Synthetic il-2 polypeptides and conjugates, compositions, and uses thereof

Abstract

Interleukin-2 (IL-2) promotes the proliferation, differentiation, activation, and survival of mature lymphocytes, particularly T cells. IL-2 facilitates physiologic antigen-specific acquired cellular immune responses, and can promote innate host defenses by activating natural killer (NK) cells. The synthetic IL-2 polypeptides lack disordered loops found in wild-type (WT) IL-2 and show increased stability and expression. The synthetic polypeptides have increased IL-2R beta binding, they maintain similar activity and phenotypes compared to recombinant human interleukin-2 (rhIL-2), which contains a cysteine to serine mutation at position 125 of WT IL-2.

Description

SYNTHETIC IL-2 POLYPEPTIDES AND CONJUGATES, COMPOSITIONS, AND USES THEREOFFIELD

[0001] The present disclosure provides synthetic IL-2 polypeptides, protein conjugates or fusion proteins thereof, and compositions comprising thereof.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 572,531, filed April 1, 2024, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled BIOTN_42867_601_SequenceListing.xml (Size: 56,722 bytes; and Date of Creation: March 28, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0004] Interleukins are a group of cytokine signaling molecules that are expressed and secreted by and regulate the activation, differentiation, proliferation, maturation, migration, and adhesion of immune cells (e.g., leukocytes, often lymphocytes). The function of the immune system primarily depends on interleukins, and deficiencies or genetic mutations in interleukins have been associated with autoimmune diseases or immune deficiency.

[0005] Interleukin-2 (IL-2) promotes the proliferation, differentiation, activation, and survival of mature lymphocytes, particularly T cells. IL-2 facilitates physiologic antigen-specific acquired cellular immune responses, and can promote innate host defenses by activating natural killer (NK) cells. IL-2 controls diverse biological functions (e.g., speed, magnitude, and duration of the T-cell immune response) by binding with IL-2 receptors (IL-2R) including an alpha subunit (also known as IL-2Ra or CD25), a beta subunit (also known as IL-2RP or CD122), and a gamma subunit (also known as IL-2Ry, yc, or CD132). Genetic deficiencies of TL-2 or IL-2R a or 0 chains can result in immunodeficiency soon after birth, with a later accumulation of activated T cells that do not function properly. These phenotypic changes cannot be compensated by other cytokines.SUMMARY

[0006] Embodiments of the present disclosure include synthetic polypeptides which bind interleukin 2 receptor subunit beta (IL-2R beta). In some embodiments, the synthetic polypeptides have less than 50% identity to SEQ ID NO: 12.

[0007] In some embodiments, the polypeptides comprise: a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of RDLISNINVI (SEQ ID NO: 2), EDLISN1NVI (SEQ ID NO: 3), RDTISNINVI (SEQ ID NO: 4), RDLIENINVI (SEQ ID NO: 5), RDTIENINV (SEQ ID NO: 6), EDLIENINVI (SEQ ID NO: 7), EDTISNINV (SEQ ID NO: 8), or EDTIENINV (SEQ ID NO: 9); and a third domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10) or LNRWITFAQ (SEQ ID NO: 11).

[0008] In some embodiments, the first, second, and third domains may be in any order N- terminal to C-terminal within the synthetic polypeptide. In some embodiments, the first, second, and third domains from N-terminal to C-terminal in the synthetic polypeptide are ordered as: first- second-third or first-third-second. In some embodiments, each domain is separated by a spacer polypeptide. In some embodiments, each spacer polypeptide is individually 10 to 60 amino acids in length.

[0009] In some embodiments, the synthetic polypeptides comprise one or more additional amino acids N-terminal and C-terminal to the first, second, and third domains.

[0010] In some embodiments, the synthetic polypeptides comprise: a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of RDLISNINVI (SEQ ID NO: 2); and a third domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10).

[0011] In some embodiments, the synthetic polypeptides comprise an amino acid sequence having at least 70% identity to one of SEQ ID NOs: 13-26. In some embodiments, the synthetic polypeptides comprise an amino acid sequence of any of SEQ ID NOs: 13-26.

[0012] In some embodiments, the synthetic polypeptides comprise at least one of EDLISNINVI (SEQ ID NO: 3), RDTISNINVI (SEQ ID NO: 4), RDLIENINVI (SEQ ID NO: 5), RDTIENINV (SEQ ID NO: 6), EDLIENINVI (SEQ ID NO: 7), EDTISNINV (SEQ ID NO: 8), and EDTIENINV (SEQ ID NO: 9).

[0013] In some embodiments, the synthetic polypeptides comprise one or more amino acid substitutions compared to SEQ ID NO: 18. In some embodiments, the one or more amino acid substitutions comprise substitutions of C34, E83, R86, L88, S90, and combinations thereof as compared to SEQ ID NO: 18. In some embodiments, the one or more amino acid substitutions comprise C34A, E83K, R86E, L88T, S90E, and combinations thereof as compared to SEQ ID NO: 18. In some embodiments, the one or more amino acid substitutions further comprise at least one additional amino acid substitution as compared to SEQ ID NO: 18.

[0014] In some embodiments, the synthetic polypeptides comprise an amino acid sequence having at least 70% identity to one of SEQ ID NOs: 27-33. In some embodiments, the synthetic polypeptides comprise an amino acid sequence of any of SEQ ID NOs: 27-33.

[0015] In some embodiments, the synthetic polypeptides are 90 to 130 amino acids in length.

[0016] In some embodiments, the synthetic polypeptides are thermostable.

[0017] In some embodiments, the synthetic polypeptides have increased stability in cell culture media as compared to recombinant human IL-2.

[0018] In some embodiments, the synthetic polypeptides have fully or partially reduced binding to interleukin 2 receptor subunit alpha (IL-2R alpha) as compared to recombinant human IL-2.

[0019] Additional embodiments of the present disclosure include protein conjugates or fusion proteins comprising a synthetic polypeptide as disclosed herein linked to at least one moiety. In some embodiments, the at least one moiety is a protein, a protein domain, a polypeptide, or a peptide. In some embodiments, the at least one moiety comprises an antibody or fragment thereof. In some embodiments, the at least one moiety is polyethylene glycol (PEG).

[0020] Other embodiments of the present disclosure include compositions comprising a synthetic polypeptide, protein conjugate, or fusion protein as disclosed herein and a carrier. In some embodiments, the composition is a cell culture media or component thereof.

[0021] Further embodiments of the present disclosure include methods for in vitro or ex vivo T cell or NK cell differentiation, stimulation and / or expansion. In some embodiments the methods comprise contacting a T cell, an NK cell, or a precursor or progenitor cell thereof, with a synthetic polypeptide, protein conjugate or fusion protein, or composition as disclosed herein.

[0022] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGS. 1A-1B: FIG. 1 A shows the structure of IL-2 (black) with the IL-2 receptors alpha (bottom left), beta (left), and gamma (top) receptors from the protein data bank (ID: 2B5I). This was overlayed with the AlphaFold structure of an exemplary synthetic IL-2 (white). FIG. IB shows the same structure as FIG. 1 A, but with the receptors removed to better visualize the difference in the IL-2. The synthetic IL-2 structure maintains the helices that interact with the beta and gamma receptors, however the disordered loops in IL-2 that are in proximity to the alpha receptor are replaced with helices in the synthetic IL-2.

[0024] FIG. 2: Screening of initial synthetic IL-2 polypeptides by binding the extracellular domain of IL-2R beta with an Avi Tag and staining with a streptavidin fluorophore followed by measuring mean fluorescence intensity (MFI) with flow cytometry. This was done in two experiments that are separated by recombinant human interleukin-2 (rhIL-2) in the graph. MFI is interpreted as a read out of binding affinity.

[0025] FIG. 3: Synthetic IL -2 polypeptides that had high IL-2R beta binding and high expression in E. coli were selected to test for binding data for all three receptors, (IL-2R alpha, IL- 2R beta and IL-2R gamma). Binding was measured by adding recombinant version of the extracellular domain for each receptor with an Avi Tag and staining with a streptavidin fluorophore and reading with flow cytometry. MFI is a read out of binding affinity.

[0026] FIG. 4: Surface plasma resonance (SPR) assays were performed on synthetic IL -2 constructs. The decreased KD (dissociation constant) for IL-2R beta was much stronger for the synthetic IL-2 variants compared to rhIL-2 (455 nM), except for ABG6-M3.

[0027] FIG. 5: STAT5 phosphorylation staining in YT cells (malignant hematopoietic cell line that model NK cells). STAT5 phosphorylation is a measurement of IL-2 signal transduction. YT cells with and without CD25 expression (IL2R alpha) were stained with an antibody targeting STAT5 phosphorylation. rhIL-2 had the strongest phosphorylation when cells expressed CD25, however, synthetic IL-2 (ABG6-M1) had no change in staining when CD25 was knocked out.

[0028] FIG. 6: NK92 proliferation data. NK92 has decreased CD25 expression and proliferation is dependent on IL-2 stimulation. The first plot shows NK92 proliferation after 4 days in culture. The second plot includes an IL-2R alpha blocking antibody to isolate the effect of IL- 2R beta and gamma. Column C indicates the EC50 with ABG6-M2 having the strongest activity.

[0029] FIG. 7: IL-2 stimulation of NK92 cells and measurement of INF-gamma secretion. Column C is ED50 and column D is max response. Both synthetic IL-2’s stimulated more INF- gamma secretion than rhIL-2.

[0030] FIG. 8: CD4 and CD8 positive isolated human T cells were stimulated with CD3 / CD28 Dynabeads and IL-2. ABG6-M1 had similar fold expansion to rhIL-2.

[0031] FIG. 9: CD8 positive isolated human T cells were stimulated with CD3 / CD28 Dynabeads and IL-2. ABG6-M1 had slightly increased expansion to rhIL-2, likely due to CD8+ T cells having lower CD25 expression.

[0032] FIG. 10: Day 12 T cell phenotypes. Synthetic IL-2 shows similar equivalency for phenotypes as compared to rhIL-2. CM: central memory CD45RA-CCR7+, Naive: CD45RA+CCR7+, TEMRA: CD45RA+CCR7- terminally differentiated effector memory cells, EM: effector memory CD45RA-CCR7- cells.

[0033] FIG. 11 : Comparison of solubility of rhIL-2 versus a synthetic IL-2 (ABG6-M1) when expressed in BL21 DE3 E. coli. Proteins were induced with IPGT (+) and harvested from lysed cells. Total protein was aliquoted before separating the aggregate from the soluble by centrifugation. Majority of rhIL-2 is insoluble, which is shown by the band at 14.5 kDa in the aggregate lane, while majority of the synthetic IL2 is found in the soluble fraction (band at 10 kDa).

[0034] FIGS. 12A and 12B: Stability of an exemplary synthetic IL-2 (ABG6-M1) and rhIL-2 in cell culture media. ABG6-M1 and rIL-2 were incubated in DMEM media at 37 °C for 10 days at various concentration (FIG. 12A) or for 10 days, 1 month, and 2 months at 10 ng / mL (FIG. 12B) and activity was assessed through a proliferation bioassay in NK92 cells. The Specific Activity % of control was determined with the control comprising rIL-2 stored at 4°C without any incubation.DETAILED DESCRIPTION

[0035] Disclosed herein are synthetic IL-2 polypeptides, protein conjugates or fusion proteins thereof, and compositions comprising thereof. The synthetic IL-2 polypeptides lack disordered loops found in wild-type (WT) IL-2 and show increased stability and expression. One consequence of replacing disordered loops in IL-2 is the loss of IL-2R alpha binding, however, because the synthetic polypeptides have increased IL-2R beta binding, they maintain similar activity and phenotypes compared to recombinant human interleukin-2 (rhIL-2), which contains a cysteine to serine mutation at position 125 of WT IL -2. Additionally, low IL-2R alpha binding has theadvantage of biasing cells with low IL-2R alpha expression which has potential benefits when used for immune oncology cell therapies as they do not support Treg cell expansion. For example, T regulatory cells express high IL-2Ra and thus would proportionally expand less with the disclosed synthetic IL-2 polypeptides, which could be desirable for certain outcomes. The synthetic IL-2 polypeptides are also thermostable. The extended shelf life of the synthetic IL-2 polypeptides facilitates automated cytokine addition in cell cultures and superior function in closed cell culture systems. In contrast, WT IL-2 requires freezing for stability.

[0036] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions

[0037] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell and tissue culture, molecular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0038] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0039] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, thenumbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0040] The term “antibody,” as used herein, refers to a protein which is naturally used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Typically, an antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C- terminal constant (CHI, CH2, and CHS) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda (X), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0041] The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the 0 sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds ), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

[0042] The framework regions are connected by three CDRs. As discussed above, the three CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding. The CDRs form loops connecting, and in some cases comprising part of, the beta-sheet structure formed by the framework regions. While the constant regions of the light and heavy chains are not directly involved in binding of the antibody to an antigen, the constant regions can influence the orientation of the variable regions. The constant regions alsoexhibit various effector functions, such as participation in antibody-dependent complement- mediated lysis or antibody-dependent cellular toxicity via interactions with effector molecules and cells.

[0043] As used herein, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (Ka) of at least 106M’1(e.g., >106M1, >107M’1, >108M’1, >109M’1, >1O10M’1, >10nM1, >1012M’1, >1013M’1, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

[0044] As such, antibodies, as used herein refers to monoclonal antibodies, monospecific antibodies (e g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, anti -idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21 :484-490 (2014)), and include single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. Antibodies can be of any type(for example, IgG, IgE, TgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0045] The terms “fragment of an antibody,” “antibody fragment,” and “antigen-binding fragment” of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains, (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.

[0046] ‘ ‘Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0047] The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0048] The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

[0049] As used herein, “conjugate” refers to the linking of two or more moi eties or molecules to each other by covalent or non-covalent interactions. More specifically, the terms “protein conjugate” refer to a protein or polypeptide that has been modified by the addition of another moiety or molecule (e.g., another peptide, protein, or polypeptide).

[0050] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target may occur by any means known to the skilled artisan.

[0051] The term “ex vivo” as used herein refers to cells that have been removed from a living organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0052] The term “exogenous,” when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial means, or in relation a cell refers to a cell which was isolated and subsequently introduced to other cells or to an organism by artificial means. An exogenous nucleic acid may be from a different organism or cell, or it may be one ormore additional copies of a nucleic acid which occurs naturally within the organism or cell. An exogenous cell may be from a different organism, or it may be from the same organism.

[0053] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595- 8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or nonnucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0054] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. The peptide or polypeptide may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide,” “oligopeptide,” “protein,” and “peptide” are used interchangeably herein. The peptide may be produced byrecombinant genetic technology or chemical synthesis. The peptide may be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, and size exclusion), or by any other standard techniques known in the art.

[0055] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), norvaline (found in the antifungal peptide of Bacillus subtilis), and N- formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include P-amino acids (P3and p2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring- substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D- amino acids, alpha-methyl amino acids andN-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. According to certain embodiments, a peptide inhibitor comprises an intramolecular bond between two amino acid residues present in the peptide inhibitor. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other as compared to when not bonded to each other. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state. For example, the amino acid residue homoSerine (hSer) in its unbonded form may take the form of 2-aminobutyric acid (Abu) when participating in an intramolecular bond according to the present invention.

[0056] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.

[0057] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or °F for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the peptide.

[0058] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four- character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N- methylglycine), Aib (u-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3- diaminopropanoic acid), y-Glu (y-glutamic acid), Gaba (y-aminobutanoic acid), P-Pro (pyrrolidine-3 -carboxylic acid), and 8 Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), hPro (P-homoproline), phPhe (P-homophenylalanine) and Bip (P,P diphenylalanine), and Ida (Iminodiacetic acid).

[0059] As used herein, the terms “percent sequence identity” or “percent identity” refer to the percentage of amino acids in an amino acid sequence, that is identical with the corresponding amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) andFASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389- 3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0060] The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to macromolecules (e.g., nucleic acid molecules or polypeptides) mean that the macromolecule is at least substantially free from at least one other component with which it is naturally associated in nature and as found in nature, and / or the macromolecule is associated with at least one other component with which it is not naturally associated in nature and / or that there is one or more changes in, for example, the nucleic acid or amino acid sequence as compared with such sequence as it is found in nature.

[0061] A “subject” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such as a mouse model. Likewise, the subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., humans and non-humans). Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like.

[0062] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Synthetic Polypeptides

[0063] Disclosed herein are synthetic polypeptides that bind interleukin 2 receptor subunit beta (IL-2R beta). The synthetic polypeptides comprise a first domain, a second domain, and a third domain arranged in any order about the length of the synthetic polypeptide and, optionally, separated by spacer polypeptides.

[0064] In some embodiments, the amino acid sequence of the nuclease lacks significant identity or significant amino acid homology with wild-type IL-2 (SEQ ID NO: 12) or recombinant interleukin-2 products, for example, SEQ ID NO: 34. Accordingly the synthetic polypeptide does not comprise an amino acid sequence of SEQ ID NO: 12 or 34. For example, the synthetic polypeptide may comprise less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, or less than 20%) sequence identity to SEQ ID NO: 12.

[0065] In some embodiments, the synthetic polypeptide comprises a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1), or an amino acid sequence having one, two, or three amino acid substitutions as compared to SEQ ID NO: 1. In some embodiments, the synthetic polypeptide comprises a second domain having an amino acid sequence of RDLISNINVI (SEQ ID NO: 2), EDLISNINVI (SEQ ID NO: 3), RDTISNINVI (SEQ ID NO: 4), RDLIENINVI (SEQ ID NO: 5), RDTIENINV (SEQ ID NO: 6), EDLIENINVI (SEQ ID NO: 7), EDTISNINV (SEQ ID NO: 8), or EDTIENINV (SEQ ID NO: 9), or an amino acid sequence having one or two amino acid substitutions as compared to any of SEQ ID NOs: 2-9. For example, in some embodiments, the arginine at position 1 of any of SEQ ID NOs: 2 or 4-6 is substituted with another amino acid. In some embodiments, the arginine at position 1 of any of SEQ ID NOs: 2 or 4-6 is substituted with a positively charged amino acid (e.g., glutamate, aspartate). In some embodiments, the serine at position 5 of any of SEQ ID NOs: 2-4 and 8 is substituted with another amino acid. In some embodiments, the serine at position 5 of any of SEQ ID NOs: 2-4 and 8 is substituted with a positively charged amino acid (e.g., glutamate, aspartate). In some embodiments, the leucine at position 3 of any of SEQ ID NOs: 2, 3, 5, and 6 is substituted with another amino acid. In some embodiments, the leucine at position 3 of any of SEQ ID NOs: 2, 3, 5, and 6 is substituted with an uncharged amino acid (e.g., glycine, alanine, valine, leucine, isoleucine, methionine, serine, threonine).

[0066] In some embodiments, the synthetic polypeptide comprises a second domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10) or LNRWITFAQ (SEQ ID NO: 11), oran amino acid sequence having one or two amino acid substitutions as compared to any of SEQ ID NOs: 10 or 11. For example, in some embodiments, the cysteine at position 8 of SEQ ID NO: 10 is substituted with another amino acid. In some embodiments, the cysteine at position 8 of SEQ ID NO: 10 is substituted with an uncharged amino acid (e.g., glycine, alanine, valine, leucine, isoleucine, methionine, serine, threonine).

[0067] In some embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1), or an amino acid sequence having one, two, or three amino acid substitutions as compared to SEQ ID NO: 1 ; a second domain having an amino acid sequence of RDLISNINVI (SEQ ID NO: 2), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 2; and a third domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 10.

[0068] In certain embodiments, the synthetic polypeptide comprises a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of RDLISNINVI (SEQ ID NO: 2); and a third domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10).

[0069] In some embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1), or an amino acid sequence having one, two, or three amino acid substitutions as compared to SEQ ID NO: 1 ; a second domain having an amino acid sequence of EDLISNINVI (SEQ ID NO: 3), RDTISNINVI (SEQ ID NO: 4), RDLIENINVI (SEQ ID NO: 5), RDTIENINV (SEQ ID NO: 6), EDLIENINVI (SEQ ID NO: 7), EDTISNINV (SEQ ID NO: 8), or EDTIENINV (SEQ ID NO: 9), or an amino acid sequence having one or two amino acid substitutions as compared to any of SEQ ID NOs: 3-9, or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 3-9; and a third domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10) or LNRWITFAQ (SEQ ID NO: 11), or an amino acid sequence having one or two substitutions as compared to any of SEQ ID NOs: 10 or 11.

[0070] In certain embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1), or an amino acid sequence having one, two, or three amino acid substitutions as compared to SEQ ID NO: 1, and a third domain having an amino acid sequence of LNRWITFAQ (SEQ ID NO: 11), or an amino acid sequencehaving one or two amino acid substitutions as compared to SEQ ID NO: 11 . In select embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1) and a third domain having an amino acid sequence of LNRWITFAQ (SEQ ID NO: 11).

[0071] In certain embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1), or an amino acid sequence having one, two, or three amino acid substitutions as compared to SEQ ID NO: 1 ; a second domain having an amino acid sequence of EDLIENINVI (SEQ ID NO: 7), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 7; and a third domain having an amino acid sequence of LNRWITFAQ (SEQ ID NO: 11), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 11.

[0072] In select embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of EDLIENINVI (SEQ ID NO: 7); and a third domain having an amino acid sequence of LNRWITFAQ (SEQ ID NO: 11).

[0073] In certain embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1), or an amino acid sequence having one, two, or three amino acid substitutions as compared to SEQ ID NO: 1 ; a second domain having an amino acid sequence of RDTIENINV (SEQ ID NO: 6), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 6; and a third domain having an amino acid sequence of LNRWITFAQ (SEQ ID NO: 11), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 11.

[0074] In select embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of RDTIENINV (SEQ ID NO: 6); and a third domain having an amino acid sequence of LNRWITFAQ (SEQ ID NO: 11).

[0075] In certain embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1), or an amino acid sequence having one, two, or three amino acid substitutions as compared to SEQ ID NO: 1 ; a second domain having an amino acid sequence of EDTIENINV (SEQ ID NO: 9), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 9; and a third domain having anamino acid sequence of LNRWITFAQ (SEQ ID NO: 11), or an amino acid sequence having one or two amino acid substitutions as compared to SEQ ID NO: 11.

[0076] In select embodiments, the synthetic polypeptides comprise a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of EDTIENINV (SEQ ID NO: 9); and a third domain having an amino acid sequence of LNRWITFAQ (SEQ ID NO: 11).

[0077] The first, second, and third domains can be arranged in an order along the length of the synthetic polypeptide. In some embodiments, the first domain is the most N-terminal of the first, second, and third domains. In some embodiments, the second domain is the most C-terminal of the first, second, and third domains. In some embodiments, the third domain is the most C-terminal of the first, second, and third domains. In certain embodiments, the first, second, and third domains from N-terminal to C-terminal in the synthetic polypeptide are ordered as first-second-third. In certain embodiments, the first, second, and third domains from N-terminal to C-terminal in the synthetic polypeptide are ordered as first-third-second.

[0078] In some embodiments, each of the first, second, and third domains are separated by a spacer polypeptide. The spacer polypeptides comprise amino acid sequences of which the length and identity allows the synthetic polypeptide to adapt a confirmation in which the first, second, and third domains are substantially arranged as the helices which interact with IL-2R beta as shown in FIGS. 1A and IB.

[0079] In some embodiments, each spacer polypeptide is individually 10 to 60 amino acids in length. In some embodiments, the synthetic polypeptide comprises one spacer polypeptide 10 to 20 amino acids in length and a second spacer polypeptide 30 to 60 amino acids in length. For example, the spacer polypeptide between the first and second domains may be 10 to 20 amino acids in length, whereas the spacer polypeptide between the second and third domain may be 30 to 60 amino acids in length. In some embodiments, each spacer polypeptide individually comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 35-61. In some embodiments, each spacer polypeptide individually comprises an amino acid sequence of any of SEQ ID NOs: 35-61.

[0080] In some embodiments, the synthetic polypeptide comprises one or more additional amino acids N-terminal and C-terminal to the first, second, and third domains. Thus, the synthetic polypeptide may comprise one or more additional amino acids N-terminal to the most N-terminalof the first, second, and third domains, and / or one or more additional amino acids C-terminal to the most C-terminal of the first, second, and third domains. In some embodiments, the synthetic polypeptide comprises 1 to 10 amino acids N-terminal to the most N-terminal of the first, second, and third domains. In some embodiments, the synthetic polypeptide comprises more than 10 amino acids N-terminal to the most N-terminal of the first, second, and third domains. In some embodiments, the synthetic polypeptide comprises 1 to 10 amino acids C-terminal to the most C- terminal of the first, second, and third domains. In some embodiments, the synthetic polypeptide comprises more than 10 amino acids C-terminal to the most C-terminal of the first, second, and third domains.

[0081] In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 70% identity to one of SEQ ID NOs: 13-26. In some embodiments, the synthetic polypeptide comprises an amino acid sequence of SEQ ID NOs: 13-26.

[0082] In some embodiments, the synthetic polypeptide comprises one or more amino acid substitutions compared to SEQ ID NO: 18. In some embodiments, the one or more amino acid substitutions are at positions C34, E83, R86, L88, S90, and combinations thereof as compared to SEQ ID NO: 14. In some embodiments, the one or more amino acid substitutions further comprise at least one additional amino acid substitution as compared to SEQ ID NO: 18.

[0083] In certain embodiments, the one or more amino acid substitutions include substitutions at positions R86 and S90. In certain embodiments, the one or more amino acid substitutions include substitutions at positions C34, R86, and S90.

[0084] In certain embodiments, the one or more amino acid substitutions include substitutions at positions L88 and S90. In certain embodiments, the one or more amino acid substitutions include substitutions at positions C34, L88, and S90.

[0085] In certain embodiments, the one or more amino acid substitutions include substitutions at positions C34, R86, L88, and S90.

[0086] In some embodiments, the one or more amino acid substitutions comprise C34A, E83K, R86E, L88T, S90E, and combinations thereof as compared to SEQ ID NO: 18. In certain embodiments, the one or more amino acid substitutions comprise R86E and S90E. In certain embodiments, the one or more amino acid substitutions comprise C34A, R86E, and S90E. In certain embodiments, the one or more amino acid substitutions include substitutions at positions L88T and S90E. In certain embodiments, the one or more amino acid substitutions includesubstitutions at positions C34A, L88T, and S90E. Tn certain embodiments, the one or more amino acid substitutions include substitutions at positions C34A, R86E, L88T, and S90E.

[0087] In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 70% identity to one of SEQ ID NOs: 27-33. In some embodiments, the synthetic polypeptide comprises an amino acid sequence of SEQ ID NOs: 27-33.

[0088] The synthetic polypeptide may be any length necessary to allow for adoption of the confirmation as described above and in FIGS. 1A and IB, and facilitate the increased stability, purification yield, and other properties, as described elsewhere herein, as the disclosed exemplary polypeptides of SEQ ID NO: 13-33. In some embodiments, the synthetic polypeptide is 90 to 130 amino acids in length. The synthetic polypeptide may be 90 to 100, 90 to 110, 90 to 120, 100 to 110, 100 to 120, 100 to 130, 110 to 120, 110 to 130, or 120 to 130 amino acids in length.

[0089] The synthetic polypeptides may show increased stability and expression as compared to rhIL-2. In some embodiments, the synthetic polypeptide is thermostable. A “thermostable” polypeptide is a polypeptide which under certain conditions, e.g., at certain temperature and / or for certain periods of time, has an increased retention of activity relative to a reference polypeptide or maintains activity at increased temperature conditions as compared to native, lower temperature, conditions. In some embodiments, the synthetic polypeptide retains the structure / conformation necessary for full or nearly full activity after a period of time (e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes) at increased temperatures. In some embodiments, the synthetic polypeptide has increased thermostability as compared to rh IL-2. As such, the synthetic polypeptide may retain more activity than rhIL-2 after being exposed to high heat conditions for the same period of time, the synthetic polypeptide may retain activity at higher temperatures as compared to rhIL-2, and / or the synthetic polypeptide may retain activity after increased length of duration at any given high temperature as compared to rhIL-2.

[0090] The synthetic polypeptides may show stability in cell culture media. As such, the synthetic peptides may show increased retention of activity over a period of time in cell culture media at any temperature. The synthetic peptides may show increased retention of activity in cell culture media at conditions suitable for use in cell culture (e.g., temperature, CO2 concentrations, in the presence of other cell culture components and / or cells) and suitable concentrations for the proposed use. In some embodiments, the synthetic polypeptide has increased stability in cellculture media as compared to rh IL-2. The synthetic polypeptide may retain more activity than rhIL-2 after being exposed to cell culture conditions for the same period of time, the synthetic polypeptide may retain activity at lower concentration in cell culture media as compared to rhlL- 2, and / or the synthetic polypeptide may retain activity after increased length of duration in cell culture media as compared to rhIL-2.

[0091] The synthetic polypeptides may have decreased binding to interleukin 2 receptor subunit alpha (IL-2R alpha). In some embodiments, the synthetic polypeptide does not bind to IL-2R alpha, e.g., the synthetic polypeptides exhibit similar or decreased binding characteristics as a negative control known to not bind IL-2R alpha. In some embodiments, the synthetic polypeptide has reduced binding characteristics to IL-2R alpha as compared to rhIL-2. In some embodiments, the synthetic polypeptide at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% decreased binding characteristics to IL-2R alpha as compared to rhIL-2.

[0092] Any of the synthetic polypeptides described herein may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25) additional amino acid substitutions as compared to those mutations specifically contemplated and disclosed herein. In some embodiments, the synthetic polypeptides described herein may comprise one or more amino acid substitutions outside of the first, second, and third domains.

[0093] An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He ), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0094] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. Afunctional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same subgroup. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.

[0095] In some embodiments, a synthetic polypeptide is covalently or non-covalently linked to at least one moiety. Accordingly, also provided herein are protein conjugates comprising a synthetic polypeptide as described herein and at least one moiety. For example, the synthetic polypeptide may be fused to a moiety that provides for tagging or visualization (e.g., GFP). The synthetic polypeptide may be fused to a moiety that has another functionality or activity useful to target to certain cells (e.g., therapeutic agents, antibodies). The synthetic polypeptide may be fused to a moiety that provides increased efficacy to the synthetic polypeptide (e.g., half-life extension moieties). The synthetic polypeptide may be fused to a moiety that masks the function of the synthetic polypeptide except under certain conditions or in certain locations (e.g., inactivating domains). For example, the synthetic polypeptide may be linked to polyethylene glycol (PEG) molecules, albumin, an Fc region or domain to a protein of interest, an antigen-binding domain of an antibody, an antibody which targets a certain cell or blocks an unwanted activity or interaction, toxins, NKG2D, and the like.

[0096] The synthetic polypeptide can be linked to the moiety using standard chemical conjugation techniques. Methods of chemical conjugation of peptides are known in the art.Common conjugation strategies are generally based on side-chain modification of lysine or cysteine. Noncanonical amino acids may facilitate introduction of chemically orthogonal handles at predefined sites in a given protein sequence, e.g., for oxime ligation or click chemistry methods. In addition, a wide variety of methods are available for site-specific protein modification with varying degrees of versatility. Exemplary chemical linker systems include, but are not limited to, the carbodiimide (EDC), the thiol-maleimide, the succinimidyl 3-(2- pyridyldithiojpropi onate (SPDP) and the periodate systems.

[0097] Besides chemical strategies, enzymatic conjugations may be suitable, as they enable highly controlled modification of protein (e.g., antibodies) through specific peptide tags. Exemplary enzymatic processes include, but are not limited to, sortase ligation, subtiligase- catalyzed ligation, phosphopantetheinyl transferase, tyrosinase, and transglutaminase. The components of the protein conjugates may include other moieties or linkers (e.g., amino acids) to which the substrates for the enzymatic reaction are attached, provided the enzymatic reaction can proceed. Accordingly, protein conjugates may retain the other moieties or linkers to separate the components.

[0098] When the moiety is a protein, a protein domain, a polypeptide, a peptide, or the like, the protein conjugate can also be produced as a contiguous protein (e.g., a fusion protein) using genetic engineering techniques.

[0099] The fusion proteins are not limited by orientation or directionality of the synthetic polypeptide and the at least one moiety. For example, any single moiety may be fused to the N- terminus or C-terminus of the synthetic polypeptide, in any orientation, e.g., N-terminus to N- terminus, C-terminus to C-terminus, N-terminus to C-terminus, or C-terminus to N-terminus, and directly or indirectly (e.g., fused to another moiety fused to the synthetic polypeptide).

[0100] The synthetic polypeptides may be fused to the moieties via a linker polypeptide. The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that thepreferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use, including but not limited to, glycine-serine polymers, glycine-alanine polymers, and alanine-serine polymers.

[0101] Any of the synthetic polypeptides or fusion proteins may further comprise a localization or signal sequence, a sequence tag (e.g., a tag for detection, purification, and / or monitoring expression), a protein transduction domain sequence, or a combination thereof. In some embodiments, the synthetic polypeptides or fusion proteins may comprise an epitope tag (e.g., 3xFLAG tag, an HA tag, a Myc tag, and the like). The localization or signal sequence, sequence tag, and protein transduction domain sequence may be at the N-terminus, C-terminus, or a combination thereof of the corresponding synthetic polypeptide or fusion protein and may be linked with a linker polypeptide.Nucleic Acids

[0102] Also disclosed herein are nucleic acids encoding the synthetic polypeptides or protein conjugates or fusion proteins thereof as described herein. The nucleic acids may be DNA, RNA, or combinations thereof. In some embodiments, the nucleic acids comprise one or more vectors.

[0103] In certain embodiments, the nucleic acids are engineered for codon-optimization. It will be appreciated altering codons to those most frequently used in the cells or subject of interest allows for maximum expression. Such modified nucleic acid sequences are commonly described in the art as “codon-optimized.” In some embodiments, the nucleic acid sequence is considered codon-optimized if at least about 60% (e.g., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98%) of the codons encoded therein are preferred codons to the subject of interest.

[0104] The present disclosure also provides for DNA segments encoding the synthetic polypeptides or protein conjugates or fusion proteins thereof disclosed herein, vectors containing these segments, and cells containing the vectors. The vectors may be used to propagate the DNA segment in an appropriate cell and / or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0105] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode the synthetic polypeptides or protein conjugates or fusion proteins thereof as disclosed herein. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell.

[0106] Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding components of the present system into cells. Such methods can be used to administer nucleic acids encoding components of the present system to cells in culture. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno- associated and herpes simplex viral vectors.

[0107] In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that any or all of the synthetic polypeptides or protein conjugates or fusion proteins thereof may be removed from the cells under certain conditions. For example, this may allow for DNA integration by transforming bacteria of interest, but then being left with engineered strains that have no memory of the plasmids or vectors used for the integration.

[0108] Drug selection strategies may be adopted by positively selecting for cells that underwent DNA integration. A donor nucleic acid may contain one or more drug-selectable markers within the cargo. Then presuming that the original donor plasmid is removed, drug selection may be used to enrich for integrated clones. Colony screenings may be used to isolate clonal events.

[0109] A variety of viral constructs may be used to deliver the synthetic polypeptides or protein conjugates or fusion proteins thereof or compositions thereof to the targeted cells. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7( 1): 33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[0110] In one embodiment, a DNA segment encoding a synthetic polypeptide or protein conjugate or fusion protein thereof is contained in a plasmid vector that allows expression of the protein(s) and subsequent isolation and purification produced by the recombinant vector. Accordingly, the proteins disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.

[0111] To construct cells that express a synthetic polypeptide or protein conjugate or fusion protein thereof, expression vectors for stable or transient expression may be constructed via conventional methods as described herein and introduced into cells. For example, nucleic acids encoding a synthetic polypeptide or protein conjugate or fusion protein thereof may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors / plasmids / viral vectors should be suitable for integration and replication in eukaryotic cells.

[0112] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in prokaryotic cells. Promoters that may be used include T7 RNA polymerase promoters, constitutive E. coli promoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms. The system may be used with various bacterial hosts.

[0113] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6: 187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0114] Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissuespecific, or species specific. In addition to the sequence sufficient to direct transcription, apromoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human betaactin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EFl -a) promoter with or without the EFl -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0115] Moreover, inducible and tissue specific expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence capable of driving expression of the desired protein operably linked thereto.

[0116] The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e g., tissue-specific regulatory elements are used to express the nucleic acid).Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0117] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5 ’-and 3 ’-untranslated regions for mRNA stability and translation efficiency from highly-expressed genes like a-globin or -globin; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.

[0118] When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[0119] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a cellwhether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate coprecipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0120] Any of the vectors comprising a nucleic acid sequence that encodes a synthetic polypeptide or protein conjugate or fusion protein thereof is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA 110(6): 2082-2087(2013) incorporated herein by reference); or viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid encoding the components of the present system is a DNA molecule. In some embodiments, the nucleic acid encoding the components of the present system is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the components of the present system is an RNA molecule, which may be electroporated to cells.

[0121] Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1 : 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459(1 -2):70-83), incorporated herein by reference.Compositions

[0122] Also disclosed herein are compositions comprising a synthetic polypeptide or protein conjugate or fusion protein thereof as described herein or a nucleic acid molecule comprising a sequence encoding the synthetic polypeptide or protein conjugate or fusion protein thereof.

[0123] In some embodiments, the composition is a cell culture medium. Cell culture medium refers to any media for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.

[0124] The compositions may further comprise excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0125] Excipients and carriers may include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and / or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, preservatives, and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

[0126] The compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and / or preservatives, commonly found in proteinaceous compositions.

[0127] The disclosed synthetic polypeptides or protein conjugates or fusion proteins thereof may be individually or as a group entrapped in microcapsules (for example, liposome, albumin microspheres, microemulsions, nanoparticles and nanocapsules), in macroemulsions, or in sustained-release preparation. The disclosed compositions or components thereof may be in a liposome and combined with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulations include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, and bile acids. Preparation of such liposomal formulations is within the level of skill in the art.

[0128] The disclosed compositions or components thereof may be individually or as a group prepared in a hydrogel. The term “hydrogel” herein refers to a specific type of gel in which water- swellable polymeric matrices that can absorb a substantial amount of water in a three-dimensional network of macromolecules held together by covalent or noncovalent crosslinks.Methods

[0129] The disclosure also provides methods for in vitro or ex vivo differentiation, stimulation and / or expansion of immune cells (e.g., T cells, NK cells, and B cells). The methods described herein can be used to produce immune cells that have commercial or clinical applications.

[0130] “ Differentiation” refers to a process by which cells differentiate from one cell type (e.g., a multipotent, totipotent, or pluripotent differentiable cell) to another cell type such as a target differentiated cell (e g., a T cell). Oftentimes as a result of differentiation the potency or proliferation of a cell is decrease or the cell is moved to a more developmentally restricted state. “Proliferation” refers to increasing cell division, either symmetric or asymmetric division of cells. “Expansion” refers to increasing the number of cells as compared to the number originally present, for example, as the outcome of cell division and cell death. “Stimulation” or “activation” refers to inducing a change in the biologic state of a cell (e.g., T cells and NK cells) which can result in expression of activation markers, production of cytokines, increases in autophagy, proliferation and / or cytotoxicity to target cells.

[0131] The methods comprise contacting an immune cell (e.g., a T cell, a NK cell), or a precursor or progenitor cell thereof, with a synthetic polypeptide, a protein conjugate or fusion protein thereof, or a composition comprising thereof, as described herein.

[0132] In some embodiments, the methods comprise adding the synthetic polypeptide, protein conjugate or fusion protein thereof, or composition comprising thereof, to a buffer or cell culture medium containing the immune cell (e.g., a T cell, a NK cell), or a precursor or progenitor thereof. Alternatively, or in addition, in some embodiments, the methods comprise adding a buffer or cell culture medium comprising a synthetic polypeptide or a protein conjugate or fusion protein thereof to a population of immune cells (e.g., cells under culture conditions). Thus, also disclosed herein are buffers or cell culture media comprising a synthetic polypeptide or a protein conjugate or fusion protein thereof, as disclosed herein.

[0133] A vessel used for the contacting and subsequent culturing of the immune cell (e.g., a T cell, a NK cell), or a precursor or progenitor thereof can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, Grex®culture vessels, culture bag, and roller bottle, as long as it is capable of culturing the cells therein. The cells may be cultured in any volume (e.g., at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein) depending on the needs of the culture. The vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The vessel may be part of an open or closed system. The length of time in culture, as well as other components of the cell culture medium and cell culture conditions (e.g., temperature, CO2) are those which promote cell growth and allow for the desired degree of differentiation, stimulation and / or expansion.

[0134] In some embodiments, the methods also comprise contacting the immune cell (e.g., a T cell, aNK cell), or a precursor or progenitor thereof with one or more additional cytokines. The cytokine may include, but is not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, midkine, and the like.

[0135] In some embodiments, the methods further comprise stimulating the immune cell with one or more stimulating agents to produce a population of activated immune cells under a suitablecondition. Any combination of one or more stimulating agents can be used to produce a population of activated cells including, but not limited to, an antibody or functional fragment thereof which targets a T-cell stimulatory or co-stimulatory molecule (e.g., anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody, or a functional fragment thereof), or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TP A), phytohaemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or a natural ligand to a stimulatory or costimulatory molecule.

[0136] As used herein, the term “T cell” or “T lymphocyte” refers to a cell expressing CD3 (CD3+) and a T Cell Receptor (TCR+). T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), CD4+CD8+ T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use include naive T cells and memory T cells. Also encompassed are “NKT cells,” a specialized population of T cells that express a semi-invariantT-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+ and CD8- cells. Also encompassed are “gamma-delta T cells ( / 8 T cells),” a small subset of T cells possessing a distinct TCR on their surface composed of a " / -chain and a 8-chain rather than a- and 0-TCR chains. Also encompassed are “regulatory T cells” or “Tregs”, which refer to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs cells are typically transcription factor Foxp3 -positive CD4+ T cells and can also include transcription factor Foxp3 -negative regulatory T cells that are IL-10- producing CD4+ T cells.

[0137] The T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a subject (e.g., mammal). If obtained from a subject, the T cell can be obtained from numerous sources, including but not limited to bone marrow, blood, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+ double positive T cells, CD8+ T cells (e.g., cytotoxic Tcells), CD4+ helper T cells, e.g., Thl and Th2 cells, peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infdtrating cells, memory T cells, naive T cells, and the like. The T cell may be a CD8+ T cell or a CD4+ T cell.

[0138] The T cell may be a genetically engineered T cell. In some embodiments, the genetically engineered T cell may be genetically engineered before and / or after the disclosed methods. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) or engineered / exogenous T cell receptor. CARs and engineered TCRs can confer an arbitrary specificity onto an immune effector cell. In some embodiments, the T cell has been genetically engineered to knock out an endogenous receptor, or other molecule that affects a cell function of interest (e.g., knocks out an undesired function to confer increased safety or efficacy of engineered cell). In some embodiments, the methods further comprise genetically engineering the T cells.

[0139] Natural killer cells (NK cells) refer to differentiated lymphocytes. NK cells are characterized by their ability to bind to and kill cells that fail to express “self’ MHC / HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. As with the T cells the NK cells can be a cultured NK cell, e.g., a primary NK cell, or a NK cell from a cultured cell line, or a NK cell obtained from a subject, and may be genetically engineered. In some embodiments, the methods further comprise genetically engineering the NK cells.

[0140] Precursor or progenitor cells are less differentiated cells or undifferentiated cells that have the potential to differentiate into the cell type of interest (e.g., a T cell, a NK cell). Depending on cell differentiation, precursor cells could be multipotent, pluripotent and totipotent, whereas progenitor cells can be unipotent or oligopotent. Precursor or progenitor cells include stem cells (e.g., embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), cells which are more differentiated and specified as compared to stem cells (e.g., descendants of stem cells).

[0141] In certain embodiments, the stem or progenitor cells may be selected from embryonic stem cells, hematopoietic stem or progenitor cells, cells isolated from bone marrow, cord blood, peripheral blood, thymus, or the progenitor cells may have been differentiated from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) in vitro. Stem or progenitor cells herein may be, but are not limited to, ESCs, induced pluripotent stem cells or tissue stem cells (also calledtissue-specific stem cell, or somatic stem cell). Stem or progenitor cells from primary tissue or ESC or iPSC may be from human or non-human animals (e.g., mouse) in origin.

[0142] Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos.Tissue stem cells are present at particular locations in tissues and have an undifferentiated intracellular structure. Therefore, the pluripotency of tissue stem cells is typically low. Tissue stem cells are separated into categories, based on the sites from which the cells are derived, such as the dermal system, the digestive system, the bone marrow system, the nervous system, and the like. Tissue stem cells in the bone marrow system include hematopoietic stem cells, mesenchymal stem cells, and the like.

[0143] Hematopoietic stem or progenitor cells are cells that are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation and include hematopoietic stem cells, multipotential hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).

[0144] Induced pluripotent stem cells (iPS cells or iPSCs) are pluripotent stem cells artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors.

[0145] The precursor or progenitor cells may be genetically modified. In some embodiments, the stem or progenitor cells express an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR), or both. In some embodiments, the precursor or progenitor cells express an exogenous invariant natural killer T cell (iNKT) associated TCR. In some embodiments, the stem or progenitor cells express an exogenous antigen-specific TCR or have an exogenous genetic modification of genes that modulate differentiation, expansion or function. In some embodiments, the methods further comprise genetically modifying the precursor or progenitor cells.Kits

[0146] Also within the scope of the present disclosure are kits that include the synthetic polypeptides, protein conjugates, fusion proteins, and compositions thereof as disclosed herein.

[0147] For example, the kits may contain one or more reagents or components useful, necessary, or sufficient for practicing any of the methods described herein, such as, administration reagents, cell culture media or components thereof, negative and positive control samples (e.g., cells, template DNA), cells, containers (e.g., cell culture vessels), detection and analysis instruments, software, instructions, and the like.

[0148] The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of methods to differentiate, stimulate and / or expand T cells orNK cells. The instructions generally include information as to effective quantity of the synthetic polypeptides, protein conjugates, fusion proteins, and compositions disclosed herein and conditions for contacting the T cells or NK cells with the disclosed synthetic polypeptides, protein conjugates, fusion proteins, and compositions. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert.

[0149] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. The packaging may be singleuse or multi-use packages. As described above, kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0150] The kit will typically be provided with its various components in one or more packages, e g., a fiber-based, a cardboard, polymeric, or a Styrofoam box. The enclosure(s) can be configured so as to maintain a temperature differential between the interior and the exterior, for example, to provide insulating properties to keep the reagents at a preselected temperature for a preselected time. The packaging can be air-tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.Sequences

[0151] The various polypeptides referenced in the present disclosure include the following.

[0152] Exemplary spacer polypeptides referenced in the present disclosure include the following.Examples

[0153] The following are examples of the present invention and are not to be construed as limiting.Example 1

[0154] Starting with the IL-2 structure 2B5I which includes IL-2 in complex with the three receptors: alpha, beta, and gamma, the structure of IL-2 that interacts with beta and gamma was maintained while removing the rest of the structure (FIGS. 1A and IB). This trimmed version of IL-2 was then input into a generative diffusion model (RFdiffusion) to generate a backbone for the removed parts of IL-2. Multiple structures were generated and ordered for expression in E. coli and purified with IMAC column.

[0155] Recombinant human interleukin-2 (rhIL-2) requires multiple lots and refolding when produced in E. coli, introduced time consuming steps to production and requiring validation for each lot. All synthetic IL-2s had high yields and the majority of the product was soluble in E. coli, thereby eliminating large amounts of manufacturing time and cost compared to rhIL-2 production.Example 2

[0156] The synthetic IL -2 constructs were screened for binding to IL-2R beta using mammalian display and flow cytometry (FIG. 2). The synthetic IL-2 constructs that had greater activity compared to rhIL-2 were screened using the same method for IL-2R beta, alpha, and gamma, individually (FIG. 3). Variants of ABG6 were made containing one or more of amino acid substitutions at C34, E83, R86, S90. All of these ABG6-derived variants had good expression in E. coli and the majority of the product was found in the soluble fraction. Two point mutations,S90E and R86E, in the ABG6 sequence increased IL-2R beta binding and had increased potency in cell proliferation. Two synthetic IL -2 constructs, ABG6-M1 (having S90E substitution in ABG6 background) and ABG6-M5 (having C34A, R86E, and S90E substitutions in ABG6 background), were tested for binding to IL-2R beta using surface plasmon resonance and had low nanomolar affinity compared to rhIL-2 which has approximately 500 nM affinity (FIG. 4). Additionally, using SPR the affinity to IL-2R alpha was not detectable.

[0157] To test protein function, activation of the STAT5 pathway in YT cells, a tumor cell line with NK cell like phenotype and killing activity was used. This assay showed lower phosphorylation compared to rhIL-2 but stronger activity when CD25 was knocked out from the genome (FIG. 5). Additionally, NK92 cells were activated and showed similar proliferation to rhIL-2. When IL-2R alpha was blocked with an antibody, greater proliferation was observed (FIG. 6). Using the same cells, IFN-gamma secretion was monitored and much higher activity was observed with the synthetic IL-2 compared to rhIL-2 (FIG. 7). Increased IFN-gamma facilitates antitumor mechanisms which boosts the synthetic IL-2’s application in cell therapies. The most common cell therapy requires expanding engineered T cells. CD8 and CD4 / CD8 positive T cells were isolated and their expansion characteristics were compared to rhIL-2 (FIGS. 8 and 9). Phenotypes of stimulated and expanded T cells were tested and no differences were found in the expression of different T cell phenotypes between the rhIL-2 and ABG6-M1 (FIG. 10).

[0158] The synthetic IL-2 polypeptides also have greater thermostability than rhIL-2. Thermostability was determined by heating 1.5 mL tubes with 100 pL of IL-2 ABG6-M1 and rhIL-2 at 4° C or 95° C for 15 min. After which they were tested for their ability to induce NK92 proliferation. ED50 values were determined by calculating the concentration of protein at half the max proliferation. The dose response curve was generated by measuring cell proliferation at concentrations of protein ranging from 0.015 to 30 ng / mL. ABG6-M1 had no loss in activity while rhIL-2 lost 60% of its activity (Table 1).Table 1:

[0159] The synthetic IL-2 polypeptides also have greater stability in cell culture media than rhIL-2. Specific activity was assessed through a Proliferation bioassay in NK92 cells wherein cells were treated with ABG6 Ml or IL-2 at the indicated concentrations for 72 hours. Cell proliferation was measured using a Cell Counting Kit-8 assay (CCK-8; APExBIO). As shown in FIG. 12A, following incubation at 37°C for 10 days at concentrations of 50, 25, or 10 ng / mL, the specific activity for ABG6-M1 was greater, and comparable to a control stored at 4°C without any incubation, at each concentration tested compared to rhIL-2. rhIL-2 has specific activities of less than 50% of the control at each concentration. When comparing the stability of 10 ng / mL of ABG6-M1 and rhIL-2 over time, up to two months, the specific activity of ABG6-M1 remained relatively constant and similar to the control (FIG. 12b). Whereas the specific activity of rhIL-2 decreased over time, with specific activities well below 50% of the control even after 10 days, as in FIG. 12A). This data indicates that the synthetic IL-2 polypeptides as well as being thermostable would also be particularly well-suited for use in bulk media formulations for cell therapies and result in enhanced cost efficiency and reduction of labor during cell production.

[0160] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0161] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

CL IMSWhat is claimed is:

1. A synthetic polypeptide which binds interleukin 2 receptor subunit beta (IL-2R beta), wherein the synthetic polypeptide comprises: a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of RDLISNINVI (SEQ ID NO: 2), EDLISNINVI (SEQ ID NO: 3), RDTISNINVI (SEQ ID NO: 4), RDLIENINVI (SEQ ID NO: 5), RDTIENINV (SEQ ID NO: 6), EDLIENINVI (SEQ ID NO: 7), EDTISNINV (SEQ ID NO: 8), or EDTIENINV (SEQ ID NO: 9); and a third domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10) or LNRWITFAQ (SEQ ID NO: 11).

2. The synthetic polypeptide of claim 1, wherein the synthetic polypeptide has less than 50% identity to SEQ ID NO: 12.

3. The synthetic polypeptide of claim 1 or 2, wherein the first, second, and third domains may be in any order N-terminal to C-terminal within the synthetic polypeptide.

4. The synthetic polypeptide of any one of claims 1-3, wherein the first, second, and third domains from N-terminal to C-terminal in the synthetic polypeptide are ordered as: first-second- third or first-third-second.

5. The synthetic polypeptide of any one of claims 1-4, wherein each domain is separated by a spacer polypeptide.

6. The synthetic polypeptide of claim 5, wherein each spacer polypeptide is individually 10 to 60 amino acids in length.

7. The synthetic polypeptide of any one of claims 1-6, wherein the synthetic polypeptide comprises one or more additional amino acids N-terminal and C-terminal to the first, second, and third domains.

8. The synthetic polypeptide of any one of claims 1-7, wherein the synthetic polypeptide comprises: a first domain having an amino acid sequence of LQLEHLLLDLQM (SEQ ID NO: 1); a second domain having an amino acid sequence of RDLISNINVI (SEQ ID NO: 2); anda third domain having an amino acid sequence of LNRWITFCQ (SEQ ID NO: 10).

9. The synthetic polypeptide of any one of claims 1-8, wherein the synthetic polypeptide comprises an amino acid sequence having at least 70% identity to one of SEQ ID NOs: 13-26.

10. The synthetic polypeptide of any one of claims 1-9, wherein the synthetic polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 13-26.

11. The synthetic polypeptide of any one of claims 1-7, wherein the synthetic polypeptide comprises at least one of: EDLISNINVI (SEQ ID NO: 3), RDTISNINVI (SEQ ID NO: 4), RDLIENINVI (SEQ ID NO: 5), RDTIENINV (SEQ ID NO: 6), EDLIENINVI (SEQ ID NO: 7), EDTISNINV (SEQ ID NO: 8), and EDTIENINV (SEQ ID NO: 9).

12. The synthetic polypeptide of any one of claims 1-7 or 11, wherein the synthetic polypeptide comprises one or more amino acid substitutions compared to SEQ ID NO: 18.

13. The synthetic polypeptide of claim 12, wherein the one or more amino acid substitutions comprise substitutions of C34, E83, R86, L88, S90, and combinations thereof as compared to SEQ ID NO: 18.

14. The synthetic polypeptide of claim 12 or 13, wherein the one or more amino acid substitutions comprise C34A, E83K, R86E, L88T, S90E, and combinations thereof as compared to SEQ ID NO: 18.

15. The synthetic polypeptide of claim 13 or 14, wherein the synthetic polypeptide further comprises at least one additional amino acid substitution as compared to SEQ ID NO: 18.

16. The synthetic polypeptide of any one of claims 11-15, wherein the synthetic polypeptide comprises an amino acid sequence having at least 70% identity to one of SEQ ID NOs: 27-33.

17. The synthetic polypeptide of any one of claims 11-16, wherein the synthetic polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 27-33.

18. The synthetic polypeptide of any one of claims 1-17, wherein the synthetic polypeptide is 90 to 130 amino acids in length.

19. The synthetic polypeptide of any one of claims 1-18, wherein the synthetic polypeptide is thermostable.

20. The synthetic polypeptide of any one of claims 1-19, wherein the synthetic polypeptide has increased stability in cell culture media as compared to recombinant human IL -2.

21. The synthetic polypeptide of any one of claims 1-20, wherein the synthetic polypeptide has fully or partially reduced binding to interleukin 2 receptor subunit alpha (IL-2R alpha) as compared to recombinant human IL-2.

22. A protein conjugate or fusion protein comprising a synthetic polypeptide of any of claims 1- 21 linked to at least one moiety.

23. The protein conjugate or fusion protein of claim 22, wherein the at least one moiety is a protein, a protein domain, a polypeptide, or a peptide.

24. The protein conjugate or fusion protein of claim 22 or 23, wherein the at least one moiety comprises an antibody or fragment thereof.

25. The protein conjugate or fusion protein of claim 22, wherein the at least one moiety is polyethylene glycol (PEG).

26. A composition comprising a synthetic polypeptide of any of claims 1-21 or a protein conjugate or fusion protein thereof and a carrier.

27. The composition of claim 26, wherein the composition is a cell culture media or component thereof.

28. A method for in vitro or ex vivo T cell or NK cell differentiation, stimulation and / or expansion comprising contacting a T cell, an NK cell, or a precursor or progenitor cell thereof, with a synthetic polypeptide of any of claims 1-21, a protein conjugate or fusion protein thereof, or a composition comprising thereof.

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