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

Abstract

The present disclosure provides engineered IL-2 polypeptides, protein conjugates, and fusion proteins, and related compositions and uses thereof. In particular, the present disclosure provides engineered IL-2 polypeptides that exhibit enhanced binding affinity for IL-2 receptor β (IL-2Rβ) and the IL-2Rβ / IL-2Rγ dimeric complex.

Description

ENGINEERED IL-2 POLYPEPTIDES AND CONJUGATES, COMPOSITIONS, AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 572,536, filedApril 1 , 2024, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0002] The content of the electronic sequence listing entitled “BIOTN_42866_601_SequenceListing.xml” (Size: 70,452 bytes; and Date of Creation: April 1, 2025) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure provides engineered IL-2 polypeptides, protein conjugates, and fusion proteins, and related compositions and uses thereof. In particular, the present disclosure provides engineered IL-2 polypeptides that exhibit enhanced binding affinity for IL-2 receptor p (IL-2Rp) and the IL-2Rp / IL-2Ry dimeric complex.BACKGROUND

[0004] Interleukins are a group of cytokine signaling molecules that are expressed and secreted by and regulate the activation and differentiation of immune cells (e.g., leukocytes, often lymphocytes), as well as proliferation, maturation, migration, and adhesion. Many aspects of the immune system depend on interleukins, and deficiencies or genetic mutations in interleukins have been associated with autoimmune diseases or immune deficiency. In particular, Interleukin-2 (IL- 2) promotes the proliferation, differentiation, activation, and survival of mature lymphocytes, including 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 CD 122), and a gamma subunit (also known as IL-2Ry, yc, or CD 132). Genetic deficiencies of IL-2 or IL-2R a or chains can result in immunodeficiency soon after birth, with a later accumulation of activated T cells that do not function properly. It is significant that these phenotypic changes cannot be compensated by other cytokines.SUMMARY

[0005] Embodiments of the present disclosure include engineered interleukin-2 (IL-2) polypeptides. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 99% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the engineered IL-2 polypeptides of the present disclosure exhibit increased binding affinity for IL-2 receptor p (IL-2Rp).

[0006] In some embodiments, the engineered IL-2 polypeptide comprises increased binding affinity for the IL-2Rp as compared to wildtype IL-2 (SEQ ID NO: 1) or compared to recombinant human IL-2 (SEQ ID NO: 2). In some embodiments, the engineered IL-2 polypeptide comprises increased binding affinity for the IL-2Rp / IL-2Ry dimeric complex compared to the IL-2Rp / IL- 2Ry / IL-2Ra trimeric complex. In some embodiments, the engineered IL-2 polypeptide comprises decreased binding affinity for IL-2Ra.

[0007] In some embodiments, the engineered IL-2 polypeptide comprises a dissociation constant (KD) for the IL-2Rp that is less than about 100 nM. In some embodiments, the engineered IL-2 polypeptide comprises a dissociation constant (KD) for the IL-2R[3 that is less than about 10 nM.

[0008] In some embodiments, the engineered IL-2 polypeptide (i) increases NK cell proliferation, (ii) increases IFNy secretion, (iii) increases expansion capacity of CD8 / CD4 T cells, (iv) increases expression of tumor homing antigens on T cells, or any combinations thereof.

[0009] In some embodiments, the engineered IL -2 polypeptide comprises at least one amino acid substitution. In accordance with these embodiments, the at least one amino acid substitution is present at amino acid position 4, 7, 12, 26, 32, 35, 45, 54, 60, 61, 62, 64, 68, 73, 74, 79, 80, 81, 83, 85, 87, 90, 95, 104, 105, 110, 112, 116, 120, 124, and / or 125, including any combinations thereof.

[0010] In some embodiments, the engineered IL-2 polypeptide comprises at least 2 amino acid substitutions, at least 3 amino acid substitutions, at least 4 amino acid substitutions, at least 5 amino acid substitutions, at least 6 amino acid substitutions, at least 7 amino acid substitutions, at least 8 amino acid substitutions, at least 9 amino acid substitutions, or at least 10 amino acid substitutions. In some embodiments, the engineered IL-2 polypeptide comprises 2 to 5 amino acid substitutions,6 to 10 amino acid substitutions, 11 to 15 amino acid substitutions, 16 to 20 amino acid substitutions, 21 to 25 amino acid substitutions, or 26 to 30 amino acid substitutions.

[0011] In some embodiments, the engineered IL -2 polypeptide comprises at least one amino acid substitution, including but not limited to, S4E, T7I, L12E, N26E, K32Q, K35V, Y45K, K54I, E60K, E61R, E61K, E62Q, K64D, E68A, A73L, Q74E, H79S, L80Q, R81D, R83D, L85T, S87E, S87D, N90K, E95T, M104P, C105T, E110D, A112L, E116Q, R120Y, F124A, and / or C125S, including any combinations thereof.

[0012] In some embodiments, the engineered IL-2 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 3-29. In some embodiments, the engineered IL- 2 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 30-35. In some embodiments, the engineered IL-2 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 36-43. In some embodiments, the engineered IL-2 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 44- 52. In some embodiments, the engineered IL-2 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 53-54. In some embodiments, the engineered IL-2 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 55- 60. In some embodiments, the engineered IL-2 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 61-70. In some embodiments, the engineered IL-2 polypeptide comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 3- 70.

[0013] In some embodiments, the engineered IL-2 polypeptide comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 3-70. In some embodiments, the engineered IL- 2 polypeptide comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 3- 70.

[0014] Embodiments of the present disclosure also include a protein conjugate or fusion protein comprising any of the engineered IL-2 polypeptides of the present disclosure 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).

[0015] Embodiments of the present disclosure also include a composition comprising any of the engineered IL-2 polypeptides of the present disclosure, or a protein conjugate or fusion protein of any of the engineered IL-2 polypeptides of the present disclosure, and a carrier.

[0016] Embodiments of the present disclosure also include an in vitro or ex vivo method for enhancing or improving one or more of properties of a T cell or NK cell, including but not limited to, T cell or NK cell differentiation, T cell or NK cell stimulation, T cell or NK cell expansion or proliferation, and / or T cell or NK cell viability or longevity. In accordance with these embodiments, the method includes contacting a T cell or NK cell with any of the engineered IL-2 polypeptides of the present disclosure, or a protein conjugate or fusion protein of any of the engineered IL-2 polypeptides of the present disclosure, or a composition comprising any of the engineered IL-2 polypeptides of the present disclosure or a protein conjugate or fusion protein thereof.

[0017] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 : Binding activity of enriched mutations (amino acid substitutions) conferring highest binding to IL-2 receptor (IL-2Rp) were combined in variants of three mutations at a time (see Table 1). Binding was measured by displaying the IL-2 variant on the surface of HEK293T cells and binding an Avi-tagged IL-2Rp and a fluorescently labeled Streptavidin. Greater MFI indicates greater receptor binding. Variants were split into 3 experiments and are indicated in the figure beginning with recombinant human IL-2 (SEQ ID NO: 2). Experiments were normalized to each other based on recombinant human IL-2 MFI.

[0019] FIG. 2: Exemplary binding activity of IL-2 variants in a subsequent round of binding experiments (see Table 1). Binding was measured by displaying the IL-2 variant on the surface of HEK293T cells and binding Avi-tagged IL-2Rp and a fluorescently labeled Streptavidin. Greater MFI indicates greater receptor binding.

[0020] FIG. 3: Exemplary binding activity of IL-2 variants in a subsequent round of binding experiments (see Table 1). Binding was measured by displaying the IL-2 variant on the surface of HEK293T cells and binding Avi-tagged IL-2Rp (white) and IL-2Ra (black) and Streptavidin. Greater MFI indicates greater receptor binding.

[0021] FIG. 4: Exemplary binding activity of IL-2 variants in a subsequent round of binding experiments (see Table 1). Binding was measured by displaying the IL-2 variant on the surface of HEK293T cells and binding Avi-tagged IL-2Rp (white) and IL-2Ra (black) and Streptavidin. Greater MFI indicates greater receptor binding.

[0022] FIG. 5: To validate IL-2 variant binding affinity for IL-2Rp determined using mammalian display, surface plasmon resonance (SPR) was performed on various IL-2 variants. In some cases, SPR can be a more accurate measurement of affinity since the molarity of the receptor and ligand are accurately measured compared to the variable expression when using mammalian display. The decreased KD (dissociation constant) was much lower for the engineered IL-2 variants compared to recombinant human IL-2 (503 nM), indcating that binding of the enginereed IL-2 variants to IL-2Rp was signifiantly stronger.

[0023] FIG. 6: Exemplary results of the effects of IL-2 variants in an NK92 cell proliferation assay. The lower the EC50 value, the more potent the variant for inducing proliferation. Two assays were conducted: one stimulating NK92 cells; and one stimulating NK92 cells with the addition of a CD25 blocking antibody. The antibody was added to simulate the proliferation of cytotoxic CD8 / CD4 T cells, which have low levels of CD25.

[0024] FIG. 7: Exemplary results of the effects of IL-2 variants on NK92 induced INFy secretion. NK92 cells were treated with the IL-2 variants for 48 hours and measured for INFy secretion. These data demonstrate that the IL-2 variants induce greater production of INFy than the recombinant human IL-2. (EC50 was not calculated for recombinant human IL-2 since it was higher than the most concentrated samples.)

[0025] FIG. 8: Exemplary results of the effect of IL-2 variants on expansion of primary T cells. Primary T cells were stimulated with CD3 / CD28 Dynabeads and grown with the indicated IL-2 variants for 19 days. These data indicate that IL-2 variants with increased binding to the IL- 2Rp / IL-2Ry dimeric complex can increase the expansion capacity of CD8 / CD4 T cells.

[0026] FIG. 9: Exemplary results demonstrating greater expansion capacity of CD4 / CD8 T cells when grown with the CD122 IL-2 variant, V3-8. V3-8 supported the same expansion capacity at 3.6 ng / mL as standard IL-2 at 14.3 ng / mL. The standard IL-2 treated T cells grown in 14.3 ng / mL reached their peak expansion on day 21, and on that day the V3-8 14.3 ng / mL cells had expanded 2.7 times. The maximal expansion difference was seen on day 28 where the V3-8 cells had expanded 6.1 times more than the standard IL-2 grown cells at 14.3ng / mL.

[0027] FIG. 10: Exemplary results of the activation of marker expression of CD4 / CD8 T cells grown with the IL-2 CD122 binding variant, V3-8. The activation markers CTLA-4, CD40L, and CD62L show similar expression profiles when grown with V3-8 in comparison to standard IL -2 C125S.

[0028] FIG. 11 : Exemplary results of exhaustion marker expression of CD4 / CD8 T cells grown with the IL-2 CD122 binding variant, V3-8. The exhaustion markers Lag-3, PD-1 and Tim-3 show similar expression when grown with V3-8 in comparison to standard IL-2 C125S. This is indicative of the cells ability to remain functional longer term in a CAR T cell therapy.

[0029] FIG. 12: Exemplary results of the viability of CD4 / CD8 T cells grown with the IL-2 CD122 binding variant, V3-8. Viability was recorded each culture day. The IL-2 CD122 binding variant V3-8 supported greater viability over the course of the experiment then the standard IL-2 C125S, even at a low dose.

[0030] FIG. 13: Exemplary results of the percentage of T cells expressing the longevity phenotype, CD45RA+CCR7+. Percentages of CD45RA+CCR7+ T cells identified in CD4 / CD8 T cells grown with the IL-2 CD122 binding variant, V3-8. T cells expressing both CD45RA and CCR7 are known to be long lived and self-renewing. Their presences correlates with the success of CAR T therapies. As can be seen here, V3-8 IL-2 supports their growth within the culture even at the higher dose of 14.3 ng / mL in comparison to standard C125S IL-2.

[0031] FIG. 14: Exemplary results demonstrating that transposed CAR T cells cultured with engineered IL-2 kill tumor cells equivalently as standard IL-2 C125S. Purified Human CD4+and CD8+T cells were TcBuster-M transposed with a CAR19. Specific killing was measured by the luciferase expression of unlysed NALM-6 target cells.

[0032] FIGS. 15A-15C: Exemplary results demonstrating that cells cultured with the engineered IL-2 polypeptides of the present disclosure released higher levels of cytotoxic factors than controls. After the 24-hour kill assay described above, media from the assay was reserved to examine the amount of cytotoxic factors Interferon gamma (INFg), Granzyme B, and Perforin present in the media.DETAILED DESCRIPTION

[0033] Disclosed herein are engineered IL-2 polypeptides, protein conjugates, and fusion proteins, and related compositions and uses thereof. In particular, the present disclosure provides engineered IL-2 polypeptides that exhibit selective and increased binding for IL-2 receptor (IL-2Rp of CD122) and the IL-2Rp / IL-2Ry dimeric complex (e.g., as compared to the IL-2Ra / IL- 2Rp / IL-2Ry trimeric complex). In some embodiments, the engineered IL-2 polypeptide variants of the present disclosure are capable of inducing greater expansion of CD8 effector cells versus recombinant human IL-2, and decreased expansion of unwanted cells like Tregs. These and other improvements have various therapeutic benefits for treatments like tumor-infiltrating (TIL) therapy and CAR-T therapy. Other engineered IL-2 polypeptides are primarily injectable drugs and not commercially available as reagents. Engineered IL-2 polypeptides may improve proliferation capacity of stimulated T and NK cells, maintaining phenotype and enhancing INFy secretion and killing properties for NK cells. There are currently no other IL-2 variants available with these functional properties.

[0034] For example, current TIL therapy workflows rely heavily on the use of IL-2 in large amounts, which can lead to longer cell culture times. The prolonged cell culture times required may also lead to T cells that begin to differentiate and experience exhaustion. Moreover, IL-2 has cellular pleiotropy and tends to preferentially expand cells with higher levels of CD25, thereby fostering less desirable traits that compromise successful TIL therapy. Furthermore, engineered IL-2 polypeptides can improve proliferation capacity of stimulated T and NK cells, maintaining phenotype and enhancing INFy secretion and killing properties for NK cells. Furthermore, engineered IL -2 polypeptides can selectively proliferate cytotoxic lymphocytes over immunosuppressive T-ReG Cells that would be unwanted in a TIL therapy.

[0035] As described further herein, experiments were conducted to build a mammalian display platform to evolve a deep scanning IL -2 mutation library to identify variants that preferentially bind to IL-2Rp and exhibit reduced binding to IL-2Ra. Although other studies have been conducted to identify IL-2 variants, none involved deep scanning mutation and display on mammalian cells. Therefore, many potentially beneficial mutations were missed. Furthermore, experiments were conducted to assess the effects of different receptor affinities on T cell / NK cell proliferation / activity. As described further herein, IL-2 polypeptide variants were created that preferentially bind to the IL-2Rp / IL-2Ry dimeric complex and have reduced IL-2Ra binding.

[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, the numbers 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 Cm) regions, and each light chain contains one N-terminalvariable (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 (A), 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 p 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 also exhibit 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., >106M’1, >107M’1, >108M’1, >109M’1, >1O10M’1, >10nM’1, >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, IgM, 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, andCHI 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 human immunoglobulin. 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 antibodiesthat 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 moieties 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 or more 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 ordouble-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 by recombinant 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 methanogenicorganisms 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 ofa peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, orDF 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 (a-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3- diaminopropanoic acid), y-Glu / -glutamic acid), Gaba (y-aminobutanoic acid), p-Pro (pyrrolidine-3 -carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), phPro (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) and FASTA 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 atleast 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] As used herein, the terms “IL-2 variant,” “IL-2 polypeptide variant,” “IL-2 mutation,” “IL-2 mutant,” and the like, generally refer to an IL-2 polypeptide that is less than 100% identical in primary amino acid sequence to a wild type IL-2 (e.g., SEQ ID NO: 1), and also comprises at least one amino acid substitution compared to a wild type IL-2 (e.g., SEQ ID NO: 1). IL-2 polypeptide variants described herein can have one or more functional and / or structural features that are considered enhancements or improvements over other IL-2 polypeptides. IL-2 polypeptide variants described herein are non-naturally occurring and are generally referred to as engineered polypeptides or engineered polypeptides.

[0100] 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.Engineered Polypeptides

[0063] Embodiments of the present disclosure pertain to engineered interleukin-2 (IL-2) polypeptides. In accordance with these embodiments, the engineered IL-2 polypeptides of thepresent disclosure have less than 99% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1.

[0064] In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 98% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 97% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 96% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 95% amino acid sequence identity with human IL -2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 90% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 85% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 80% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 70% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 60% amino acid sequence identity with human IL -2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In accordance with these embodiments, the engineered IL-2 polypeptides of the present disclosure have less than 50% amino acid sequence identity with human IL-2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1.

[0065] The engineered IL-2 polypeptides of the present disclosure exhibit various structural and functional enhancements over other IL-2 polypeptides. In some embodiments, the engineeredIL-2 polypeptides of the present disclosure exhibit increased binding affinity for IL-2 receptor p (IL-2Rp). In some embodiments, the engineered IL-2 polypeptide comprises increased binding affinity for the IL-2Rp as compared to wildtype IL-2 (SEQ ID NO: 1) or compared to recombinant human IL-2 (SEQ ID NO: 2). In some embodiments, the engineered IL-2 polypeptide comprises increased binding affinity for the IL-2Rp / IL-2Ry dimeric complex compared to the IL-2Rp / IL- 2Ry / IL-2Ra trimeric complex. In some embodiments, the engineered IL-2 polypeptide comprises decreased binding affinity for IL-2Ra.

[0066] The engineered IL-2 polypeptides may have decreased binding to IL-2Ra. In some embodiments, the engineered IL-2 polypeptide does not bind to IL-2Ra (e.g., the engineered IL-2 polypeptides exhibit similar or decreased binding characteristics as a negative control known to not bind IL-2Ra). In some embodiments, the engineered IL-2 polypeptide has reduced binding to IL-2Ra as compared to a wild type IL-2 and / or recombinant human IL-2.

[0067] In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a dissociation constant (KD) for the IL-2Rp that is less than about 100 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a dissociation constant (KD) for the IL-2Rp that is less than about 90 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less than about 80 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL- 2Rp that is less than about 70 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less than about 60 nM. In some embodiments an engineered IL-2 polypeptide of the present disclosure exhibits KD for the IL-2Rp that is less than about 50 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less than about 40 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less than about 30 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less than about 20 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less than about 10 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL- 2Rp that is less than about 5 nM.

[0068] In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 1 nM to about 100 nM. In some embodiments, anengineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 1 nM to about 75 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 1 nM to about 50 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL- 2Rp that is less from about 1 nM to about 25 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 1 nM to about 10 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 10 nM to about 100 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 25 nM to about 100 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 50 nM to about 100 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 75 nM to about 100 nM. In some embodiments, an engineered IL- 2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 5 nM to about 25 nM. In some embodiments, an engineered IL-2 polypeptide of the present disclosure exhibits a KD for the IL-2Rp that is less from about 10 nM to about 50 nM.

[0069] The engineered IL-2 polypeptides of the present disclosure exhibit various functional enhancements over other IL-2 polypeptides. In some embodiments, the engineered IL-2 polypeptide increases NK cell proliferation. In some embodiments, the engineered IL-2 polypeptide increases IFNy secretion. In some embodiments, the engineered IL-2 polypeptide increases expansion capacity of CD8 / CD4 T cells. In some embodiments, the engineered IL-2 polypeptide increases expression of tumor homing antigens on T cells. In some embodiments, the engineered IL-2 polypeptide can exert the above effects in any combinations, and may exert other effects.

[0070] In some embodiments, an engineered IL-2 polypeptide of the present disclosure comprises at least one amino acid substitution or mutation (also referred to as an IL-2 variant, an IL-2 polypeptide variant, an engineered IL-2 variant, or an engineered IL-2 polypeptide variant). In accordance with these embodiments, an amino acid substitution or mutation can be present at one or more of the following amino acid position of the IL-2 variant: 4, 7, 12, 26, 32, 35, 45, 54, 60, 61, 62, 64, 68, 73, 74, 79, 80, 81, 83, 85, 87, 90, 95, 104, 105, 110, 112, 116, 120, 124, and / or 125, including any combinations thereof.

[0071] In some embodiments, the engineered IL-2 variant comprises at least 2 amino acid substitutions, at least 3 amino acid substitutions, at least 4 amino acid substitutions, at least 5 amino acid substitutions, at least 6 amino acid substitutions, at least 7 amino acid substitutions, at least 8 amino acid substitutions, at least 9 amino acid substitutions, or at least 10 amino acid substitutions. In some embodiments, the engineered IL-2 variant comprises 2 to 5 amino acid substitutions, 6 to 10 amino acid substitutions, 11 to 15 amino acid substitutions, 16 to 20 amino acid substitutions, 21 to 25 amino acid substitutions, or 26 to 30 amino acid substitutions. Any of the engineered 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.

[0072] In some embodiments, the engineered IL-2 polypeptide variant comprises at least one amino acid substitution, including but not limited to, S4E, T7I, L12E, N26E, K32Q, K35V, Y45K, K54I, E60K, E61R, E61K, E62Q, K64D, E68A, A73L, Q74E, H79S, L80Q, R81D, R83D, L85T, S87E, S87D, N90K, E95T, M104P, C105T, E110D, A112L, E116Q, R120Y, F124A, and / or C125S, including any combinations thereof. In some embodiments, an engineered IL -2 polypeptide variant of the present disclosure comprises a combination of amino acid substitutions, such as those provided in Table 2 (e.g., SEQ ID NOs: 3-70).

[0073] In some embodiments, the above specific amino acid substitutions that can be present in an engineered IL -2 polypeptide variant of the present disclosure can be replaced with a different amino acid that has similar structural and / or functional properties. For example, 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 (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0074] 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. A functional 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.

[0075] In some embodiments, an engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 3-29. In some embodiments, an engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 3-29. In some embodiments, an engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 3-29. In some embodiments, an engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identity to any one of SEQ ID NOs: 3-29. In some embodiments, an engineered IL-2 polypeptide variant of the present disclosure comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 3-29. In some embodiments, an engineered IL-2 polypeptide variant of the present disclosure comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 3-29.

[0076] In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 30-35. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 30-35. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 30-35. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identity to any one of SEQ ID NOs: 30-35. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 30-35. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 30- 35.

[0077] In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 36-43. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 36-43. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 36-43. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identity to any one of SEQ ID NOs: 36-43. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 36-43. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 36- 43.

[0078] In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 44-52. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 44-52. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 44-52. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identityto any one of SEQ ID NOs: 44-52. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 44-52. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 44- 52.

[0079] In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 53-54. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 53-54. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 53-54. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identity to any one of SEQ ID NOs: 53-54. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 53-54. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 53- 54.

[0080] In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 55-60. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 55-60. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 55-60. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identity to any one of SEQ ID NOs: 55-60. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 55-60. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 55- 60.

[0081] In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 61-70. In someembodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 61-70. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 61-70. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identity to any one of SEQ ID NOs: 61-70. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 61-70. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 61- 70.

[0082] In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 3-70. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 75% amino acid sequence identity to any one of SEQ ID NOs: 3-70. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 3-70. In some embodiments, the engineered IL-2 polypeptide variant of the present disclosure comprises at least 85% amino acid sequence identity to any one of SEQ ID NOs: 3-70. In some embodiments, the engineered IL-2 polypeptide comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 3-70. In some embodiments, the engineered IL-2 polypeptide comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 3-70.

[0083] Embodiments of the present disclosure also include a protein conjugate or fusion protein comprising any of the engineered IL-2 polypeptides of the present disclosure 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).

[0084] In some embodiments, an engineered IL-2 polypeptide is covalently or non-covalently linked to at least one moiety. Accordingly, also provided herein are protein conjugates comprising engineered polypeptide as described herein and at least one moiety. For example, the engineered polypeptide may be fused to a moiety that provides for tagging or visualization (e.g., GFP). The engineered polypeptide may be fused to a moiety that has another functionality or activity usefulto target to certain cells (e.g., therapeutic agents, antibodies). The engineered polypeptide may be fused to a moiety that provides increased efficacy to the engineered polypeptide (e.g., half life extension moieties). The engineered polypeptide may be fused to a moiety that masks the function of the engineered polypeptide except under certain conditions or in certain locations (e.g., inactivating domains). For example, the engineered polypeptide may be linked to polyethylene glycol (PEG) molecules, albumin, an Fc region or domain to a protein of interest, an antigenbinding domain of an antibody, an antibody which targets a certain cell or blocks an unwanted activity or interaction, toxins, NKG2D, and the like.

[0085] The engineered 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-mal eimide, the succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and the periodate systems.

[0086] 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.

[0087] 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.

[0088] The fusion proteins are not limited by orientation or directionality of the engineered polypeptide and the at least one moiety. For example, any single moiety may be fused to the N- terminus or C-terminus of the engineered 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 engineered polypeptide).

[0089] The engineered 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 the preferred 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.

[0090] Any of the engineered 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 engineered 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 engineered polypeptide or fusion protein and may be linked with a linker polypeptide.Nucleic Acids

[0091] Also disclosed herein are nucleic acid molecules encoding the engineered IL-2 polypeptides or protein conjugates or fusion proteins thereof, as described further herein. The nucleic acids may be DNA, RNA, or combinations thereof. In some embodiments, the nucleic acids comprise one or more vectors.

[0092] 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 describedin 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.

[0093] The present disclosure also provides for DNA segments encoding the engineered 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.

[0094] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode the engineered 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.

[0095] 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.

[0096] 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 engineered 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.

[0097] 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, drugselection may be used to enrich for integrated clones. Colony screenings may be used to isolate clonal events.

[0098] A variety of viral constructs may be used to deliver the engineered 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.

[0099] In one embodiment, a DNA segment encoding an engineered 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.

[0100] To construct cells that express an engineered 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 an engineered 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.

[0101] 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.

[0102] 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 hereinby 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.

[0103] 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, a promoter 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.

[0104] 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 specificpromoter / 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.

[0105] 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.

[0106] 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 p-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.

[0107] 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.

[0108] 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 cell whether 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.

[0109] Any of the vectors comprising a nucleic acid sequence that encodes an engineered 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 presentsystem 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.

[0110] 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

[0111] Embodiments of the present disclosure also include a composition comprising any of the engineered IL-2 polypeptides of the present disclosure, or a protein conjugate or fusion protein of any of the engineered IL-2 polypeptides of the present disclosure, and a carrier. In accordance with these embodiments, the compositions provided herein can include an engineered IL-2 polypeptide or protein conjugate or fusion protein thereof as described herein, or a nucleic acid molecule comprising a sequence encoding the engineered IL-2 polypeptide or protein conjugate or fusion protein thereof. 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.

[0112] 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 asother 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).

[0113] 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.

[0114] The disclosed engineered 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.

[0115] 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

[0116] Embodiments of the present disclosure also include an in vitro or ex vivo method for enhancing or improving one or more of properties of a T cell or NK cell, including but not limited to, T cell or NK cell differentiation, T cell or NK cell stimulation, T cell or NK cell expansion or proliferation, and / or T cell or NK cell viability or longevity. In accordance with these embodiments, the method includes contacting a T cell or NK cell with any of the engineered IL-2 polypeptides of the present disclosure, or a protein conjugate or fusion protein of any of the engineered IL-2 polypeptides of the present disclosure, or a composition comprising any of theengineered IL-2 polypeptides of the present disclosure or a protein conjugate or fusion protein thereof.

[0117] Embodiments of the present disclosure also provide 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.

[0118] “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.

[0119] The methods comprise contacting an immune cell (e.g., a T cell, a NK cell), or a precursor or progenitor cell thereof, with an engineered polypeptide, a protein conjugate or fusion protein thereof, or a composition comprising thereof, as described herein.

[0120] In some embodiments, the methods comprise adding the engineered 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 an engineered 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 an engineered polypeptide or a protein conjugate or fusion protein thereof, as disclosed herein.

[0121] 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, G-REX® 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.

[0122] In some embodiments, the methods also comprise contacting the immune cell (e.g., a T cell, a NK 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.

[0123] 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 suitable condition. 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 (TPA), phytohaemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or a natural ligand to a stimulatory or costimulatory molecule.

[0124] 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 thatexpress a semi-invariant ap T-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 (y8 T cells),” a small subset of T cells possessing a distinct TCR on their surface composed of a y-chain and a 8-chain rather than a- and p-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.

[0125] 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 T cells), CD4+ helper T cells, e.g., Thl and Th2 cells, peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cell may be a CD8+ T cell or a CD4+ T cell.

[0126] 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.

[0127] 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 calledcytokines 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.

[0128] 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).

[0129] 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 called tissue-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.

[0130] 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.

[0131] 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 (monocytes31and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).

[0132] 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.

[0133] 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

[0134] Also within the scope of the present disclosure are kits that include the engineered polypeptides, protein conjugates, fusion proteins, and compositions thereof as disclosed herein.

[0135] 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.

[0136] 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 or NK cells. The instructions generally include information as to effective quantity of the engineered polypeptides, protein conjugates, fusion proteins, and compositions disclosed herein and conditions for contacting the T cells or NK cells with the disclosed engineered 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.

[0137] 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 componentssuch 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.

[0138] 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.Examples

[0139] The following are examples of the present invention and are not to be construed as limiting.Example 1

[0140] To identify the novel IL-2 variants of the present disclosure, deep mutational scanning was performed on human IL-2. Every position was mutated to every possible amino acid one at a time. This library was screened for IL-2 binding to an epitope on IL-2Ra and IL-2Rp (a FLAG tag used to detect expression of the variant due to the readily available antibody with strong binding). The FLAG epitope was the first screen to eliminate frameshift mutations, early stop codons, and deleterious mutations for protein stability and / or expression. Positive enrichment and negative enrichment were determined from next generation sequencing counts of positive binding cells versus negative binding cells. IL-2Ra enrichment indicated mutations that increased or decreased binding to IL-2Ra, and IL-2Rp enrichment indicated mutations that increased or decreased binding to IL2Rp.

[0141] As shown in FIGS. 1-4, experiments were conducted to assess the binding activities of the IL-2 variants of the present disclosure. Briefly, binding activities of the enriched IL-2 variants identified were analyzed for binding to IL-2Rp. IL-2 variants were combined in variants of three mutations at a time (see Table 1). Binding was measured by displaying the IL-2 variant on the surface of HEK293T cells and binding an Avi -tagged IL-2Rp and a fluorescently labeled Streptavidin. Greater MFI indicates greater binding to IL-2Rp. Variants were divided into four groups of experiments, as indicated each figure (FIG. 1 - VI variants; FIG. 2 - V2 variants; FIG.3 - V3 variants; FIG. 4 - V4 variants). Recombinant human IL-2 (SEQ ID NO: 2) was tested in each round of experiments, and data were normalized to each other based on recombinant human IL-2 MFI.

[0142] In subsequent experiments, additional IL-2 variants were created by combining the mutations that exhibited the best binding to IL-2Rp from previous screening rounds (V5 and V6 variants). These variants included iterations of the mutations found in the highest binding variants from previous screens. Individual amino acid substitutions were added and removed systematically to identify their role in binding to IL-2Rp and IL-2Ra. Many mutations were consistently added to the IL-2 variants based on their binding contributions.

[0143] As shown in Table 1 below, additional amino acid substitutions were added to IL-2 variants to increase binding to IL-2Rp (V7 variants). Surface plasmon resonance (SPR) was used to confirm that these IL-2 variants had decreased KD values, indicating stronger binding. Specifically, the L85T and S87D mutations exhibited the most significant effects in these experiments.

[0144] Table 1: IL-2 Variants.Example 2

[0145] Previous experiments were conducted to validate IL-2 variant binding affinity for IL- 2Rp using mammalian display. However, experiments in this example were performed using surface plasmon resonance (SPR) on the IL-2 variants identified as having the highest binding affinity for the IL-2Rp. In some cases, SPR can be a more accurate measurement of affinity sincethe molarity of the receptor and ligand are accurately measured compared to the variable expression when using mammalian display. As shown in FIG. 5, the decreased KD (dissociation constant) was much lower for the engineered IL-2 variants compared to recombinant human IL-2 (503 nM), indicating that binding of the enginereed IL-2 variants to IL-2Rp was signifiantly stronger.

[0146] As shown in FIG. 6, experiments were also conducted to assess the effects of IL-2 variants in an NK92 cell proliferation assay. The lower the EC50 value, the more potent the variant for inducing proliferation. Two assays were conducted: one stimulating NK92 cells; and one stimulating NK92 cells with the addition of a CD25 blocking antibody. The antibody was added to stimulate the proliferation of cytotoxic CD8 / CD4 T cells, which have low levels of CD25. These results demonstrate that several IL-2 variants were equally as potent or more potent at inducing NK92 cell proliferation than recombinant human IL-2.Example 3

[0147] Experiments were also conducted to generate IL-2 polypeptide variants with improved properties for the expansion of desired T cell phenotypes. These experiments were performed by increasing the affinity of the IL-2 variants towards IL-2Rp and decreasing affinity to IL-2Ra.

[0148] Exemplary results shown in FIG. 7 demonstrate the effects of IL-2 variants on NK92 induced INFy secretion. NK92 cells were treated with the IL-2 variants listed in Table 2 for 48 hours and measured for INFy secretion. These data demonstrated that the IL-2 variants induced greater production of INFy than the recombinant human IL-2. (EC50 was not calculated for recombinant human IL-2 since it was higher than the most concentrated samples.) Exemplary results shown in FIG. 8 demonstrate the effect of IL-2 variants on expansion of primary T cells. Primary T cells were stimulated with CD3 / CD28 Dynabeads and grown with the indicated IL-2 variants for 19 days. These data indicate that IL-2 variants with increased binding to the IL- 2Rp / IL-2Ry dimeric complex can increase the expansion capacity of CD8 / CD4 T cells. Additionally, exemplary results shown in FIG. 9 demonstrate the ability of IL-2 variants to increase the expression of tumor homing antigens on T cells. CXCR3 and ICAM-1 are tumor homing antigens that are indicative of successful tumor-infiltrating lymphocyte (TIL) therapies. T cells were grown for 7 days in the indicated IL-2 variants.

[0149] These results demonstrate that abrogating the binding to IL-2Ra and increasing binding to IL-2Rp caused increased expression of tumor homing antigens in T cells (e.g., V3-8; FIG. 9), as well as increased expression of INFy in NK92 cells (FIG. 7). However, increasing the binding to IL-2Rp while still maintaining some IL-2Ra binding resulted in increased proliferation of T cells (e.g., V5-2; FIG. 8).Example 4

[0150] Experiments were conducted to test the effects of the engineered IL-2 polypeptides of the present disclosure on various aspects of T cell function and morphology. T cells from three donors were obtained and tested with 2-3 replicates per condition. T cells were grown at standard parameters (IL-2 C125S at 50IU and 200IU equivalents of 3.6 ng / mL and 14.3ng / mL), and in LONZA X-VIVO 15 media supplemented with 10% human AB serum, n-acetylcysteine, Glutamine, and Pen / Strep. They were stimulated with THERMO CD3 / CD28 Expansion microbeads, and grown in standard cell culture plates and flasks.

[0151] As shown in FIG. 9, T cells grown with the CD 122 IL-2 variant, V3-8, demonstrated greater expansion capacity. Frozen T cells from 3 donors were thawed and stimulated with CD3 / CD28 Expansion DYNA beads and then cultured with either standard IL-2 (C125S) or the CD122 binding variant, V3-8, at 3.6ng / mL and 14.3 ng / mL, which is equivalent to 50 and 200 Intemation Units of standard IL-2. V3-8 supported the same expansion capacity at 3.6 ng / mL as standard IL-2 at 14.3 ng / mL. The standard IL-2 treated T cells grown in 14.3 ng / mL reached their peak expansion on day 21, and on that day the V3-8 14.3 ng / mL cells had expanded 2.7 times. The maximal expansion difference was seen on day 28 where the V3-8 cells had expanded 6.1 times more than the standard IL-2 grown cells at 14.3ng / mL.

[0152] Additionally, experiments were conducted to assess the effects of the IL-2 engineered polypeptides of the present disclosure on various T cell marker expression. FIG. 10 includes exemplary results of the activation of marker expression of CD4 / CD8 T cells grown with the IL- 2 CD122 binding variant, V3-8. Cells were assayed via flow cytometry with Biolegend CTLA-4 BV605, Biolegend CD40L BV785, and R&D Systems CD62L PE. The activation markers CTLA- 4, CD40L, and CD62L show similar expression profiles when grown with V3-8 in comparison to standard IL-2 C125S. Cells were grown as described above.

[0153] Experiments were also conducted to assess the expression of exhaustion markers from T cells exposed to V3-8. FIG. 11 provides exemplary results of exhaustion marker expression of CD4 / CD8 T cells grown with V3-8. The exhaustion markers Lag-3, PD-1 and Tim-3 show similar expression when grown with V3-8 in comparison to standard IL-2 C125S, which is indicative of the cells ability to remain functional for longer term in CAR T cell therapy. Cells were grown as described above. Cells were assayed via flow cytometry with Biolegend antibodies (Lag-3 BV510, Tim-3 BV650, and PD-1 PE-Cy7).

[0154] Experiments were conducted to assess the effects of the IL-2 engineered polypeptides of the present disclosure on T cell viability. Exemplary results of the viability of CD4 / CD8 T cells grown with the IL-2 CD122 binding variant, V3-8, are provided in FIG. 12. Viability was recorded each culture day. The IL-2 CD122 binding variant V3-8 supported greater viability over the course of the experiment then the standard IL-2 C125S, even at a low dose. Cells were grown as described above, and viability was Measured via AO / PI on a Cellaca cell counter.

[0155] Additionally, experiments were conducted to assess the effects of the IL-2 engineered polypeptides of the present disclosure on T cell longevity. Exemplary results of the percentage of T cells expressing the longevity phenotype, CD45RA+CCR7+ are provided in FIG. 13. Percentages of CD45RA+CCR7+ T cells identified in CD4 / CD8 T cells grown with the IL-2 CD122 binding variant, V3-8. T cells expressing both CD45RA and CCR7 are known to be long lived and self-renewing. Their presences correlates with the success of CAR T therapies. As can be seen here, V3-8 IL-2 supports their growth within the culture even at the higher dose of 14.3 ng / mL in comparison to standard C125S IL-2. Cells were assayed via flow cytometry using Biolegend antibodies (CD45RA PE-Dazzle and CCR7 BV711).Example 5

[0156] Experiments were conducted to assess the cytotoxic effects of CAR T cells cultured with the engineered IL-2 polypeptides of the present disclosure. As shown in FIG. 14, transposed CAR T cells cultured with engineered IL-2 polypeptides killed tumor cells equivalently as standard IL- 2 C125S. Purified Human CD4+and CD8+T cells were TcBuster-M transposed with a CAR19. T cells were grown out for 12 days in media containing 10 ng / mL dose of either Animal-free Recombinant Human IL-2 as the standard IL-2 protein shown or engineered Recombinant Human IL-2. Conditions were refreshed every 2-3 days. CAR expressing T cells were harvested and co-cultured with human NALM-6 tumor cells using Promega’s™ Luciferase Assay System for 24 hours at variable Effector (T cells) to Target (Nalm-6 tumor cells). Specific killing was measured by the luciferase expression of unlysed NALM-6 target cells.

[0157] Additionally, after the 24-hour kill assay described above, media from the assay was assessed to determine the amount of the cytotoxic factors Interferon gamma (INFg), Granzyme B, and Perforin present in the media. Samples were analyzed using SimplePlex assays on the Ella instrument from ProteinSimple. The exemplary results provided in FIGS. 15A-15C demonstrate that cells cultured with the engineered IL -2 polypeptides of the present disclosure released higher levels of cytotoxic factors than controls.

[0158] Thus, taken together, these data demonstrate that the IL-2 variants of the present disclosure having increased binding to the IL-2 Receptor Beta (CD 122) and decreased or no binding to the alpha receptor (CD25) confer enhanced expansion of CD4 / CD8 T cells without increased expression of exhaustion markers (Lag-3, PD-1, Tim-3), but with comparable activation marker expression. The IL-2 variants of the present disclosure also confer and maintain a long- lived phenotype (CD45RA+CCR7+) and increased cell viability over extended culture time, which are advantageous characteristics for cell therapies, including CAR T applications.Sequences

[0159] Table 2: The various polypeptides referenced in the present disclosure include the following.

[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

CLAIMSWhat is claimed is:

1. An engineered interleukin-2 (IL-2) polypeptide comprising less than 99% amino acid sequence identity with human IL -2 (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1; wherein the engineered IL-2 polypeptide comprises an increased binding affinity for IL-2 receptor p (IL-2Rp).

2. The engineered IL-2 polypeptide of claim 1 , wherein the polypeptide comprises increased binding affinity for the IL-2Rp as compared to wildtype IL-2 (SEQ ID NO: 1) or compared to recombinant human IL-2 (SEQ ID NO: 2).

3. The engineered IL-2 polypeptide of claim 1 or claim 2, wherein the polypeptide comprises increased binding affinity for the IL-2Rp / IL-2Ry dimeric complex compared to the IL-2Rp / IL-2Ry / IL-2Ra trimeric complex.

4. The engineered IL-2 polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises decreased binding affinity for IL-2Ra.

5. The engineered IL-2 polypeptide of any one of claims 1 to 4, wherein the polypeptide comprises a dissociation constant (KD) for the IL-2Rp that is less than about 100 nM.

6. The engineered IL-2 polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises a dissociation constant (KD) for the IL-2Rp that is less than about 10 nM.

7. The engineered IL-2 polypeptide of any one of claims 1 to 6, wherein the polypeptide (i) increases NK cell proliferation, (ii) increases IFNy secretion, (iii) increases expansion capacity of CD8 / CD4 T cells, (iv) increases expression of tumor homing antigens on T cells, or any combinations thereof.

8. The engineered IL-2 polypeptide of any one of claims 1 to 7, wherein the at least one amino acid substitution is present at amino acid position 4, 7, 12, 26, 32, 35, 45, 54, 60, 61, 62, 64, 68, 73, 74, 79, 80, 81, 83, 85, 87, 90, 95, 104, 105, 110, 112, 116, 120, 124, 125, or any combinations thereof.

9. The engineered IL-2 polypeptide of any one of claims 1 to 8, wherein the polypeptide comprises at least 2 amino acid substitutions, at least 3 amino acid substitutions, at least 4 amino acid substitutions, at least 5 amino acid substitutions, at least 6 amino acid substitutions, at least 7 amino acid substitutions, at least 8 amino acid substitutions, at least 9 amino acid substitutions, or at least 10 amino acid substitutions.

10. The engineered IL-2 polypeptide of any one of claims 1 to 9, wherein the polypeptide comprises 2 to 5 amino acid substitutions, 6 to 10 amino acid substitutions, 11 to 15 amino acid substitutions, 16 to 20 amino acid substitutions, 21 to 25 amino acid substitutions, or 26 to 30 amino acid substitutions.

11. The engineered IL-2 polypeptide of any one of claims 1 to 10, wherein the at least one amino acid substitution comprises S4E, T7I, L12E, N26E, K32Q, K35V, Y45K, K54I, E60K, E61R, E61K, E62Q, K64D, E68A, A73L, Q74E, H79S, L80Q, R81D, R83D, L85T, S87E, S87D, N90K, E95T, M104P, C105T, El 10D, Al 12L, El 16Q, R120Y, F124A, C125S, or any combinations thereof.

12. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 3-29.

13. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 30-35.

14. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 36-43.

15. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 44-52.

16. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 53-54.

17. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 55-60.

18. The engineered IL-2 polypeptide of any one of claims 1 to 11, wherein the polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 61-70.

19. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 3-70.

20. The engineered IL-2 polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 3-70.

21. The engineered IL-2 polypeptide of any one of claims 1 to 11, wherein the polypeptide comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 3-70.

22. A protein conjugate or fusion protein comprising the engineered IL-2 polypeptide of any one 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 the engineered IL-2 polypeptide of any one of claims 1-21 or the protein conjugate or fusion protein of any one of claims 22-25, and a carrier.

27. An in vitro or ex vivo method for stimulating differentiation of T cells or NK cells comprising contacting a T cell or NK cell with the engineered IL-2 polypeptide of any one of claims 1-21, the protein conjugate or fusion protein of any one of claims 22-25, or the composition of claim 26.

28. An in vitro or ex vivo method for expanding T cells or NK cells comprising contacting a T cell or NK cell with the engineered IL-2 polypeptide of any one of claims 1-21, the protein conjugate or fusion protein of any one of claims 22-25, or the composition of claim 26.

29. An in vitro or ex vivo method for increasing viability of T cells or NK cells comprising contacting a T cell or NK cell with the engineered IL-2 polypeptide of any one of claims 1-21, the protein conjugate or fusion protein of any one of claims 22-25, or the composition of claim 26.

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