD127 ^low subset of CD4 ⁺ cells. Anti-IL-2 IgG, P5, P6, and S4 proteins inhibited human IL-2 pSTAT5 signaling at shown concentrations (Figures 6A-D).

[00146] Polypeptides of the invention (P4, P5 and P6) were not as effective at inhibiting murine IL-2R signaling (data not shown). This is believed to be due to weaker binding to the mouse IL-2R as compared to hIL-2R. S4 was determined to have a Kd of 70.6 nM by biolayer interferometry (371 nM measured by yeast display) to the mouse IL-2 beta receptor.

Table 5

EXAMPLE 8: Polypeptides P4, P5 and P6 demonstrate little to no pSTAT5 signaling in T cells

[00147] Human pan T cells were purified from whole blood and rested overnight by culturing in X-VIVO 15 media (Lonza) without human IL-2. The following day, T cells were enumerated and plated at 100,000 cells per well in 96 well plates. P5, P6, and S4 were added to T cells starting at final concentration of 100 nM, titrating 6 points with 1 : 10 dilutions to 1 pM and incubated at 37 °C for 30 minutes. Cells were fixed in wells by adding formaldehyde to a final concentration of 1.5% by volume for 10 minutes at room temperature, then centrifuged and resuspended in 200 uL ice cold methanol to permeabilize the cells. Methanol was washed out twice with FACS buffer before adding antibodies (BD Biosciences) for anti-human pSTAT5 pY694 diluted 1 :20, anti-human CD4 L200 diluted 1 :50, anti-human CD8a SKI diluted 1 :20, anti-human CD25 M-A251 diluted 1 :20, and antihuman CD127 HIL-7RM21 diluted 1 : 10 in FACS buffer. Cells were incubated with antibody for 1 hour before washing twice and resuspending in 100 uL FACS buffer. Cells were acquired on a Cytek Aurora flow cytometer (Cytek Biosciences) and analyzed with FlowJo Software (FlowJo LLC) to determine the percentage of cells demonstrating phosphorylated STAT5. Live cells were gated on FSC and SSC plots. CD4 ⁺ cells and CD8 ⁺ cells were gated on live cells, and regulatory T cells were defined as the CD25 ⁺ CD127 ^low subset of CD4 ⁺ cells. P5, P6, and S4 proteins showed negligible pSTAT5 signaling at shown concentrations (Figures 7A-B, 8A-D). EXAMPLE 9: Polypeptide P4 demonstrate little to no pSTAT5 signaling in PBMC and

NK Cells

[00148] Human PBMCs or NK cells were purified from whole blood and rested overnight by culturing in X-VIVO 15 media (Lonza) without human IL-2. The following day, PBMCs were enumerated and plated at 200,000 cells per well in 96-well plates. NK cells were enumerated and plated at 50,000 cells per well in 96-well plates. S4 was added to cells starting at final concentration of lOOnM, titrating 6 points with 1 : 10 dilutions to 1 pM and incubated at 37 °C for 30 minutes. Cells were fixed in wells by adding formaldehyde to a final concentration of 1.5% by volume for 10 minutes at room temperature, then centrifuged and resuspended in 200 uL ice cold methanol to permeabilize the cells. Methanol was washed out twice with FACS buffer before adding antibody for anti-human pSTAT5 pY694 (BD Biosciences) diluted 1 :20 in FACS buffer. Cells were incubated with antibody for 1 hour before washing twice and resuspending in 100 uL FACS buffer. Cells were acquired on a Cytek Aurora flow cytometer (Cytek Biosciences) and analyzed with FlowJo Software (FlowJo LLC) to determine the percentage of cells demonstrating phosphorylated STAT5. Live cells were gated on SSC and FSC plots. S4 protein showed negligible pSTAT5 signaling in both PBMCs and NK cells at shown concentrations (Figure 9 A-B).

EXAMPLE 10: Polypeptide P4 inhibits IL-15 signaling in T cells

[00149] Human pan T cells were purified from whole blood and rested overnight by culturing in X-VIVO 15 media (Lonza) without human IL-2. The following day, the T cells were enumerated and plated at 100,000 cells per well in 96 well plates. Anti-IL-15 and S4 were added to T cells starting at final concentration of lOOnM, titrating 8 points with 1 : 10 dilutions to 0.01 pM and incubated at 37 °C for 30 minutes. Recombinant human IL-15 (R&D Systems) was then added to cultures at a final concentration of 5 pM. Cultures were incubated at 37 °C for 30 minutes before fixing cells in wells by adding formaldehyde to a final concentration of 1.5% by volume. Cells were fixed for 10 minutes at room temperature, centrifuged, and resuspended in 200 uL ice cold methanol to permeabilize the cells. Methanol was washed out twice with FACS buffer before adding antibodies (BD Biosciences) for anti-human pSTAT5 pY694 diluted 1 :20, anti-human CD4 L200 diluted 1 :50, anti-human CD8a SKI diluted 1 :20, anti-human CD25 M-A251 diluted 1 :20, and antihuman CD127 HIL-7RM21 diluted 1 : 10 in FACS buffer. Cells were incubated with antibody for 1 hour before washing twice and resuspending in 100 uL FACS buffer. Cells were acquired on a Cytek Aurora flow cytometer (Cytek Biosciences) and analyzed with FlowJo Software (FlowJo LLC) to determine the percentage of cells demonstrating phosphorylated STAT5. Live cells were gated on FSC and SSC plots. CD4 ⁺ cells and CD8 ⁺ cells were gated on live cells, and regulatory T cells were defined as the CD25 ⁺ CD127 ^low subset of CD4 ⁺ cells. Anti-IL-2 IgG, P5, P6, and S4 proteins inhibited human IL- 15 pSTAT5 signaling at shown concentrations (Figures 10A-D).

EXAMPLE 11: Polypeptides S4, P5 and P6 are hyperstable.

[00150] Far-ultraviolet circular dichroism measurements were carried out on S4, P5 and P6 at pH 7.4 using an CHIRASCAN spectrometer VI 00 (Applied Photophysics). Protein samples were measured in PBS buffer at protein concentrations of 0.2 mg/mL, using a 0.1 mm path-length cuvette. Temperature unfolding curves were obtained from 20 to 98 °C by monitoring the absorption signal at 222 nm (steps of 0.5 °C per min, 30 s of equilibration by step). Wavelength scans (200-250 nm) were collected at 20 °C, 98 °C, and again at 20 °C after refolding.

[00151] The shape and signal of the scans indicate that the proteins are structured at 20°C (two minima at 208 and 222 nm with MRE <= -20 deg cm2 dmol' ¹ ), unfold at 98°C and refold after decreasing the temperature to the initial value. The unfolding curves were fit using a Van't Hoff equation-based model to calculate the melting temperatures (Tm). The Tm values obtained for all proteins are similar or higher than those calculated for thermophilic proteins (~80 °C), indicating that they are folded even at high temperatures, at pH 7.4. See Figures 11A-C and 12A-C.

EXAMPLE 12: Polypeptides of the present invention are tolerant to amino acid substitutions

[00152] As taught in the present application, amino acid substitutions can be tolerated at multiple positions. S4 variants with mutations at select positions were made and tested for binding to IL-2Rp. Positions that were mutated include: I22F, P29L, A30V, K3E, and S54G, position numbering in accordance with SEQ ID NO:57. All variants retained beta binding activity (data not shown). EXAMPLE 13: Polypeptide S4 was optimized in order to increase its affinity to mouse IL-2RB

[00153] In order to identify variants with improved mouse-human cross-reactivity, two different IL-2RB interface variant libraries were constructed- one by error-prone PCR (GeneMorph II, Agilent), and one by rational combination of potentially affinity-improving mutations into a combinatorial library (IDT). The resulting clones were tested for improved binding to IL-2RB. The protein variants are shown in Table 6. All variants retained beta binding activity (data not shown).

Table 6

[00154] Based on the binding affinities calculated by yeast display for the variants shown in Table 6, new polypeptides Cl, C2, C3, and C4 were designed. Binding of polypeptides C1-C4 to mouse IL2 receptor beta (mIL2RB) was analyzed by biolayer interferometry. Biotinylated mIL2RB molecules were immobilized to Streptavidin sensors (SA, ForteBio) at 2 pg/mL in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk). After baseline measurement in the binding buffer alone, the binding kinetics were monitored by dipping the biosensors in wells containing two-fold dilutions of the corresponding protein (100 to 1.6 nM) for 300sec (association) and then dipping the sensors back into baseline wells for 600 sec (dissociation). Data were collected at 25 °C in an Octet RED96 (ForteBio) and processed using the instrument’s integrated software; kinetics were globally fit using a 1 : 1 binding to calculate the reported KD values (see Table 7).

Table 7

[00155] Five mutations were made to Cl in positions not directly involved in binding to IL- 2RB or IL-2RBG (R89K, A38C, K75C, E31K, and D59K). Binding of the resultant polypeptides, C9, CIO, C7, to mouse IL-2 receptor beta was performed as described for Cl- C4. Binding of C9, CIO and C7 to human IL2 receptor beta (hIL2RB) was also analyzed using the same technique. Biotinylated hIL2RB molecules were immobilized to Streptavidin sensors (SA, ForteBio) at 2 pg/mL in binding buffer. After baseline measurement in the binding buffer alone, the binding kinetics were monitored by dipping the biosensors in wells containing two-fold dilutions of the corresponding protein (20 to 0.62 nM) for 1000 sec (association) and then dipping the sensors back into baseline wells for 1000 sec (dissociation). See Table 8.

Table 8

[00156] As shown in Table 8 and the figures, polypeptides Cl, C9, CIO, and C7 demonstrated high binding affinity to hIL-2RB (Fig. 13A-13D), no detectable binding to hIL-2RG (Fig. 14A-14D), and inhibited binding of hIL-2 to hIL-2RBG (Fig. 15A-15D). In a subsequent experiment it was found that mutating any single gamma-abolishing mutation of C7 did not restore gamma binding (data not shown).

[00157] Optimized polypeptides Cl, C7, C9, and CIO along with polypeptides S4, P5 and P6 were tested for their ability to inhibit IL-2R signaling in T cells as described in Example 7. See Table 9 below and Figures 16A-C. At 100 nM, P6, S4, Cl, C7, C9, and CIO all showed 90% or higher inhibition of pSTAT5 signaling from IL-2 in effector T cell types. IC50s ranged from 300-2000 pM in CD4+ cells and 50-500 pM in CD8+ cells. P5 did not reach an inhibition plateau at 100 nM. As compared to CD4 and CD8 T cells, human Treg pSTAT5 signaling was resistant to antagonism by C7 with an IC50 of 10,2625 pM as compared to 215 for CD4+ T cells and 42 for CD8+ T cells (data not shown). Table 9

[00158] Multi -valent serial duplications of Cl, C7, and CIO were constructed as well as C- terminal and N-terminal Fc fusions of C7 and C8. See Table 10.

Table 10

[00159] Polypeptides S4, C7, and C8 and fusion proteins of C7 and C8 were tested for their ability to inhibit IL-15 signaling in T cells as described in Example 10. See Table 11 below and figures 17A-C. S4 showed significantly lower inhibition of IL- 15 signaling compared to other test articles. IC50s for C7-NFc and C8-Fc were much lower when compared to corresponding polypeptides without Fc fusion, which can be explained by the increased avidity of the Fc-fusion homodimer. IC50s for signaling inhibition in Tregs were much higher for C7-CFc, C7-NFc, and C8 (data not shown), showing that effector cell signaling is more effectively blocked by these compounds relative to Tregs

Table 11

[00160] Polypeptides C7 and C8 were tested for their ability to inhibit IL- 15 and IL-2R signaling in T cells as described in the examples above (except for a starting concentration of 250nM and 1 :5 serial dilutions). See Table 12 below and figures 18A-C.

Table 12

EXAMPLE 14: Exemplary polypeptides of the present invention are hyperstable including at extreme pH environments and demonstrate resistance to protease cleavage

[00161] Far-ultraviolet circular dichroism measurements were carried out on S4, Cl, C9, CIO and C7 at pH 8.0, 6.0, 4.0 and 2.0, using an CHIRASCAN spectrometer V100 (Applied Photophysics). Protein samples were measured in citrate-phosphate buffer at protein concentrations of 0.2 mg/mL, using a 0.1 mm path-length cuvette. Temperature induced- unfolding curves were obtained from 20 °C to 98 °C by monitoring the absorption signal at 222 nm for pH 8.0 and 6.0, as well as 230 nm for pH 4.0 and 2.0 (steps of 1.0 °C per min, 30 s of equilibration by step). The unfolding curves were fit using a Van't Hoff equationbased model to calculate the melting temperatures (T _m ). Aggregation was measured by monitoring the static light scattering (SLS) signal at 266 nm, using an Uncle spectrometer (Unchained labs).

[00162] Pepsin proteolysis experiments were carried out on S4, Cl, C9, CIO and C7; to this end, protein samples at 0.25 mg/mL were incubated in 0.01 N HC1 + 1 % HC1, at 37 °C with pepsin at 0.025 mg/mL (10: 1 protein to protease ratio). Enzymatic digestion was stopped by adding 5 uL of 1 M Tris pH 8.0 to 25 uL reaction at incubation times of 0, 10, 20, 30, 45, 60, 90, 180 and 360 min, and freezing at -80°C. Samples were analyze by mass spectrometry (Thermo Scientific) and the plot describing the amount of intact protein vs the incubation time was fit to a one-phase decay model to calculate the half-life values. [00163] Notably, the thermal unfolding of polypeptides S4, Cl, C7, C9, and CIO have high thermostability, at pH 4.0 they show aggregation and apparent melting temperatures higher than 60 °C and Tm values above 70 °C at pH 2.0. See Figure 19A-D, 20A-D, 21 A-D, 22A- D, and 23 A-D. The kinetic stability of S4, Cl, C9, CIO and C7 as measured by pepsin proteolysis experiments is shown in Table 13 below; each with a half-life over 5 hours.

Table 13

EXAMPLE 15: Polypeptide C7i-Fc demonstrated anti-inflammatory activity in a murine delayed-type hypersensitivity (DTH) model

[00164] Biological anti-inflammatory activity of C7i-Fc was assessed using a murine delayed-type hypersensitivity (DTH) model. Mice were ordered from Charles River Laboratories (Wimington, MA) and groups of 12 were dosed 5 times over 7 days on days - 1, 0, 1, 6, and 7 with both C7 and C7-Fc at 100 ug and 10 ug. PBS was used as a control. Mice were immunized with lOug ovalbumin (ova) in 25 uL PBS mixed with 25 uL AddaVax (50uL total volume) on Day 0 and injected intradermally at the base of tail. Foot pad thickness was measured on Day 7 before ova challenge to serve as baseline swelling control. An Ova challenge was performed on day 7 with 20 ug ova in 20uL PBS in the right foot pad, while the left foot pad received 20uL of plain PBS. Foot pad thickness was measured on Day 8, 24 hours after challenge. Results (data not shown): On visual inspection, right foot (ova challenge) of vehicle mice and mice receiving low dose (10ug) of C7i or C7i-Fc were very swollen and inflamed. Left feet (PBS challenge) of all mice showed no swelling or redness. The 200ug Combo7i-Fc treatment group showed very little change in foot pad thickness after challenge and very little inflammation (statistically significant (p=0.0002) difference compared to vehicle mice). The dosing of C7i-Fc during ovalbumin immunization and challenge using the DTH model shows significant reduction of foot pad swelling. The high dose of 7i showed some trends in reducing foot pad swelling, but without statistical significance likely due to the shorter half-life of this compound in vivo as compared to the Fc version. EXAMPLE 16: Polypeptide C7i-Fc increased survival in a murine graft versus host disease model

[00165] The ability of polypeptide C7i to treat Graft verse host disease (GVHD) was assessed in a xenograft system consisting of human cells adoptively transferred into NSG murine hosts. Briefly, sub-1 ethally irradiated NSG mice were engrafted with PBMC and treated with C7i-Fc at two different doses and compared against a reference compound and vehicle control. Primary readouts were survival, body weight loss, body condition scoring, and disease activity index. Dosing began on Day 0 and was continued through Day 39, with mice receiving therapy every three days according to Table 14. Mice were graded 3 times weekly for evidence of GVHD by assessment of five clinical parameters: weight loss, posture (hunching), activity, fur texture, skin integrity and paleness. The coding is shown in Table 15. Mice that lost greater than 20% body weight or deceased due to disease were taken off study.

[00166] Treatment with Fc-C7i provided improved disease activity index (“DAI”) scores with a sum of 41 for the vehicle group and a group average of 5.13, a sum of 35 for the 20 mg/kg Fc-C7i group and a group average of 4.38, and a sum of 36 for the 2 mg/kg Fc-C7i group and a group average of 4.5. 2 mice in each Fc-C7i group were high outliers in DAI scoring. Fc-C7i prevented weight loss in a dose dependent manner as shown in Figure 24A and increased survival in a dose dependent manner as shown in Figure 24B

Table 14

Table 15

EXAMPLE 17: Polypeptide C7i inhibited proliferation of PBMC cells and inhibited IFN-gamma production

[00167] Approximately IxlO ⁵ huPBMC cells were plated in the interior 60 wells of a 96- well round bottom plate in lOOul of X- VIVO 15 media (Lonza). Cells were stimulated lOOul of 2x serial dilutions of IL2 (Aknon) and anti-CD3 mAb (UCHT1, Biolegend), 2x serial dilutions of C7i or 2x serial dilutions of C7i fixed IL2 and anti-CD3 (InM, 30pM final). Cells were incubated at 37 °C, 5% CO2 for 6 days. On day 6 plates are spun at 400g for 4 minutes and lOOul of supernatant is removed for IFN gamma quantitation. An equal volume of (lOOul) of CellTiter-Glo Luminescent Cell viability reagent (Promega) is added to each well. Mix by gentle pipetting, transfer full volume to a 96 well white solid bottom plate and incubated at room temperature for 10 minutes with light orbital shaking (250rpm). Luminescence quantified with a spectrax i3x multimode plate reader (Molecular Devices). As demonsrated in Figure 25, anti-CD3 +IL-2 induced robust PBMC proliferation with an IL-2 EC90 of roughly InM, whereas C7i potently inhibited proliferation of PBMCs stimulated with 1 nM IL-2 and 30 pM anti-CD3 with an IC50 of 70 pM and C7i cultured alone did not induce proliferation of PBMC cells at any concentration.

[00168] Human IFN gamma levels in supernatant were determined by huIFN gamma AlphaLisa assay (Perkin Elmer). 5ul of sample or standard were transferred to a 96 well half-area white plate (Perkin Elmer) and 20ul of Acceptor bead and biotinylated antibody mix (25ug/ml, 2.5nM) were added and gently mixed. Samples were incubated at room temperature for 60 minutes, followed by the addition of 25ul of Streptavidin Donor Beads (80ug/ml). Samples were incubated for an additional 30 minutes at room temperature (dark) and plates read with a spectrax i3x multimode plate reader (Molecular Devices). As demonstrated in Figure 26, anti-CD3 +IL-2 induced robust IFN-gamma production with an IL-2 EC90 of roughly O.lnM, whereas C7i potently inhibited IFN-gamma production of PBMCs stimulated with 1 nM IL-2 and 30 pM anti-CD3 with an IC50 of 8.1 nM and C7i cultured alone did not induce IFN-gamma production at any concentration.

EXAMPLE 18: C7i is functional in gut following oral administration in mice

[00169] Mice were orally gavaged with a single dose of 0.5 mg C7i and samples of the stomach, colon, and plasma were collected at 1 hour, 4 hours, and 24 hours post dose (n=3 mice/ timepoint). Blood was processed to plasma and clean tissue lysate prepared for test article quantification via Meso Scale Discovery platform. Detection of drug in tissue lysate was dependent on ligand capture step, indicating functional protein.

[00170] C7i was detected and stable in the low pH of the stomach for at least 24 hours post dose. Further, C7i was stable during GI transit and was detected in the terminal end of the GI tract (i.e., colon) for at least 24 hours following oral administration, with an apparent peak 4 hours post dose.