Designed cytokine compositions and methods of use

Abstract

The present disclosure provides an engineered cytokine comprising alpha helices H1, H2, H3, and H4, from amino terminus to carboxy terminus, a first loop (L1) connects H1 and H4, a second loop (L2) connects H4 and H2, and a third loop (L3) connects H2 and H3, and wherein the polypeptide binds to the IL-2 receptor βγ (IL-2Rβγ).

Description

Related Applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [1] This application claims the benefit of U.S. Provisional Application No. 63 / 298,623, filed January 11, 2022, U.S. Provisional Application No. 63 / 479,176, filed January 9, 2023, U.S. Provisional Application No. 63 / 479,177, filed January 9, 2023; and U.S. Provisional Application No. 63 / 479,178, filed January 9, 2023, all of which are incorporated herein in their entireties. [Technical field]

[0002] Field of Disclosure [2] This disclosure relates to the fields of immunology, gene therapy, protein design, cell signaling, biologics and cell therapy. [Background technology]

[0003] [3] IL-2 has shown promise as an anti-cancer immunotherapy, but efficacy and safety are limited by dose-limiting toxicity due to preferential stimulation of Treg cells and due to IL-2Rα binding. The present disclosure provides a non-naturally occurring engineered cytokine as a solution to this unmet need in the art. Summary of the Invention

[0004] [4] The present disclosure provides non-naturally occurring engineered cytokines with enhanced function and improved stability when compared to wild-type IL-2. Named for their dual function of signaling through both IL-2 and IL-15 receptors, the non-naturally occurring engineered cytokines of the present disclosure, also referred to herein as IL-2 / 15 cytokines, reduce preferential Treg stimulation, enhance T cell subtype targeting, stabilize protein folding, and reduce immunogenicity. In some embodiments, the engineered cytokines eliminate preferential Treg stimulation. In some embodiments, the engineered cytokines eliminate immunogenicity. In some embodiments, the engineered cytokines provide enhanced function and improved stability with reduced post-translational modifications compared to post-translational modifications made to wild-type IL-2. In some embodiments, the engineered cytokines provide enhanced function and improved stability without post-translational modifications.

[0005] [5] The disclosure provides an engineered cytokine comprising alpha helices H1, H2, H3, and H4, where, from amino terminus to carboxy terminus, a first loop (L1) connects H1 and H4, a second loop (L2) connects H4 and H2, and a third loop (L3) connects H2 and H3, and where the polypeptide binds to IL-2 receptor βγ (IL-2Rβγ), also referred to as IL-2 / 15Rβγ. In some embodiments, the engineered cytokine does not bind to IL-2 receptor alpha (IL-2Rα).

[0006] [6] In some embodiments of the designed cytokines of the present disclosure, the designed cytokine comprises one or more of the sequences of SEQ ID NO:1 to SEQ ID NO:350. [7] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises one or more of: (a) the sequences of SEQ ID NO:1 through SEQ ID NO:350; and (b) a sequence having at least 70% identity to the sequence of (a).

[0007] [8] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises a sequence of SEQ ID NO:1-SEQ ID NO:38 or SEQ ID NO:150-SEQ ID NO:350. [9] In some embodiments of the designed cytokines of the present disclosure, the L3 loop comprises the sequence QSKNFHLR (SEQ ID NO: 132).

[0008]

[10] In some embodiments of the designed cytokines of the present disclosure, the H1 helix comprises the sequence of SEQ ID NO:39 to SEQ ID NO:51.

[11] In some embodiments of the designed cytokines of the present disclosure, the H2 helix comprises the sequence of SEQ ID NO:52 to SEQ ID NO:77.

[0009]

[12] In some embodiments of the designed cytokines of the present disclosure, the H3 helix comprises the sequence of SEQ ID NO:78 to SEQ ID NO:97.

[13] In some embodiments of the designed cytokines of the present disclosure, the H4 helix comprises the sequence of SEQ ID NO:98 to SEQ ID NO:101.

[0010]

[14] In some embodiments of the designed cytokines of the present disclosure, the L1 loop comprises the sequences of SEQ ID NO:102 to SEQ ID NO:111.

[15] In some embodiments of the designed cytokines of the present disclosure, the L2 loop comprises the sequences of SEQ ID NO:112 to SEQ ID NO:131.

[0011]

[16] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises a sequence of SEQ ID NO:1-SEQ ID NO:38 or SEQ ID NO:150-SEQ ID NO:350.

[17] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK.

[0012]

[18] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0013]

[19] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0014]

[20] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTLTAGGSLSGDLKHLQNLSEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0015]

[21] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSVDPEELAKELQKLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0016]

[22] In some embodiments of the designed cytokines of the present disclosure, the polypeptide is operably linked to a targeting moiety. In some embodiments, the polypeptide comprises a targeting moiety. In some embodiments, a fusion protein comprises a polypeptide and a targeting moiety. In some embodiments, the targeting moiety binds to a component of the tumor microenvironment (TME). In some embodiments, the targeting moiety binds to one or more of the T cell surface glycoprotein CD8 (also known as cluster of differentiation 8), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1; also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin mucin receptor 3 (TIM3). In some embodiments, the targeting moiety binds to CD8. In some embodiments, the targeting moiety binds to PD-1. In some embodiments, the targeting moiety binds to PD-L1. In some embodiments, the targeting moiety comprises an antibody, an antibody mimetic, or a functional fragment thereof. In some embodiments, the targeting moiety comprises one or more of a monoclonal antibody, an antigen-binding fragment (Fab), a single chain variable fragment (scFv), a domain antibody, a heavy chain (VH) and a light chain (VL) domain of an immunoglobulin (Ig) polypeptide or the genes encoding them, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and a nanobody. In some embodiments, the targeting moiety comprises an scFv. In some embodiments, the targeting moiety comprises a VHH.

[0017]

[23] In some embodiments of the designed cytokines of the present disclosure, the polypeptide is operably linked to a tether. In some embodiments, the polypeptide comprises a tether. In some embodiments, a fusion protein comprises a polypeptide and a tether. In some embodiments, the tether comprises one or more of a nucleic acid sequence, an amino acid sequence, a small molecule. In some embodiments, the tether comprises a sequence isolated or derived from a transmembrane sequence. In some embodiments, the tether comprises the sequence PLFIPVAVMVTAFSGLAFIIWLARRLKKGKK.

[0018]

[24] In some embodiments of the designed cytokines of the present disclosure, the polypeptide is operably linked to a second cytokine or a second designed cytokine. In some embodiments, the polypeptide comprises a second cytokine or a second designed cytokine. In some embodiments, a fusion protein comprises the polypeptide and a second cytokine or a second designed cytokine.

[0019]

[25] In some embodiments of the designed cytokines of the present disclosure, the second cytokine comprises a sequence isolated or derived from one or more of an IL-2 polypeptide, an IL-12 polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, an IL-21 polypeptide, an IL-23 polypeptide, an interferon alpha polypeptide, an interferon beta polypeptide, an interferon gamma polypeptide, and an interferon omega polypeptide. In some embodiments, the polypeptide comprises a first targeting moiety and the second cytokine comprises a second targeting moiety. In some embodiments, the first targeting moiety and the second targeting moiety are the same. In some embodiments, the first targeting moiety and the second targeting moiety are not the same.

[0020]

[26] In some embodiments of the designed cytokines of the present disclosure, the second designed cytokine comprises any one or more of the sequences of SEQ ID NO:1-SEQ ID NO:38 or SEQ ID NO:150-SEQ ID NO:350. In some embodiments, the polypeptide comprises a first targeting moiety and the second designed cytokine comprises a second targeting moiety. In some embodiments, the first targeting moiety and the second targeting moiety are identical. In some embodiments, the first targeting moiety and the second targeting moiety are not identical.

[0021]

[27] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises a first tether and the second cytokine comprises a second tether. In some embodiments, the first tether and the second tether are the same. In some embodiments, the first tether and the second tether are not the same.

[0022]

[28] In some embodiments of the designed cytokines of the present disclosure, the polypeptide comprises a first tether and the second designed cytokine comprises a second tether. In some embodiments, the first tether and the second tether are the same. In some embodiments, the first tether and the second tether are not the same.

[0023]

[29] The present disclosure provides a nucleic acid encoding a designed cytokine of the present disclosure or a fusion protein comprising a designed cytokine of the present disclosure. In some embodiments, the nucleic acid further comprises a regulatory element capable of driving expression of the designed cytokine. In some embodiments, the regulatory element comprises a promoter. In some embodiments, the promoter comprises a minimal promoter. In some embodiments, the minimal promoter comprises a sequence isolated or derived from one or more of minimal promoter 1 ("minP1"), YB-TATA, and human beta globin. In some embodiments, minP1 comprises a sequence of AGAGGGTATATAAAAGCTCGACTTCCAG. In some embodiments, the minimal promoter comprises a sequence of TAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC. In some embodiments, the minimal promoter comprises a sequence of CTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC. In some embodiments, the promoter is inducible. In some embodiments, the regulatory element comprises a response element. In some embodiments, the regulatory element comprises a non-coding or non-translated sequence isolated or derived from one or more of NFAT, NFkB, REL, RELA, IRF2, GATA3 and ATF3. In some embodiments, the regulatory element comprises a non-coding or non-translated sequence isolated or derived from a GATA3 gene. In some embodiments, the regulatory element comprises a non-coding or non-translated sequence isolated or derived from RELA. In some embodiments, the response element comprises a repeat sequence.

[0024]

[30] The present disclosure provides a vector comprising a nucleic acid of the present disclosure. In some embodiments, the vector comprises an expression vector. In some embodiments, the vector comprises a delivery vector. In some embodiments, the vector further comprises a sequence encoding an exogenous receptor. In some embodiments, the exogenous receptor comprises an antigen binding moiety. In some embodiments, the exogenous receptor comprises a T cell receptor (TCR). In some embodiments, the exogenous receptor comprises a chimeric antigen receptor (CAR). In some embodiments, the antigen is expressed in or secreted within one or more of tumor cells, cancer cells, components of the TME, and the TME.

[31] In some embodiments of the present disclosure, the vector is a non-viral vector. In some embodiments, the non-viral vector comprises one or more of a plasmid, a nucleic acid, a polymer, a micelle, a polymersome, an exosome, a lysosome, a nanoparticle, and any combination thereof.

[0025]

[32] In some embodiments of the present disclosure, the vector is a viral vector. In some embodiments, the viral vector comprises a sequence isolated or derived from a viral, lentiviral, or lentiviral vector sequence.

[0026]

[33] The present disclosure provides a cell comprising an engineered cytokine of the present disclosure. The present disclosure provides a cell comprising a nucleic acid of the present disclosure. The present disclosure provides a cell comprising a vector of the present disclosure. In some embodiments of the present disclosure, the cell is a mammalian cell. In some embodiments of the present disclosure, the cell is a human cell. In some embodiments of the present disclosure, the cell is a primary cell. In some embodiments of the present disclosure, the cell is a cultured cell. In some embodiments, the cultured cell is an immortalized cell. In some embodiments, the cell is present ex vivo or in vitro. In some embodiments, the cell is present in vivo. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a stem or progenitor cell capable of producing an immune cell. In some embodiments, the stem cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), or a dedifferentiated immune cell. In some embodiments, the immune cell is a T lymphocyte (T cell), a B lymphocyte (B cell), a macrophage, or a natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is an alpha beta T cell. In some embodiments, the T cell is a gamma delta T cell. In some embodiments, the immune cell is a NK cell.

[0027]

[34] The present disclosure provides a composition comprising an engineered cytokine of the present disclosure. The present disclosure provides a composition comprising a nucleic acid of the present disclosure. The present disclosure provides a composition comprising a vector of the present disclosure. The present disclosure provides a composition comprising a cell of the present disclosure.

[0028]

[35] The present disclosure provides pharmaceutical compositions comprising one or more of: (1) a designed cytokine of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, and a cell of the present disclosure; and (2) a pharma- ceutically acceptable carrier.

[0029]

[36] The present disclosure provides for the use of an engineered cytokine of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of the present disclosure, or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for the treatment of a disease or condition. In some embodiments, the disease or disorder comprises a cancer or a subtype thereof. In some embodiments, the cancer or a subtype thereof comprises a liquid cancer. In some embodiments, the cancer or a subtype thereof comprises a hematological cancer. In some embodiments, the cancer or a subtype thereof comprises a solid cancer.

[0030]

[37] The present disclosure provides an engineered cytokine of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of the present disclosure, or a pharmaceutical composition of the present disclosure for use in treating a disease or condition. In some embodiments, the disease or disorder comprises a cancer or a subtype thereof. In some embodiments, the cancer or a subtype thereof comprises a liquid cancer. In some embodiments, the cancer or a subtype thereof comprises a hematological cancer. In some embodiments, the cancer or a subtype thereof comprises a solid cancer.

[0031]

[38] The present disclosure provides a method of treating a disease or disorder comprising administering to a subject an effective amount of a designed cytokine of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of the present disclosure, or a pharmaceutical composition of the present disclosure, wherein the severity of a sign or symptom of the disease or disorder is reduced, thereby treating the disease or disorder. In some embodiments, the disease or disorder comprises a cancer or a subtype thereof. In some embodiments, the cancer or a subtype thereof comprises a liquid cancer. In some embodiments, the cancer or a subtype thereof comprises a hematological cancer. In some embodiments, the cancer or a subtype thereof comprises a solid cancer.

[0032]

[39] The present disclosure provides a method of preventing a disease or disorder comprising administering to a subject an effective amount of an engineered cytokine of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of the present disclosure, or a pharmaceutical composition of the present disclosure, wherein the onset or recurrence of a sign or symptom of the disease or disorder is delayed or inhibited, thereby preventing the disease or disorder. In some embodiments, the disease or disorder comprises a cancer or a subtype thereof. In some embodiments, the cancer or a subtype thereof comprises a liquid cancer. In some embodiments, the cancer or a subtype thereof comprises a hematological cancer. In some embodiments, the cancer or a subtype thereof comprises a solid cancer. [Brief description of the drawings]

[0033] [Figure 1A]

[40] Schematic diagram representing the naturally occurring form of IL-2 (left) and an exemplary non-naturally occurring engineered cytokine of the present disclosure, where one or more loops have been redesigned to alter the binding of the engineered cytokine to naturally occurring or endogenous IL-2 receptor beta (IL-2Rβ) or IL-2 receptor gamma (IL-2Rγ, also known as the common gamma chain receptor), but not IL-2 receptor alpha (IL-2Rα). As shown in this figure, in some embodiments, an exemplary non-naturally occurring engineered cytokine of the present disclosure contains sequences isolated or derived from the helices of the IL-2 cytokine, which have the connectivity of an engineered cytokine that follows the 1-4-2-3 plan depicted herein, or a structure of four helices connected by loops that lacks an interface for binding to IL-2 receptor alpha and therefore lacks the ability to bind and / or activate IL-2 receptor alpha. [Figure 1B]

[41] A series of schematic diagrams depicting a diagram according to FIG. 1A with an updated diagram on the left half highlighting the interactions with each subunit of the IL-2 receptor that enable IL-15 function, and a diagram on the right half showing the correspondence between the abstract and ribbon diagrams. As represented in this diagram, not only does the designed cytokine lack an interface for binding and / or activating the alpha subunit of the IL-2 receptor, but "helix 4" of the designed cytokine binds to the gamma subunit of the IL-2 receptor, which is shared with the IL-15 receptor, and "helix 1" and "helix 3" of the designed cytokine bind to the IL-2 receptor beta, which is also shared with the IL-15 receptor. As a result, the designed cytokine of the present disclosure has function through the IL-2 receptor and / or the IL-15 receptor. [Diagram 2]

[42] Schematic diagram depicting T cell subtype-specific IL-2 WT signaling. As shown in Figures 1A-1B, the engineered cytokines of the present disclosure signal through one or more of the beta and gamma subunits of the IL-2 receptor. [Figure 3A]

[43] Schematic diagram showing that the designed cytokine of the present disclosure can target specific cell subtypes as a beta / gamma binding agent.Furthermore, the designed cytokine of the present disclosure can be linked to or contain a targeting moiety.Meanwhile, the targeting moiety can bind to any target, including, but not limited to, antigens present or expressed within cells, or other components of the tumor microenvironment (TME), such as CD8 or PDL1. [Figure 3B]

[44] Schematic diagram showing that the designed cytokines of the present disclosure may target specific cell subtypes as a result of (1) being designed as beta / gamma binders, (2) being targeted to cell subtypes, (3) being tethered to T cells or subtypes thereof (e.g., alpha-beta, gamma-delta, CD8+, CD4+, natural killer T cells (NKT cells)), or any combination thereof. The targeted designed cytokine may be linked to or include a targeting moiety that may bind to any target, including, but not limited to, antigens present or expressed within the cell, or other components of the tumor microenvironment (TME), such as CD8 or PDL1. The tethered designed cytokine may be linked to or include a tether, which may include a nucleic acid, amino acid, small molecule, or any combination thereof, and which may link the designed cytokine to a cell expressing the designed cytokine (e.g., a T cell, a NK cell, or other immune cell) or to any component of the cell. In some embodiments, the use of a targeting moiety may be suitable for localizing the designed cytokine or the cells expressing it to a solid tumor or to the TME for localized signaling from the designed cytokine (as opposed to systemic signaling from the designed cytokine). In some embodiments, the use of a tether may be suitable for minimizing bystander activity of cells proximal to either the target cell or the cells expressing the designed cytokine. In some embodiments, the use of a tether may be suitable for treating liquid or hematological tumors. In some embodiments, the use of a tether may be suitable for use with T cells, where the bystander cells may be natural killer (NK) cells. [Figure 4]

[45] Schematic diagram showing that the engineered cytokines of the present disclosure lack an interface for binding and / or activating the alpha subunit of the IL-2 receptor. Furthermore, removal of this interface retains the natural interface for binding to the IL-2 receptor beta and IL-2 receptor gamma subunits shared between the IL-2 receptor and the IL-15 receptor. The design of short structured loops between the helices enhances protein stability when compared to the loop length, loop structure and / or protein stability of wild-type (WT) IL-2. [Figure 5A]

[46] A pair of graphs demonstrating that among 38 (38) engineered cytokines tested alone along with WT IL-2 and a negative control, a common structural arrangement of the engineered cytokines abolishes IL-2 receptor alpha binding in all engineered cytokines (top), while retaining signaling through the beta and gamma subunits of the IL-2 receptor (the gamma subunit is shared with the IL-15 receptor). [Figure 5B]

[47] is a series of graphs demonstrating that the engineered cytokines of the present disclosure bind to IL-2 / 15Rβγ with very low nanomolar affinity. For each plot shown, the affinity (measured in nanomolar (nm) concentrations) of either WT IL2 (top) or engineered cytokines (bottom) when contacted with either IL-1Rα (left) or IL-2 / 15Rβγ (right) is shown as a function of time (measured in seconds (s)). Compositions of either WT IL-2 or engineered cytokines were tested from 0.9 nM to 3000 nM to generate these plots. [Figure 6]

[48] ​​A series of graphs demonstrating that the activity of the designed cytokines of the present disclosure can be tuned over a wide range. As used throughout this disclosure, the designed cytokines can be "tuned" to perform optimally within either a desired cell type, a desired TME, a desired target cell, and / or a desired cytokine receptor (e.g., the ratio of activity between IL-2 and IL-15). The initial design of the designed cytokine can include a sequence of the present disclosure or a sequence having at least 70% identity thereto. The optimized design of the designed cytokine can include a sequence of the present disclosure or a sequence having at least 70% identity thereto. The optimized design of the designed cytokine can include a sequence derived from a sequence of the present disclosure generated according to the teachings provided by this disclosure. Top plot: Cell proliferation was assessed by measuring the percentage of phosphorylated signal transduction and activator of transcription 5 (STAT5) in CD8+ T cells after stimulation with antigen as a function of the concentration (concentrations measured as nanograms per milliliter (ng / ml)) of either WT IL-2 or the provided designed cytokine. The designed cytokines of the present disclosure may be "optimized" as shown in the top right plot to have a desired activity profile. In contrast to IL-2Rα, with regard to activity of IL-2 / 15Rβγ binding, the two bottom plots show that the designed cytokines each bind to IL-2 / 15Rβγ with different activity, whereas the designed cytokines do not bind to IL-2Rα. In this figure, Design 1 = Designed Cytokine No. 169, Design 2 = Designed Cytokine No. 175, and Design 3 = Designed Cytokine No. 153. [Figure 7]

[49] A series of graphs demonstrating that the engineered cytokines do not have regulatory T cell (Treg) preference when compared to WT IL-2, but the engineered cytokines preserve NK and CD8+ T cell activity. WT IL-2 (top plots) or the engineered cytokines (bottom plots) were contacted with CD8+ T cells, NK cells or Tregs, and cell proliferation was assessed by measuring the percentage of phosphorylated signal transduction and activator of transcription 5 (STAT5) in CD8+ T cells after stimulation with antigen as a function of the concentration (shown as nanomolar (nM) concentration) of either WT IL-2 or the engineered cytokine provided. Two cell donors are shown (left and right plots). WT IL-2 favors Treg activation, while the engineered cytokines show reduced Treg potency that eliminates WT IL-2 Treg preference. [Figure 8]

[50] is a graph demonstrating in vitro cell proliferation in the presence of 1 nanomolar (nM) of WT IL-2 or engineered cytokines. Measurements on the Y-axis are on a relative scale where cell proliferation measured as shown in Figure 7 is normalized to WT activity (maintained at a value of 1.0). Within either a population of mixed peripheral blood mononuclear cells (PBMCs) or isolated populations of NK cells or activated CD8+ T cells, respectively, the engineered cytokines eliminate the Treg preference exhibited by WT IL-2 while retaining the ability to expand NK and CD8+ T cells. [Figure 9]

[51] A series of graphs demonstrating that the engineered cytokines do not have regulatory T cell (Treg) preference when compared to WT IL-2, but the engineered cytokines preserve NK and CD8+ T cell activity. For each plot, either WT IL-2 (top) or the engineered cytokines (bottom) were provided at nanomolar (nM) concentrations to mixed PBMCs (left), isolated NK cells (middle), and CD3 / 28 activated T cells in vitro, and fold proliferation was measured. Of note, the bottom left plot shows a significant right shift in activity with respect to Treg cells, as the engineered cytokines have reduced potency with respect to Treg cells when compared to WT IL-2. [Figure 10]

[52] Schematic and graph demonstrating enhanced activity of T cells expressing both the disclosed CAR and engineered cytokines. The schematic represents the experimental design used to generate the data displayed in the graph. Mock-transfected cells, cells transfected with only a chimeric antigen receptor (CAR), cells transfected with a CAR in combination with a WT IL-2 construct, or cells transfected with a construct providing a CAR and the disclosed engineered cytokines were repeatedly stimulated with plate-bound antigen and fold proliferation was assessed after each round. [Figure 11]

[53] Schematic and graphs demonstrating that T cell activation induced secretion of an engineered cytokine using an inducible promoter capable of conditionally driving expression of the engineered cytokine in response to antigen receptor signaling (e.g., CAR or TCR) or cellular context (stimulated T cell). Modulation of the expression and / or secretion of the engineered cytokine of the present disclosure reduces systemic exposure in vivo and provides a favorable safety profile for the engineered cytokine. [Figure 12]

[54] Schematic and graphs demonstrating the incorporation of short structured loops within a designed cytokine increases protein stability of the designed cytokine when compared to WT IL-2. The schematic on the left represents the structural differences of the loops in WT IL-2 and the designed cytokine of the present disclosure. The graph represents protein unfolding / instability (measured by intrinsic fluorescence) as a function of increasing molar concentration of guanidine hydrochloride ([GdnHCl(M)]), an agent that denatures / unfolds proteins. The higher stability of the designed cytokine (circle data points) is indicated by lower fluorescence (right shift) at higher concentrations compared to WT IL-2. As used throughout this disclosure, the designation "WT IL-2 internal" means that we are describing WT IL-2 with an identical sequence to the commercially available WT IL-2 protein synthesized by the applicant. [Figure 13]

[55] is a graph demonstrating that two exemplary designed cytokines of the present disclosure exhibit IL-15-like effects on NK cells in vitro. In this experiment, the activity of commercially purchased WT IL-2 and internally produced WT IL-2 was compared. WT IL-15 polypeptide was used as a positive control and as a basis for comparison to IL-15-like as opposed to IL-2-like effects. [Figure 14A]

[56] A series of schematic diagrams demonstrating that in some embodiments, the engineered cytokines of the present disclosure can be fused to a targeting moiety, or stated another way, a fusion protein can include an engineered cytokine and a targeting moiety. The rightmost diagram represents signaling in trans. [Figure 14B]

[57] is a graph demonstrating that the use of a targeting moiety to localize the physical presence and / or signaling activity of a designed cytokine, for example to the TME, does not impair any activity of the designed cytokine, its receptor binding, or signaling therefrom. In this graph, the percentage of total cells that bind the designed cytokine is shown as a function of nanomolar (nM) protein concentration of the targeting moiety bound to the designed cytokine. [Figure 14C]

[58] is a series of graphs demonstrating the use of targeting moieties with four exemplary designed cytokines of the present disclosure. In each plot, the cell proliferation activity of each targeted designed cytokine is measured as the percentage of phosphorylated PSTAT5+ cells as a function of the nanomolar concentration of the targeted designed cytokine provided. The designation "VHH-1" is meant to represent two VHH targeting moieties with different sequences in contrast to "VHH-2." [Figure 15]

[59] A series of schematic diagrams of two exemplary designed cytokines of the present disclosure showing the structural differences between IL-2 and each of the designed cytokines in contrast to WT IL-2. Additionally, the diagram reflects structural modifications to each of the designed cytokines that result in functional changes to the protein. Each of the designed cytokines contains a helix initiator (e.g., lysine (L)) in front of the H4 helix, while the "conjugation" designed cytokine, which more easily conjugates, for example, to a targeting moiety, has an extended H2 helix and a shorter loop between the H4 helix and the H2 helix. Both designed cytokines have a substitution three amino acids from the H2 inter-helical loop, but the substitution itself differs between these two designed cytokines. [Figure 16]

[60] A series of graphs demonstrating that designed cytokine No. 201 shows no detectable binding to IL2Rα, whereas WT IL-2 binds to IL2Rα with nanomolar affinity. [Figure 17A]

[61] Figure 6 is a graph showing two replicate experiments (previous experiment shown in Figure 6) side-by-side, demonstrating that the activity of an exemplary designed cytokine can be tuned over a wide range. [Figure 17B]

[62] Figure 17B is a graph representing a backward repeat of the experiment shown in Figure 6 and Figure 17A. [Figure 18]

[63] A series of graphs demonstrating the binding affinity (by Octet) to IL-2Rβγ of WT IL-2 (left plot) and either of two exemplary designed cytokines (middle and right plots). [Figure 19]

[64] is a series of graphs demonstrating the cell proliferation potential of three exemplary designed cytokines compared to WT IL-2 in three cell types (NK cells, Treg cells and CD8+ T cells). [Figure 20]

[65] A coordinate pair of a schematic and graph demonstrating sist-targeting and trans-targeting of engineered cytokines of the present disclosure by altering the binding preference of the targeting moiety. As shown in the top row of the schematic and graphs above, PD-1 and CD8 mediate sist-targeting of activated T cells by engineered cytokines of the present disclosure. As shown in the bottom row of the schematic and graphs below, PD-L1 mediates sist-targeting and trans-presentation of engineered cytokines of the present disclosure to activated cells. [Figure 21A]

[66] Schematic diagram showing the experimental design used to generate the data provided in Figures 21B and 21C. Briefly, CAR-T cells were sequentially loaded with H1975 tumor cells in the presence of recombinant engineered cytokines, engineered cytokines + target binding domains (added separately in equimolar ratios), or targeted engineered cytokines: the targeted engineered cytokine No. 169 fused to a VHH binding domain that binds either PD-L1, PD-1, or CD8. [Figure 21B]

[0023] FIG. 67 is a pair of graphs representing two different T cell donors demonstrating the killing activity of the PDL1 targeted engineered cytokines of the present disclosure. [Figure 21C]

[0023] FIG. 68 is a pair of graphs representing two different T cell donors demonstrating the killing activity of the PDL1 targeted engineered cytokines of the present disclosure. [Figure 21D]

[0023] FIG. 69 is a pair of graphs representing two different T cell donors demonstrating the killing activity of the CD8-targeted engineered cytokines of the present disclosure. [Figure 22A]

[70] Figure 1 is a graph demonstrating the cell proliferation activity of designed cytokines derived from designed cytokine No. 153 in natural killer cells. [Figure 22B]

[71] Figure 1 is a graph demonstrating the cell proliferation activity of engineered cytokines derived from engineered cytokine No. 153 in CD8+ T cells. [Figure 23A]

[72] A series of schematic diagrams depicting two-vector (2V), single-vector (1V) and autoregulatory circuits incorporating inducible engineered cytokines. In these schematic diagrams, the term "DC" is meant to describe the engineered cytokine. In some embodiments and according to these diagrams, the expression of the inducible engineered cytokine is under the control of one or more response elements (RE arrays) and promoter sequences, which may be minimal promoter sequences (pMin). The marker and CAR elements in each 2V and 1V diagram are under the control of the MND promoter. In the autoregulatory diagram, the expression of the CAR and engineered cytokine is under the control of an inducible promoter that is active during the period of cell stimulation. [Figure 23B]

[73] Schematic diagram showing the experimental design used to generate the data shown in Figure 23C. In this experiment, CAR-T cells containing the vector or circuit depicted in 23A were stimulated with plate-bound antigen. WT IL-2 in control or secreted inducible engineered cytokines was measured in the supernatant. [Figure 23C]

[74] Figure 13 is a graph demonstrating that inducible engineered cytokines (DCs) show increased expression versus either control, CAR only (no engineered cytokines) or no antigen stimulation. [Figure 24]

[75] is a series of graphs demonstrating that exemplary inducible engineered cytokines retain target cell killing activity better than the CAR-only control after three successive rounds of stimulation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034]

[76] The present disclosure provides "designed cytokines," which are non-naturally occurring polypeptides of the disclosure that exhibit the functional benefits of IL-2 and IL-15, according to embodiments illustrated in the figures and sequences provided herein. Thus, the "designed cytokines" may be referred to as "IL-2 / 15 polypeptides" or "IL-2 / 15." These terms are interchangeable with "designed cytokines."

[0035]

[77] The present disclosure provides engineered cytokines that preferably exhibit one or more of the following attributes: (1) reducing or eliminating binding of the engineered cytokine to the alpha subunit of the IL-2 receptor (IL-2Rα), optionally without post-translational modifications to the engineered IL-2 polypeptide; (2) binding to the beta and / or gamma subunit of the IL-2 receptor (IL-2Rβ / γ) or retaining binding of the engineered IL-2 polypeptide to the beta and / or gamma subunit of the IL-2 receptor (IL-2Rβ / γ); (3) preferentially or more potently stimulating T cell(s) expressing CD8 (CD8 T cells) over stimulating regulatory T cell(s) (Tregs); (4) preferentially or more potently stimulating natural killer (NK) cells over CD8 T cells; (5) enhancing activity of T cells that express or secrete an engineered IL-2 polypeptide of the present disclosure; and (6) detecting in vitro or in vivo the activity of the engineered IL-2 polypeptide. exhibit stable folding into a protein in vivo, and (7) exhibit reduced, minimal, or undetectable levels of immunogenicity when administered to a subject (e.g., a mouse, another animal species, or a human patient) when compared to the level of immunogenicity exhibited by wild-type IL-2 protein under the same circumstances. Alternatively, or in addition, the engineered cytokines of the present disclosure may (1) bind to the IL-15 receptor (IL-15R), its beta subunit, or gamma subunit, and / or (2) compete with IL-15 cytokines, including WT IL-15, for binding to the IL-15R, its beta subunit, or gamma subunit.

[0036]

[78] In some embodiments, the designed cytokine of the present disclosure (1) localizes expression, translation, production and / or secretion of the designed IL-2 polypeptide of the present disclosure to the tumor, target cells and / or tumor microenvironment (TME), (2) targets the TME, and / or (3) maintains localization in the TME. In some embodiments of the designed cytokine of the present disclosure, including those in which the polypeptide provides targeting to, localizes to and / or maintains localization in the TME, the designed cytokine comprises a targeting moiety. In some embodiments, the targeting moiety comprises a nucleic acid, an amino acid, or a combination thereof that specifically binds to a target on or having a lymph node, a tumor, a tumor microenvironment, a site of malignancy or a site of metastasis, or in any case a cell thereof. In some embodiments, the fusion protein or targeting moiety comprises an antibody, an antibody mimetic, or a functional fragment thereof. In some embodiments, the targeting moiety comprises an scFv, a VH, or a VHH. In some embodiments, the targeting moiety comprises an scFv, VH, or VHH that specifically or selectively binds to the T cell surface glycoprotein CD8 (also known as Cluster of Differentiation 8), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1; also known as Cluster of Differentiation 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), T cell immunoglobulin mucin receptor 3 (TIM3).

[0037]

[79] In some embodiments of the designed cytokines of the present disclosure, the designed cytokine comprises a "tether" for operably linking the designed cytokine to (1) a cell expressing the designed cytokine or (2) a cell that delivers the designed cytokine to a target cell, immune synapse, and / or TME. In some embodiments, the tether comprises DNA, RNA, amino acids, or any combination thereof. In some embodiments, the tether comprises a defined secondary structure. In some embodiments, the tether has a rigid structure. In some embodiments, the tether has a flexible conformation. In some embodiments, the tether has an elastic conformation. In some embodiments, the tether comprises a cleavable sequence. In some embodiments, the tether comprises a transmembrane sequence or a membrane anchor sequence. In some embodiments, the tether is optionally linked to the designed cytokine by a linker sequence. In some embodiments, the tether is optionally linked to the designed cytokine by a linker sequence that includes a "GS" linker. In some embodiments, the tether comprises the sequence "PLFIPVAVMVTAFSGLAFIIWLARRLKKGKK".

[0038]

[80] In some embodiments of the designed cytokines of the present disclosure, the construct comprises an inducible promoter capable of expressing the designed cytokine and a sequence encoding the designed cytokine. In some embodiments, the construct may further comprise a sequence encoding a targeting moiety. Alternatively, or in addition, in some embodiments, the construct may further comprise a sequence encoding a tether. In some embodiments, the construct may further comprise a linker located between the sequence encoding the designed cytokine and one or more of the sequence encoding the targeting moiety and the sequence encoding the tether.

[0039]

[81] In some embodiments of the designed cytokines of the present disclosure, a fusion protein comprises the designed cytokine and either a targeting moiety or a tether. In some embodiments of the fusion protein, a linker is positioned between the designed cytokine and either the targeting moiety or the tether.

[0040]

[82] In some embodiments of the designed cytokines of the present disclosure, the immune cell expresses the designed cytokine of the present disclosure. In some embodiments, the immune cell secretes the designed cytokine. In some embodiments, the designed cytokine comprises a tether and contacts the immune cell. In some embodiments, the tethered designed cytokine contacts the plasma membrane of the immune cell or a component thereof. In some embodiments, the tethered designed cytokine contacts the outer surface of the plasma membrane of the immune cell.

[0041]

[83] In some embodiments, the engineered cytokines of the present disclosure can be expressed in a vector alone or in combination with one or more antigen receptors (e.g., T cell receptor (TCR), chimeric antigen receptor (CAR), or any combination thereof). In some embodiments, the vector comprises a viral vector. In some embodiments, the vector comprises a non-viral vector. In some embodiments, the vector consists of a single vector. In some embodiments, the viral vector comprises a sequence comprising a sequence encoding the engineered cytokine and a sequence encoding an antigen receptor (e.g., T cell receptor (TCR), chimeric antigen receptor (CAR), or any combination thereof). In some embodiments, the single viral vector comprises a sequence comprising a sequence encoding the engineered cytokine and a sequence encoding an antigen receptor (e.g., T cell receptor (TCR), chimeric antigen receptor (CAR), or any combination thereof). In some embodiments, the viral vector comprises a sequence isolated or derived from a lentiviral vector. In some embodiments, the viral vector comprises a lentiviral vector.

[0042] Function of engineered cytokines

[84] The present disclosure provides engineered cytokines that exhibit the functional benefits of IL-2 and IL-15. The present disclosure provides engineered cytokines that preferably exhibit one or more of the following attributes: (1) reducing or eliminating binding of the engineered cytokine to the alpha subunit of the IL-2 receptor (IL-2Rα), optionally without post-translational modifications to the engineered IL-2 polypeptide; (2) binding to the beta and / or gamma subunit of the IL-2 receptor (IL-2Rβ / γ) or retaining binding of the engineered IL-2 polypeptide to the beta and / or gamma subunit of the IL-2 receptor (IL-2Rβ / γ); (3) preferentially or more potently stimulating T cell(s) expressing CD8 (CD8 T cells) over stimulating regulatory T cell(s) (Tregs); (4) preferentially or more potently stimulating natural killer (NK) cells over CD8 T cells; (5) enhancing activity of T cells that express or secrete the engineered IL-2 polypeptide of the present disclosure; and (6) detecting the activity of the engineered IL-2 polypeptide in vitro or in vivo. exhibit stable folding into a protein in vivo, and (7) exhibit reduced, minimal, or undetectable levels of immunogenicity when administered to a subject (e.g., a mouse, another animal species, or a human patient) when compared to the level of immunogenicity exhibited by wild-type IL-2 protein under the same circumstances. Alternatively, or in addition, the engineered cytokines of the present disclosure may (1) bind to the IL-15 receptor (IL-15R), its beta subunit, or gamma subunit, and / or (2) compete with IL-15 cytokines, including WT IL-15, for binding to the IL-15R, its beta subunit, or gamma subunit.

[0043]

[85] Both wild-type IL-2 and IL-15 stimulate signaling through IL-2 / 15Rβ (also referred to as IL-2Rβ) and the common IL-2 / 15Rγ chain (also referred to as IL-2Rγ), and both IL-2 and IL-15 bind to and activate signaling through the heterodimeric βγ receptor complex IL-2 / 15Rβγ (also referred to as IL-2Rβγ), and their unique biology is driven primarily through their respective interactions with IL-2Rα (cis presentation) and IL-15Rα (trans presentation). The engineered IL-2 polypeptides of the present disclosure (also known as the IL-2 / 15 polypeptides of the present disclosure) agonize the βγ receptor pair shared by IL-2 and IL-15 while avoiding IL-2Rα and IL-15Rα. Thus, a designed IL-2 / 15 polypeptide of the present disclosure may exhibit both IL-2 and IL-15 activity when expressed by or contacted with different cell types.

[0044]

[86] IL-2 and IL-15 stimulate various types of lymphocytes and natural killer cells. Among the different functions between these two cytokines, IL-2 mediates the homeostasis of regulatory T cells and regulates the differentiation of helper T (TH). In addition, IL-15 mediates the proliferation of CD8 memory T cells, NK cells, and NK T cells. The designed IL-2 / 15 polypeptides of the present disclosure may exhibit one or more activities of IL-2 and IL-15 in a particular cell type.

[0045] Tailored or optimized engineered cytokines

[87] The engineered cytokines of the present disclosure may be "tuned" or "optimized" with respect to cytokine activity. The engineered cytokines of the present disclosure may be "tuned" or "optimized" to exhibit a favorable ratio of IL-2 activity to IL-15 activity. In some embodiments, the engineered cytokines may be tuned to achieve a particular threshold of activity of a wild-type IL-2 polypeptide and a particular threshold of activity of a wild-type IL-15 polypeptide. In some embodiments, tuning of the IL-2 / 15 polypeptides of the present disclosure does not include changes to the structure of the engineered cytokine (e.g., the 1-4-2-3 arrangement of its helices). In some embodiments, tuning of a designed cytokine of the present disclosure includes one or more of: (1) modifying the sequence of the designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof); (2) modifying the physical length of the folded designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof); (3) inserting a new sequence into the existing sequence of the designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof); and / or (4) removing a sequence of the designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof). In some embodiments, a tuned designed cytokine of the present disclosure includes a sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage in between, identity when compared to a designed cytokine of the present disclosure that has not been subjected to the tuning process. In some embodiments, a tuned designed cytokine of the present disclosure includes a sequence isolated or derived from an IL-15 sequence.

[0046]

[88] The present disclosure provides designed cytokines (previously known as "IL-2 / 15 polypeptides") that can be "tuned" or "optimized" with respect to any activity of the cytokine to exhibit a threshold of activity in one or more cell types. In some embodiments, tuning or optimizing a designed cytokine of the present disclosure for use in a cell type does not include changes to the structure of the designed cytokine (e.g., the 1-4-2-3 arrangement of its helices). In some embodiments, optimizing a designed cytokine of the present disclosure for use in a cell type includes one or more of: (1) modifying the sequence of the designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof); (2) modifying the physical length of the folded designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof); (3) inserting new sequence into the existing sequence of the designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof); and / or (4) removing sequence of the designed cytokine or a portion thereof (e.g., an alpha helix, a loop, or a combination thereof). In some embodiments, the optimized engineered cytokines of the present disclosure comprise sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of identity in between, when compared to an engineered cytokine of the present disclosure that has not been subjected to an optimization process. In some embodiments, the optimized engineered cytokines of the present disclosure comprise sequences isolated or derived from IL-15 sequences.

[0047] Designed cytokine combinations

[89] In some embodiments of the present disclosure, the designed cytokine may be used in combination with or operably linked to a second cytokine or a second designed cytokine. In some embodiments, the designed cytokine and the second cytokine or the second designed cytokine may be independently modified, regulated, and / or targeted. In some embodiments, the designed cytokine and the second cytokine or the second designed cytokine may be coordinated in one or more of the modification, regulation, and / or targeting.

[0048]

[90] In some embodiments, the designed cytokines of the present disclosure may be used in combination with any cytokine, whether naturally occurring or modified. In some embodiments, the designed cytokines of the present disclosure may be used in combination with one or more of an IL-2 polypeptide, an IL-12 polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, an IL-21 polypeptide, an IL-23 polypeptide, and an interferon polypeptide (including, but not limited to, an interferon alpha, beta, gamma, and / or omega polypeptide). In some embodiments, the designed cytokines of the present disclosure may be used in combination with an IL-2 polypeptide. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an IL-12 polypeptide. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an IL-15 polypeptide. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an IL-18 polypeptide. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an IL-21 polypeptide. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an IL-23 polypeptide. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide. In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon beta polypeptides. In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon gamma polypeptides. In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon omega polypeptides. In some embodiments, the second cytokine comprises a wild-type sequence. In some embodiments, the second cytokine does not comprise a wild-type sequence. In some embodiments, the second cytokine comprises one or more modifications to alter the activity of the cytokine on one or more cytokine receptors.

[0049]

[91] In some embodiments, a designed cytokine of the present disclosure may be used in combination with a second designed cytokine of the present disclosure to produce a combination of the first designed cytokine and the second designed cytokine.

[0050]

[92] In some embodiments of the present disclosure, the designed cytokine may be expressed with a second cytokine or a second designed cytokine of the present disclosure. In some embodiments, expression may be simultaneous. In some embodiments, expression may be sequential. In some embodiments, expression may be regulated or inducible by use of an inducible promoter of the present disclosure.

[0051]

[93] In some embodiments of the present disclosure, the designed cytokine may be secreted with a second cytokine or with a second designed cytokine of the present disclosure. In some embodiments, the secretion may be simultaneous. In some embodiments, the secretion may be sequential. In some embodiments, the secretion may be regulated or inducible by use of an inducible promoter of the present disclosure.

[0052]

[94] In some embodiments of the present disclosure, the designed cytokine may be operably linked to a second cytokine or a second designed cytokine of the present disclosure. In some embodiments, the designed cytokine and the second cytokine or the second designed cytokine may be operably linked by a linker sequence. In some embodiments, the linker sequence comprises a nucleic acid, an amino acid, a small molecule, or any combination thereof. In some embodiments, the linker is rigid. In some embodiments, the linker is flexible. For example, the linker sequence may comprise a "GS" sequence of any length.

[0053]

[95] In some embodiments, the designed cytokine and the second cytokine or the second designed cytokine may be operably linked by a targeting moiety of the present disclosure. In some embodiments, the designed cytokine and the second cytokine or the second designed cytokine may be operably linked to the same targeting moiety. In some embodiments, a composition comprising a designed cytokine and a second cytokine or the second designed cytokine may comprise the same targeting moiety. In some embodiments, the designed cytokine may comprise a first targeting moiety and the second cytokine or the second designed cytokine may comprise a second targeting moiety. In some embodiments, the first targeting moiety and the second targeting moiety bind to the same target. In some embodiments, the first targeting moiety and the second targeting moiety do not bind to the same target. In some embodiments, the first targeting moiety and the second targeting moiety are identical. In some embodiments, the first targeting moiety and the second targeting moiety are not identical.

[0054]

[96] In some embodiments, the designed cytokine and the second cytokine or the second designed cytokine may be operably linked by a tether of the present disclosure. In some embodiments, the designed cytokine and the second cytokine or the second designed cytokine may be operably linked to the same tether. In some embodiments, a composition comprising a designed cytokine and a second cytokine or the second designed cytokine may comprise the same tether. In some embodiments, the designed cytokine may comprise a first tether and the second cytokine or the second designed cytokine may comprise a second tether. In some embodiments, the first tether and the second tether bind to the same target. In some embodiments, the first tether and the second tether do not bind to the same target. In some embodiments, the first tether and the second tether are identical. In some embodiments, the first tether and the second tether are not identical.

[0055]

[97] In some embodiments, the engineered cytokine is operably linked to one or more of an IL-2 polypeptide, an IL-12 polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, an IL-21 polypeptide, an IL-23 polypeptide, and an interferon polypeptide (including, but not limited to, interferon alpha, beta, gamma, and / or omega polypeptides). In some embodiments, the engineered cytokine is operably linked to an IL-21 polypeptide.

[0056] Targeted engineered cytokines

[98] In some embodiments of the present disclosure, the engineered cytokine targets, localizes to, or remains active at one or more of a target cell, a target cell type, an organ, a lymph node, a tumor (including, but not limited to, liquid tumors, hematological cancers, and solid tumors), a biological microenvironment, a tumor microenvironment (TME), a site of malignancy, a site of metastasis, a site of tumor angiogenesis, and any combination thereof. In some embodiments, the fusion protein comprises an engineered cytokine that targets, localizes to, or remains active at one or more of a target cell, a target cell type, an organ, a lymph node, a tumor (including, but not limited to, liquid tumors, hematological cancers, and solid tumors), a biological microenvironment, a tumor microenvironment (TME), a site of malignancy, a site of metastasis, a site of tumor angiogenesis, and any combination thereof. In some embodiments, the engineered cytokine comprises a targeting moiety. In some embodiments, the engineered cytokine is operably linked to a targeting moiety. In some embodiments, the designed cytokine is operably linked to the targeting moiety by one or more of covalent bonds, non-covalent bonds, hybridization, dimerization, complex formation, linkers and tethers. In some embodiments, the designed cytokine is operably linked to the targeting moiety by a linker comprising one or more of a nucleic acid sequence, an amino acid sequence, a small molecule (organic or inorganic), and any combination thereof. In some embodiments, the designed cytokine is operably linked to the targeting moiety by a tether comprising one or more of a nucleic acid sequence, an amino acid sequence, a small molecule (organic or inorganic), and any combination thereof. In some embodiments, the designed cytokine is operably linked to the targeting moiety by a tether that is attached to one or more components of a target cell, a target cell type, an organ, a lymph node, a tumor (including but not limited to liquid tumors, blood cancers, and solid tumors), a biological microenvironment, a tumor microenvironment (TME), a site of a malignant tumor, a site of metastasis, a site of tumor vascularization, and any combination thereof.In some embodiments, the engineered cytokine is operably linked to the targeting moiety by a tether that is attached to a component of an immune cell that expresses or secretes the engineered cytokine.

[0057]

[99] In some embodiments of the present disclosure, the targeting moiety comprises a nucleic acid, amino acid, or combination thereof that specifically binds to one or more components of a target cell, a target cell type, an organ, a lymph node, a tumor (including, but not limited to, liquid tumors, hematological cancers, and solid tumors), a biological microenvironment, a tumor microenvironment (TME), a site of malignancy, a site of metastasis, a site of tumor vascularization, and any combination thereof. In some embodiments, the targeting moiety comprises a binding domain, a protein scaffold, an antibody, an antibody mimic, and / or a functional fragment thereof. In some embodiments, the targeting moiety comprises a sequence that may be isolated or derived from any species, including, but not limited to, human, non-human primate, rodent (including, but not limited to, mouse), and camelid species. In some embodiments, the targeting moiety comprises a sequence that may be humanized, chimeric, recombinant, non-naturally occurring, modified (e.g., to include synthetic nucleic acids or amino acids), and optimized (e.g., to reduce immunogenicity and / or aggregation during manufacturing).

[0058]

[0100] In some embodiments of the present disclosure, the targeting moiety comprises an antibody, including, but not limited to, a monoclonal antibody, an antigen binding fragment (Fab), a single chain variable fragment (scFv), a domain antibody, one or more of the heavy (VH) and light (VL) chain domains of an immunoglobulin (Ig) polypeptide or the genes encoding them, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and a nanobody. In some embodiments, the targeting moiety comprises an scFv, a VH or a VHH. In some embodiments, the targeting moiety comprises an scFv, VH, or VHH that specifically or selectively binds to targets including, but not limited to, T cell surface glycoprotein CD8 (also known as Cluster of Differentiation 8), programmed cell death-protein 1 (PD-1), programmed death-ligand 1 (PD-L1; also known as Cluster of Differentiation 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin mucin receptor 3 (TIM3).

[0059]

[0101] In some embodiments of the present disclosure, the targeting moiety comprises an antibody mimetic, including but not limited to one or more of engineered protein scaffolds, monobodies, affibody molecules, adnectin molecules, affimer molecules, affitin molecules, affilin molecules, alphabody molecules, anticalin molecules, aptamer molecules, atrimer molecules, avimer molecules, DARPin molecules, finomers, armadillo repeat protein molecules, Kunitz domain inhibitor molecules, knottin molecules, designed ankyrin repeat protein molecules, nanophytin molecules, and centirin molecules. In some embodiments, the targeting moiety comprises an antibody mimetic that specifically or selectively binds to targets including, but not limited to, T cell surface glycoprotein CD8 (also known as cluster of differentiation 8), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1; also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin mucin receptor 3 (TIM3).

[0060] Inducible engineered cytokines

[0102] The present disclosure provides nucleic acids encoding the non-naturally occurring polypeptides of the present disclosure. In some embodiments, a promoter or an inducible promoter capable of driving expression in a mammalian cell controls expression of a nucleic acid encoding a designed cytokine of the present disclosure.

[0061]

[0103] In some embodiments, the inducible promoter of the present disclosure comprises a minimal promoter. In some embodiments, the minimal promoter of the present disclosure comprises a sequence isolated or derived from one or more of minimal promoter 1 ("minP1"), YB-TATA and human beta globin. In some embodiments, the minimal promoter comprises one or more of "minP1" having the sequence of AGAGGGTATATAAAAGCTCGACTTCCAG, "minP2" having the sequence of TAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC, and "minP3" having the sequence of CTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC.

[0062]

[0104] In some embodiments, an inducible promoter of the disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with one or more of: BACH2, BARX1, BATF, ELF1, ELF2, Elf4, Elk1, ERF, ETV1, Fli1, FOXP1, GABPA, GATA3, IRF1, IRF2, IRF5, IRF7, IRF9, MAF, MAFF, Maz, Mef2d, MLX, MYB, NFAT, NFATC3, NFkB, NR4A1, Nur77, PATZ1, REL, RELA, RORa, RORg, RORgt, STAT2, Tbox, TFEB, TOX, USF1, ZBTB2, ZKSCAN3, ZNF12, ZNF140, ZNF263, ZNF282, ZNF304, ZNF398, ZNF708, and ZNF75D. In some embodiments, the inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with NFAT, NFkB, REL, RELA, IRF2, GATA3 and ATF3. In some embodiments, the inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with NFAT. In some embodiments, the inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with NFkB. In some embodiments, the inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with REL. In some embodiments, the inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with RELA. In some embodiments, the inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with IRF2. In some embodiments, the inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with GATA3. In some embodiments, an inducible promoter of the present disclosure comprises a sequence isolated or derived from a coding or non-coding sequence of a gene related to ATF3.

[0063]

[0105] In some embodiments, an inducible promoter of the present disclosure comprises a response element and / or an enhancer sequence. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with one or more of: BACH2, BARX1, BATF, ELF1, ELF2, Elf4, Elk1, ERF, ETV1, Fli1, FOXP1, GABPA, GATA3, IRF1, IRF2, IRF5, IRF7, IRF9, MAF, MAFF, Maz, Mef2d, MLX, MYB, NFAT, NFATC3, NFkB, NR4A1, Nur77, PATZ1, REL, RELA, RORa, RORg, RORgt, STAT2, Tbox, TFEB, TOX, USF1, ZBTB2, ZKSCAN3, ZNF12, ZNF140, ZNF263, ZNF282, ZNF304, ZNF398, ZNF708, and ZNF75D. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from a coding or non-coding sequence of a gene associated with one or more of NFAT, NFkB, REL, RELA, IRF2, GATA3 and ATF3. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from NFAT. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from NFkB. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from REL. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from RELA. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from IRF2. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from GATA3. In some embodiments, the response element and / or enhancer sequence comprises a sequence isolated or derived from ATF3.

[0064]

[0106] In some embodiments, the inducible promoter of the present disclosure comprises two or more response elements and / or enhancer sequences. In some embodiments, the inducible promoter of the present disclosure comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 response elements and / or enhancer sequences. In some embodiments, the repeated response elements and / or enhancer sequences are identical. In some embodiments, the repeated response elements and / or enhancer sequences are not identical.

[0065]

[0107] In some embodiments, the inducible promoter of the present disclosure does not include a combination of (1) nuclear factor of activated T cells (NFAT) sequence, interferon regulatory factor 4 (IRF4) sequence, activator protein 1 (AP-1)-IRF complex element (AICE) sequence, or interferon stimulated response element (ISRE) sequence, and (2) human beta globin sequence. In some embodiments, the inducible promoter of the present disclosure does not include the sequence GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGAATTCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC. As used throughout this disclosure, the AICE sequence includes or consists of a sequence isolated or derived from IRF4 or IRF8, each with BATF. In some embodiments, the AICE sequence may be derived from the untranslated regions of IRF4, IRF8, and / or BATF. As used throughout this disclosure, the ISRE sequence includes the consensus sequence of "YAGTTTC(A / T)YTTTYCC" (where "Y" is either C or T).

[0066]

[0108] In some embodiments, the inducible promoter of the present disclosure does not include a combination of (1) an NFAT sequence and (2) a YB-TATA sequence. In some embodiments, the inducible promoter of the present disclosure does not include the sequence: GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGAATTCCTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC.

[0067]

[0109] In some embodiments, the inducible promoter of the present disclosure comprises one or more transcription factor binding motif(s). In some embodiments, the inducible promoter of the present disclosure comprises a concatemer of two or more repeat sequences, where the repeat sequences may comprise one or more transcription factor binding motif(s). In some embodiments, the two or more repeat sequences are identical. In some embodiments, the two or more repeat sequences are not identical. In some embodiments, the concatemer comprises a linking sequence located between the repeat sequences. In some embodiments, the linking sequence may comprise one or more of TACGCT, TGATCT, TGCTTT, and TGCCCGT.

[0068]

[0110] In some embodiments, the inducible promoters of the present disclosure that include concatemers bind more than one unique transcription factor. Alternatively, or in addition, in some embodiments, the inducible promoters of the present disclosure that include concatemers bind the same transcription factor at more than one site.

[0069]

[0111] In some embodiments, an inducible promoter of the present disclosure that comprises one or more transcription factor binding motifs, optionally organized as a concatemer, comprises a sequence according to the consensus sequence provided in Table 16.

[0070]

[0112] In some embodiments, an inducible promoter of the present disclosure comprising one or more transcription factor binding motifs, optionally organized as a concatemer, comprises a sequence selected from any one or more of the sequences in Table 18.

[0071]

[0113] In some embodiments, an inducible promoter of the present disclosure comprising a concatemer comprises a sequence according to any one or more of the sequences in Table 19.

[0114] In some embodiments, the inducible promoter of the present disclosure comprises a pairing of an enhancer sequence and a promoter sequence (EP pairing). In some embodiments, the inducible promoter of the present disclosure comprising one or more EP pairings comprises a sequence in Table 17.

[0072]

[0115] In some embodiments, the designed cytokines of the present disclosure are operably coupled to any of the nucleic acid constructs disclosed in U.S. Provisional Application Nos. 63 / 479,176, 63 / 479,177, and 63 / 479,178, the entireties of which are incorporated herein by reference.

[0073] Designed cytokine structures

[0116] The present disclosure provides a non-naturally occurring engineered cytokine (previously referred to as an interleukin-2 / 15 (IL-2 / 15) polypeptide) comprising alpha helices H1, H2, H3, and H4, where, from amino terminus to carboxy terminus, a first loop (L1) connects H1 and H4, a second loop (L2) connects H4 and H2, and a third loop (L3) connects H2 and H3. In some embodiments, the engineered cytokine binds to the beta (β) and / or gamma (γ) subunits of the IL-2 receptor. In some embodiments, the engineered cytokine binds to the IL-2 receptor β / γ heterodimer (IL-2Rβ / γ) as a heterodimeric receptor.

[0074]

[0117] In some embodiments of the designed cytokines of the present disclosure, the designed cytokine comprises one or more of the sequences of SEQ ID NO:1 through SEQ ID NO:350.

[0118] In some embodiments of the designed cytokines of the present disclosure, the designed cytokine comprises one or more of the sequences of SEQ ID NO:1-SEQ ID NO:38 or SEQ ID NO:150-SEQ ID NO:350.

[0075]

[0119] In some embodiments of the designed cytokines of the present disclosure, the designed cytokines comprise sequences isolated or derived from IL-2 polypeptides. In some embodiments, the IL-2 polypeptides are wild-type polypeptides. In some embodiments, the IL-2 polypeptides comprise the sequence of SEQ ID NO: 500 below. In some embodiments, the designed cytokines of the present disclosure may be used in combination with IL-2 polypeptides, including IL-2 polypeptides having the sequence of SEQ ID NO: 500 below.

[0076]

[0120] Native human (hIL-2) is (SEQ ID NO:500): 1 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE 61 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 121 WITFCQSIIS TLT Contains an array of.

[0077]

[0121] hIL-2 has four helices connected by long irregular loops: the N-terminal helix (H1) interacts with both the beta and gamma subunits, the third helix (H3) interacts with the beta subunit, the C-terminal helix (H4) interacts with the gamma subunit, and the alpha subunit interacting surface is formed by an irregular second helix (H2) and two long loops, one connecting H1 to H2 and the other connecting H3 and H4.

[0078]

[0122] In some embodiments of the designed cytokines of the present disclosure, the designed cytokines comprise sequences isolated or derived from IL-15 polypeptides. In some embodiments, the IL-15 polypeptides are wild-type polypeptides. In some embodiments, the IL-15 polypeptides comprise the sequence of SEQ ID NO: 501 below. In some embodiments, the designed cytokines of the present disclosure may be used in combination with IL-15 polypeptides, including IL-15 polypeptides having the sequence of SEQ ID NO: 501 below.

[0079]

[0123] Natural human IL-15 (hIL-15) is

[0080]

number

[0081] Contains an array of.

[0124] hIL-15 has a similar structure to hIL-2, although hIL-15 shares less than 20% sequence identity with hIL-2.

[0082]

[0125] In some embodiments, the designed cytokines of the disclosure may be used in combination with an IL-12 polypeptide having the sequence MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS, an IL-12 polypeptide comprising subunit A. In some embodiments, an engineered cytokine of the disclosure may be used in combination with an IL-12 polypeptide having the sequence: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS, an IL-12 polypeptide comprising subunit B.

[0083]

[0126] In some embodiments, designed cytokines of the disclosure may be used in combination with IL-18 polypeptides, including IL-18 polypeptides having the sequence MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED.

[0084]

[0127] In some embodiments, designed cytokines of the disclosure may be used in combination with IL-21 polypeptides, including IL-21 polypeptides having the sequence: MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

[0085]

[0128] In some embodiments, the designed cytokines of the disclosure may be used in combination with an IL-23 polypeptide having the sequence: MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP, an IL-23 polypeptide comprising subunit A. In some embodiments, an engineered cytokine of the disclosure may be used in combination with an IL-23 polypeptide having the sequence: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS, an IL-23 polypeptide comprising subunit B.

[0086]

[0129] In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 1 / 13, which is a polypeptide having the sequence: MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide, including interferon alpha 2, which is a polypeptide having the sequence: MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide, including interferon alpha 4, which is a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISHFSCLKDRHDFGFPEEEFDGHQFQKAQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD.In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide, including interferon alpha 5, which is a polypeptide having the sequence: MALPFVLLMALVVLNCKSICSLGCDLPQTHSLSNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEDTPLMNVDSILTVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSANLQERLRRKE. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide, including interferon alpha 6, which is a polypeptide having the sequence: MALPFALLMALVVLSCKSSCSLDCDLPQTHSLGHRRTMMLLAQMRRISLFSCLKDRHDFRFPQEEFDGNQFQKAEAISVLHEVIQQTFNLFSTKDSSVAWDERLLDKLYTELYQQLNDLEACVMQEVWVGGTPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSSSRNLQERLRRKE. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide, including interferon alpha 7, which is a polypeptide having the sequence: MARSFSLLMVVLVLSYKSICSLGCDLPQTHSLRNRRALILLAQMGRISPFSCLKDRHEFRFPEEEFDGHQFQKTQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDFILAVRKYFQRITLYLMEKKYSPCAWEVVRAEIMRSFSFSTNLKKGLRRKD.In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide, including interferon alpha 8, which is a polypeptide having the sequence: MALTFYLLVALVVLSYKSFSSLGCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFEFPQEEFDDKQFQKAQAISVLHEMIQQTFNLFSTKDSSAALDETLLDEFYIELDQQLNDLESCVMQEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE. In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon alpha polypeptide, including interferon alpha 10, which is a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD. In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 14, a polypeptide having the sequence: MALPFALMMALVVLSCKSSCSLGCNLSQTHSLNNRRTLMLMAQMRRISPFSCLKDRHDFEFPQEEFDGNQFQKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLMEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD.In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 17, a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQFQKTQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKILRRKD. In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 21, a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE.

[0087]

[0130] In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon beta polypeptide, including an interferon beta polypeptide having the sequence: MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN.

[0088]

[0131] In some embodiments, the designed cytokines of the present disclosure may be used in combination with interferon gamma polypeptides, including interferon gamma polypeptides having the sequence MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ.

[0089]

[0132] In some embodiments, the designed cytokines of the present disclosure may be used in combination with an interferon omega polypeptide, including an interferon omega polypeptide having the sequence: MALLFPLLAALVMTSYSPVGSLGCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSS.

[0090] Exemplary Designed Cytokine Sequences

[0133] Exemplary designed cytokines of the present disclosure include, but are not limited to, one or more of the polypeptides provided in any one of Tables 1-15. In some embodiments, a designed cytokine of the present disclosure comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 90%, 95%, 97%, 99%, or any percentage in between identity to a designed cytokine of the present disclosure. In some embodiments, a designed cytokine of the present disclosure comprises a polypeptide having the sequence of one or more of helix H1, helix H4, helix H2, helix H3 of an IL-2 / 15 polypeptide of the present disclosure, optionally where the order of the helices is H1, H4, H2, followed by H3 in the polypeptide from its amino terminus to its carboxy terminus. In some embodiments, an IL-2 / 15 polypeptide of the present disclosure comprises a polypeptide having one or more sequences of helix H1, helix H4, helix H2, helix H3 of a designed cytokine of the present disclosure, where the order of the helices is H1, H4, H2, followed by H3 in the polypeptide from its amino terminus to its carboxy terminus. In some embodiments, a designed cytokine of the present disclosure comprises a polypeptide having helix H1, helix H4, helix H2, helix H3 of a designed cytokine of the present disclosure, in that order from its amino terminus to its carboxy terminus. In some embodiments, a designed cytokine of the present disclosure comprises a polypeptide having helix H1, helix H4, helix H2, helix H3 of a designed cytokine of the present disclosure, in that order from its amino terminus to its carboxy terminus, and optionally a loop connecting each helix having substantially the same topology and / or secondary structure as the loops in the designed cytokine of the present disclosure.

[0091] [Table 1-1]

[0092] [Table 1-2]

[0093]

Table 1-3

[0094]

Table 1-4

[0095]

Table 2-1

[0096]

Table 2-2

[0097]

Table 2-3

[0098]

Table 2-4

[0099]

Table 2-5

[0100]

Table 2-6

[0101]

Table 3-1

[0102]

Table 3-2

[0103]

Table 3-3

[0104]

Table 4-1

[0105]

Table 4-2

[0106]

Table 4-3

[0107]

Table 5-1

[0108]

Table 5-2

[0109]

Table 5-3

[0110]

Table 5-4

[0111]

Table 5-5

[0112]

Table 5-6

[0113]

Table 6

[0114]

Table 7-1

[0115]

Table 7-2

[0116]

Table 8

[0117]

Table 9

[0118]

Table 10

[0119]

Table 11

[0120]

Table 12

[0121]

Table 13-1

[0122]

Table 13-2

[0123]

Table 13-3

[0124]

Table 14-1

[0125]

Table 14-2

[0126] [Table 15]

[0127] nucleic acid

[0134] In some embodiments of the present disclosure, the terms "nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide sequence", "polynucleotide", and grammatical variations thereof are used interchangeably and refer to the phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecule"), or any phosphoester analogs such as phosphorothioates and thioesters, in either single-stranded form or double-stranded helices. Single-stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible.

[0128]

[0135] In some embodiments of the present disclosure, "nucleic acid", particularly DNA or RNA molecules, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. In some embodiments of the present disclosure, "nucleic acid" includes double-stranded DNA found in, among others, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence is provided according to the usual convention of depicting the sequence from left to right in the 5' to 3' direction along the untranscribed DNA strand (i.e., the strand with sequence homology to messenger RNA or mRNA). Unless otherwise stated, all nucleic acid and nucleotide sequences are depicted from left to right in the 5' to 3' orientation.

[0129]

[0136] Nucleotides are referred to by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission: thus, "A" stands for adenine, "C" stands for cytosine, "G" stands for guanine, "T" stands for thymine, and "U" stands for uracil.

[0130]

[0137] In some embodiments of the present disclosure, the term "polynucleotide" refers to a polymer of nucleotides of any length or type, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"). It also includes modified forms, for example, by alkylation and / or by capping of the polynucleotide, and unmodified forms. More specifically, "polynucleotide" includes polyribonucleotides (containing D-ribose), including polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and mRNA, whether spliced ​​or unspliced, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers that contain a nucleotide backbone, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, as well as other synthetic sequence-specific nucleic acid polymers (but that contain the nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA).

[0131]

[0138] In some embodiments of the present disclosure, the polynucleotide comprises a DNA sequence. In some embodiments of the present disclosure, the polynucleotide comprises a DNA sequence inserted in a vector or a vector comprising the DNA sequence.

[0132]

[0139] In some embodiments of the present disclosure, the polynucleotide comprises an mRNA. In some embodiments, the mRNA is synthetic or comprises synthetic nucleotides.

[0133]

[0140] In some embodiments of the present disclosure, a polynucleotide comprises at least one non-natural, non-naturally occurring, or modified nucleic acid. In some embodiments, a polynucleotide comprises a plurality of non-natural, non-naturally occurring, or modified nucleic acids. In some embodiments, all of a certain type of nucleic acid is a non-natural, non-naturally occurring, or modified nucleic acid (e.g., all uridines in a polynucleotide can be replaced with non-natural nucleobases, such as 5-methoxyuridine).

[0134]

[0141] In some embodiments of the present disclosure, "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0142] In some embodiments of the present disclosure, "expression vector" refers to a plasmid, virus, or other nucleic acid designed for polypeptide expression in a cell. A vector or construct is used to introduce a gene into a host cell, whereby the vector interacts with a polymerase in the cell and expresses a protein encoded in the vector / construct. An expression vector can be present extrachromosomally in a cell or can be integrated into a chromosome. An expression vector may contain additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). A polynucleotide of the present disclosure may be provided as a component of an expression vector.

[0135]

[0143] In some embodiments of the present disclosure, a "cloning vector" refers to a plasmid, virus, or other nucleic acid designed to produce copies of a polynucleotide. A cloning vector may contain transcription and translation initiation sequences, transcription and translation termination sequences, and a polyadenylation signal. Such constructs usually include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof. A polynucleotide of the present disclosure may be provided as a component of a cloning vector, and the cloning vector may be used to produce a polynucleotide of the present disclosure.

[0136]

[0144] In some embodiments of the present disclosure, "code" and the like refer to the ability of a particular sequence of nucleotides in a polynucleotide (e.g., a gene, a cDNA, or an mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene, a cDNA, or an RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, may be referred to as encoding the protein or other product of that gene or cDNA.

[0137]

[0145] Unless otherwise specified, a nucleotide sequence that "encodes an amino acid sequence", e.g., a polynucleotide that "encodes" a chimeric polypeptide as defined hereinafter in this disclosure, includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0138] Polypeptides

[0146] Amino acids may be referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acid residues are abbreviated as follows, with the abbreviations shown in parentheses: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0139]

[0147] Amino acid sequences are written left to right in amino to carboxy orientation.

[0148] In some embodiments of the present disclosure, "polypeptide" may refer to a sequence of amino acid subunits. In some embodiments, a "peptide" may be less than or equal to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. "Polypeptide" refers to proteins, polypeptides, and peptides of any length, size, structure, or function. "Polypeptide," "peptide," and "protein" are used interchangeably and refer to polymers of amino acids of any length.

[0140]

[0149] The polypeptides of the present disclosure may include naturally or synthetically created or modified amino acids, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids (including, for example, synthetic amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides also include gene products, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. A polypeptide may include a single polypeptide or may be a multi-molecular complex, such as a dimer, trimer, or tetramer. The polypeptides of the present disclosure may include single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are found in multi-chain polypeptides.

[0141]

[0150] Polypeptides of the present disclosure may contain L-amino acids plus glycine, D-amino acids plus glycine (which are resistant to L-amino acid specific proteases in vivo), or a combination of D- and L-amino acids plus glycine. The described polypeptides can be chemically synthesized or recombinantly expressed.

[0142]

[0151] The polypeptides of the present disclosure may include additional residues at the N-terminus, C-terminus, internally in the polypeptide, or combinations thereof, which are not included in determining the percent identity of the polypeptides of the present disclosure relative to the reference polypeptide. Such residues may be any residues suitable for the intended use, including, but not limited to, tags.

[0143]

[0152] In some embodiments of the present disclosure, a "chimeric polypeptide" may refer to any polypeptide that is composed of a first amino acid sequence from a first source covalently or non-covalently bound to a second amino acid sequence from a second source, where the first source and the second source are not the same. In some embodiments, the not the same first source and second source may include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein may, for example, include proteins from at least two different biological sources. In some embodiments, a chimeric polypeptide may include sequences from similar proteins from two different species. In some embodiments, a chimeric polypeptide may include sequences from different proteins from the same species. A biological source may include any non-synthetically produced nucleic acid or amino acid sequence (e.g., a genomic or cDNA sequence, a plasmid or viral vector, a natural virion, or a mutant or analog of any of the above). Synthetic sources can include chemically produced proteins or nucleic acid sequences, but cannot include proteins or nucleic acid sequences produced by biological systems (e.g., solid phase synthesis of amino acid sequences). Chimeric proteins can also include proteins from at least two different synthetic sources, or from at least one biological source and at least one synthetic source. Chimeric proteins can also include a first amino acid sequence from a first source covalently or non-covalently linked to a nucleic acid from any source or a small organic or inorganic molecule from any source. Chimeric proteins can include a linker molecule between the first amino acid sequence and the second amino acid sequence, or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[0144]

[0153] In some embodiments of the present disclosure, a "fragment" of a polypeptide, or a "truncated polypeptide" may refer to an amino acid sequence of a polypeptide that is shorter than the sequence of a reference polypeptide, which may be a naturally occurring sequence. Compared to a reference polypeptide, a fragment may include an N-terminal and / or C-terminal deletion. Compared to a reference polypeptide, a fragment may include a deletion of any part of the sequence, whether or not the deletion is contiguous. A polypeptide in which internal amino acids are deleted with respect to a naturally occurring sequence is also considered a fragment. Various polypeptide components of the present disclosure may be provided as fragments or truncated versions of a reference protein.

[0145]

[0154] In some embodiments of the present disclosure, a "functional fragment" may refer to a polypeptide fragment that retains the function of the polypeptide. In some embodiments, a functional fragment of a bioactive peptide (e.g., an enzyme) retains the ability to catalyze a biological action because the functional fragment contains the catalytic domain of the enzyme. The polypeptides of the present disclosure may be provided as functional fragments or truncated versions.

[0146]

[0155] In some embodiments of the present disclosure, "amino acid substitution" may refer to replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. In some embodiments, the parent or reference sequence comprises a wild-type sequence. The amino acid may be replaced, for example, by chemical peptide synthesis or via recombinant methods known in the art. For example, replacing an amino acid residue with an alternative amino acid residue is performed by replacing a codon that codes for a first amino acid with a codon that codes for a second amino acid. One or more amino acid substitutions may be provided in the polypeptide of the present disclosure.

[0147]

[0156] In some embodiments of the present disclosure, "conservative amino acid substitution" refers to the replacement of one amino acid residue with an amino acid residue that has a chemically similar side chain. The art has defined a family of amino acid residues that have similar side chains, including acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In some embodiments, a string of amino acids can be conservatively replaced with a chemically similar string that differs in the order and / or composition of side chain family members. Conservative amino acid substitutions may be provided in various polypeptide components of the disclosure.

[0148]

[0157] In some embodiments of the present disclosure, non-conservative amino acid substitutions include (i) substitutions of an electronegative residue (e.g., Glu or Asp) with or without an electropositive side chain (e.g., Arg, His, or Lys); (ii) substitutions of a hydrophilic residue (e.g., Ser or Thr) with or without a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); iii) cysteine ​​or proline substituted for any other residue, or cysteine ​​or proline substituted for any other residue, or (iv) a residue with a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) substituted for a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly), or a residue with a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) substituted for a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly). Non-conservative amino acid substitutions may be provided in various polypeptide components of the present disclosure. The likelihood that one of the aforementioned non-conservative substitutions can change the functional properties of a protein also correlates with the location of the substitution with respect to functionally important regions of the protein, and thus some non-conservative substitutions may have little or no effect on biological properties. The various polypeptide components of the disclosure may be provided with non-conservative amino acid substitutions that do not significantly alter the functionality of the altered component.

[0149] Sequence analysis

[0158] In some embodiments of the present disclosure, "identity" refers to the overall monomer conservation between polymer molecules, e.g., between polypeptide or polynucleotide molecules. "Identical" without any further qualifiers, e.g., protein A is identical to protein B, means that the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical" is equivalent to describing them as having, e.g., "70% sequence identity."

[0150]

[0159] If a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, the molecules are identical at that position.The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap.Comparing sequences and determining the percent identity between two sequences can be accomplished using a mathematical algorithm.

[0151]

[0160] In certain embodiments, the percentage identity (%ID) of a first amino acid (or nucleic acid) sequence to a second amino acid (or nucleic acid) sequence is calculated as %ID=100(Y / Z), where Y is the number of amino acid (or nucleic acid base) residues scored as identical matches in an alignment of the first and second sequences (as aligned by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0152]

[0161] The percent identity calculation of two polypeptide sequences can be carried out, for example, by aligning the two sequences for optimal comparison.For example, gaps can be introduced into one or both of the first and second polypeptide sequences for optimal alignment, and non-identical sequences can be ignored for comparison.In certain embodiments, the length of the sequence aligned for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.The amino acids at the corresponding amino acid positions are then compared.

[0153]

[0162] The creation of sequence alignment for calculating percent sequence identity is not limited to binary sequence comparison driven solely by primary sequence data. It will also be understood that sequence alignment can be created by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to create multiple sequence alignment is T-Coffee, available at www.tcoffee.org, alternatively available, for example, from the European Bioinformatics Institute (EBI) at the website ebi.ac.uk / Tools / psa. It will also be understood that the final alignment used to calculate percent sequence identity can be curated automatically or manually.

[0154]

[0163] Suitable software programs are available from various sources for both protein and nucleotide sequence alignment. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq uses either the BLASTN or BLASTP algorithm to perform the comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from EBI. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSLE, etc. Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Please note that percent sequence identity values ​​are rounded to the nearest 0.1. For example, a value between 80.11 and 80.14 would be rounded down to 80.1, while a value between 80.15 and 80.19 would be rounded up to 80.2. Also note that length values ​​are always integers.

[0155]

[0164] In some embodiments of the present disclosure, "non-naturally occurring" refers to a polypeptide or polynucleotide sequence that does not occur in nature. In some embodiments, a non-naturally occurring sequence does not occur in nature because the sequence is altered relative to a naturally occurring sequence. In some embodiments, a non-naturally occurring sequence does not occur in nature because it is a combination of two known naturally occurring sequences that do not occur together in nature (e.g., a chimeric polypeptide). In some embodiments, a non-naturally occurring polypeptide is a chimeric polypeptide. In some embodiments, a polypeptide or polynucleotide does not occur in nature because the sequence contains a portion (e.g., a fragment) that cannot be found in nature, i.e., a novel sequence. Any of the polynucleotides described herein can be provided as a non-naturally occurring sequence, e.g., with a sequence that is altered relative to the natural sequence, or as a polynucleotide that is linked to other polynucleotides in a non-natural manner. Any of the polypeptides described herein can be provided as a non-naturally occurring sequence, e.g., with a sequence that is altered relative to the natural sequence, or as a polypeptide that is linked to other polypeptides in a non-natural manner.

[0156] treatment method

[0165] In some embodiments of the present disclosure, the term "therapeutically effective" may refer to providing a beneficial effect to the recipient, for example, providing some relief, alleviation, or reduction in at least one clinical symptom in the subject. The therapeutic effect of the present disclosure does not need to be complete or curative, as long as some benefit is provided to the subject. For example, a therapeutic regimen incorporating the polynucleotide, gene therapy vector, or cell of the present disclosure together with a small molecule of the present disclosure may be structured such that the regimen as a whole is therapeutically effective.

[0157]

[0166] In some embodiments of the present disclosure, the term "therapeutically effective amount" refers to a dose or amount of the nucleic acid, vector, polypeptide, composition, pharmaceutical composition or cell of the present disclosure that is sufficient to confer a therapeutically effective benefit to the recipient. For example, the polynucleotide, gene therapy vector or cell of the present disclosure can be administered in a therapeutically effective amount. The subject to which the polynucleotide, gene therapy vector or cell of the present disclosure has been administered can then be administered a therapeutically effective amount of the small molecule of the present disclosure, i.e., an amount sufficient to confer a beneficial effect on the recipient, taking into account the previous administration of the polynucleotide, gene therapy vector or cell.

[0158] cell therapy

[0167] In some embodiments of the present disclosure, the term "stem cell" may refer to an undifferentiated or partially differentiated cell that can differentiate into various cell types and proliferate indefinitely to produce more of the same stem cell.

[0159]

[0168] In some embodiments of the present disclosure, the term "pluripotent stem cells" (PSCs) may refer to cells that can maintain an undifferentiated state indefinitely and can differentiate into most, if not all, of the cells of the body.

[0160]

[0169] In some embodiments of the present disclosure, the term "induced pluripotent stem cells" (iPS or iPSC) may refer to pluripotent stem cells that can be generated directly from somatic cells, including, but not limited to, specialized cells such as skin or blood cells from adults.

[0161]

[0170] In some embodiments of the present disclosure, the term "multipotent" may refer to cells that can develop into more than one cell type, but are more restricted than pluripotent cells. For example, adult stem cells and umbilical cord blood stem cells may be considered multipotent.

[0162]

[0171] In some embodiments of the present disclosure, the term "hematopoietic cells" may refer to cells arising from hematopoietic stem cells (HSCs). Hematopoietic cells of the present disclosure include, but are not limited to, myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, macrophages, platelets, monocytes, natural killer cells, T lymphocytes, B lymphocytes, and plasma cells.

[0163]

[0172] In some embodiments of the present disclosure, the term "T lymphocyte" or "T cell" may refer to hematopoietic cells that normally occur in the thymus. T lymphocytes or T cells include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, gamma delta T cells, and mucosal invariant T cells.

[0164]

[0173] In some embodiments of the present disclosure, the term "mesenchyme" may refer to an animal tissue type that comprises loose cells embedded in a network of proteins and fluids, i.e., the extracellular matrix. Mesenchyme directly gives rise to most of the body's connective tissues, including bone, cartilage, and the lymphatic and circulatory systems.

[0165]

[0174] In some embodiments of the present disclosure, the term "mesenchymal cell" may refer to a cell derived from mesenchymal tissue. In some embodiments, the cells of the present disclosure may be mesenchymal cells.

[0166]

[0175] In some embodiments of the present disclosure, the term "mesenchymal stromal cells" (MSCs) may refer to spindle-shaped plastically adherent cells isolated from bone marrow, adipose, and other tissue sources that have multipotency in vitro. For example, mesenchymal stromal cells can differentiate into osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells that give rise to bone marrow adipose tissue). The term mesenchymal stromal cells has been proposed in the scientific literature to replace the term "mesenchymal stem cells." In some cases, the cells of the present disclosure may be mesenchymal stromal cells.

[0167]

[0176] In some embodiments of the present disclosure, an "autologous cell" is a cell obtained from the same individual to whom it may be administered as a therapy (the cell is autologous to the subject). Autologous cells of the present disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0168]

[0177] In some embodiments of the present disclosure, the allogeneic cells are cells obtained from an individual who is not the subject recipient of the cells as a therapy (the cells are allogeneic to the subject). The allogeneic cells of the present disclosure can be selected from a donor that is immunologically compatible with the subject of the method of the present disclosure. The allogeneic cells of the present disclosure can be modified to produce "universal" allogeneic cells suitable for administration to any subject without unintended immunogenicity. The allogeneic cells of the present disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0169]

[0178] In some embodiments of the present disclosure, the terms "transfect" or "transform" or "transducing" may refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. In some embodiments, a "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid, or a progeny of that cell.

[0170]

[0179] In some embodiments of the present disclosure, the term "cell therapy" may refer to the provision or delivery of cells to a recipient for therapeutic purposes. formulation

[0180] In some embodiments of the present disclosure, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors, or cells of the present disclosure may be administered as part of a composition together with other pharma- ceutically acceptable components, including pharma-ceutically acceptable carriers.

[0171]

[0181] In some embodiments of the present disclosure, the term "pharmaceutical acceptable salt" refers to a derivative of a small molecule of the present disclosure, where the particular compound is converted to its acid or base salt. Such pharmaceutical acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include, for example, the conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids, such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. For example, small molecules of the present disclosure may be provided as pharma- ceutically acceptable salts.

[0172]

[0182] In some embodiments of the present disclosure, the term "excipient" refers to a pharmacologically inactive ingredient that is not active in the body. See, for example, Hancock, BC, Moss, GP, & Goldfarb, DJ (2020). Handbook of pharmaceutical excipients. London: Pharmaceutical Press, the entire disclosure of which is incorporated herein by reference. The small molecules of the present disclosure may be mixed with pharma- ceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preservatives, fillers, polymers, disintegrants, glidants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, flavoring agents, lubricants, acidifiers, and dispensing agents, depending on the mode of administration and the nature of the dosage form. Such ingredients include pharma-ceutically acceptable carriers and excipients that can be used to formulate oral dosage forms. Pharmaceutically acceptable carriers include water, ethanol, polyols, vegetable oils, fats, wax polymers, including gel-forming and non-gel-forming polymers, and suitable mixtures thereof. Examples of excipients include starch, pregelatinized starch, Avicel, lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate, and Lake Blend. Examples of disintegrants include starch, alginic acid, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. For example, the small molecule, polynucleotide, gene therapy vector, or cell of the present disclosure can be provided and administered in a composition that includes a pharma-ceutically acceptable excipient.

[0173] array

[0174] [Table 16-1]

[0175] [Table 16-2]

[0176] [Table 16-3]

[0177] [Table 16-4]

[0178] [Table 16-5]

[0179] [Table 16-6]

[0180] [Table 16-7]

[0181] [Table 16-8]

[0182]

[0183] The identified consensus motifs in Table 16 include some variability within the identified sequence. In some embodiments, the identified consensus motifs in Table 16 may allow one or more nucleotide substitutions within the identified consensus motif. For example, within the consensus motifs in Table 16, "N" may allow any nucleotide to be substituted at that position, including A, G, C, T. "S" may allow either G or C to be substituted at that position. "R" may allow either A or G to be substituted at that position. Also, "W" may allow A or T to be substituted at that position.

[0183]

[0184] Table 18 below lists specific motifs identified that reduce the variability of the consensus motifs. Table 18 lists exemplary specific motifs present within the identified consensus motifs of Table 16. Transcription initiators and regulators of the present disclosure may include any one or more of the specific exemplary motifs of Table 16.

[0184] [Table 17-1]

[0185] [Table 17-2]

[0186] [Table 17-3]

[0187] [Table 17-4]

[0188] [Table 17-5]

[0189] [Table 17-6]

[0190] [Table 17-7]

[0191] [Table 17-8]

[0192] [Table 17-9]

[0193]

Table 17-10

[0194]

Table 17-11

[0195]

Table 17-12

[0196]

Table 17-13

[0197]

Table 17-14

[0198]

Table 17-15

[0199]

Table 17-16

[0200]

Table 17-17

[0201]

Table 17-18

[0202]

Table 17-19

[0203]

Table 18-1

[0204]

Table 18-2

[0205]

Table 18-3

[0206]

Table 18-4

[0207]

Table 18-5

[0208]

Table 18-6

[0209]

Table 18-7

[0210]

Table 18-8

[0211]

Table 18-9

[0212]

Table 18-10

[0213]

Table 18-11

[0214]

Table 18-12

[0215]

Table 18-13

[0216]

Table 18-14

[0217]

Table 18-15

[0218]

Table 18-16

[0219]

Table 18-17

[0220]

Table 18-18

[0221]

Table 18-19

[0222]

Table 18-20

[0223]

Table 18-21

[0224]

Table 18-22

[0225]

Table 18-23

[0226]

Table 18-24

[0227]

Table 18-25

[0228]

Table 18-26

[0229]

Table 18-27

[0230]

Table 18-28

[0231]

Table 18-29

[0232]

Table 18-30

[0233]

Table 18-31

[0234]

Table 18-32

[0235]

Table 18-33

[0236]

Table 18-34

[0237]

Table 18-35

[0238]

Table 18-36

[0239]

Table 18-37

[0240]

Table 18-38

[0241]

Table 18-39

[0242]

Table 18-40

[0243]

Table 19-1

[0244]

Table 19-2

[0245]

Table 19-3

[0246]

Table 19-4

[0247]

Table 19-5

[0248]

Table 19-6

[0249]

Table 19-7

[0250]

Table 19-8

[0251]

Table 19-9

[0252]

Table 19-10

[0253]

Table 19-11

[0254]

Table 19-12

[0255]

Table 19-13

[0256]

Table 19-14

[0257]

Table 19-15

[0258]

Table 19-16

[0259]

Table 19-17

[0260]

Table 19-18

[0261]

Table 19-19

[0262]

Table 19-20

[0263]

Table 19-21

[0264]

Table 19-22

[0265]

Table 19-23

[0266]

Table 19-24

[0267]

Table 19-25

[0268]

Table 19-26

[0269]

Table 19-27

[0270]

Table 19-28

[0271]

Table 19-29

[0272]

Table 19-30

[0273]

Table 19-31

[0274]

Table 19-32

[0275]

Table 19-33

[0276]

Table 19-34

[0277]

Table 19-35

[0278]

Table 19-36

[0279]

Table 19-37

[0280]

Table 19-38

[0281]

Table 19-39

[0282]

Table 19-40

[0283]

Table 19-41

[0284]

Table 19-42

[0285]

Table 19-43

[0286]

Table 19-44

[0287]

Table 19-45

[0288]

Table 19-46

[0289]

Table 19-47

[0290]

Table 19-48

[0291]

Table 19-49

[0292]

Table 19-50

[0293]

Table 19-51

[0294]

Table 19-52

[0295]

Table 19-53

[0296]

Table 19-54

[0297]

Table 19-55

[0298]

Table 19-56

[0299]

Table 19-57

[0300]

Table 19-58

[0301]

Table 19-59

[0302]

Table 19-60

[0303]

Table 19-61

[0304]

Table 19-62

[0305]

Table 19-63

[0306]

Table 19-64

[0307]

Table 19-65

[0308]

Table 19-66

[0309]

Table 19-67

[0310]

Table 19-68

[0311]

Table 19-69

[0312]

Table 19-70

[0313]

Table 19-71

[0314]

Table 19-72

[0315]

Table 19-73

[0316]

Table 19-74

[0317]

Table 19-75

[0318]

Table 19-76

[0319]

Table 19-77

[0320]

Table 19-78

[0321]

Table 19-79

[0322]

Table 19-80

[0323]

Table 19-81

[0324]

Table 19-82

[0325]

Table 19-83

[0326]

Table 19-84

[0327]

Table 19-85

[0328]

Table 19-86

[0329]

Table 19-87

[0330]

Table 19-88

[0331]

Table 19-89

[0332]

Table 19-90

[0333]

Table 19-91

[0334]

Table 19-92

[0335]

Table 19-93

[0336]

Table 19-94

[0337]

Table 19-95

[0338]

Table 19-96

[0339]

Table 19-97

[0340]

Table 19-98

[0341]

Table 19-99

[0342]

Table 19-100

[0343]

Table 19-101

[0344]

Table 19-102

[0345]

Table 19-103

[0346]

Table 19-104

[0347]

Table 19-105

[0348]

Table 19-106

[0349]

Table 19-107

[0350]

Table 19-108

[0351]

Table 19-109

[0352]

Table 19-110

[0353]

Table 19-111

[0354]

Table 19-112

[0355]

Table 19-113

[0356]

Table 19-114

[0357]

Table 19-115

[0358]

Table 19-116

[0359]

Table 19-117

[0360]

Table 19-118

[0361]

Table 19-119

[0362]

Table 19-120

[0363]

Table 19-121

[0364]

Table 19-122

[0365]

Table 19-123

[0366]

Table 19-124

[0367]

Table 19-125

[0368]

Table 19-126

[0369]

Table 19-127

[0370]

Table 19-128

[0371]

Table 19-129

[0372]

Table 19-130

[0373]

Table 19-131

[0374]

Table 19-132

[0375]

Table 19-133

[0376]

Table 19-134

[0377]

Table 19-135

[0378]

Table 19-136

[0379]

Table 19-137

[0380]

Table 19-138

[0381]

Table 19-139

[0382]

Table 19-140

[0383]

Table 19-141

[0384]

Table 19-142

[0385]

Table 19-143

[0386]

Table 19-144

[0387]

Table 19-145

[0388]

Table 19-146

[0389]

Table 19-147

[0390]

Table 19-148

[0391]

Table 19-149

[0392]

Table 19-150

[0393]

Table 19-151

[0394]

Table 19-152

[0395]

Table 19-153

[0396]

Table 19-154

[0397]

Table 19-155

[0398]

Table 19-156

[0399]

Table 19-157

[0400]

Table 19-158

[0401]

Table 19-159

[0402]

Table 19-160

[0403]

Table 19-161

[0404]

Table 19-162

[0405]

Table 19-163

[0406]

Table 19-164

[0407]

Table 19-165

[0408]

Table 19-166

[0409]

Table 19-167

[0410]

Table 19-168

[0411]

Table 19-169

[0412]

Table 19-170

[0413]

Table 19-171

[0414]

Table 19-172

[0415]

Table 19-173

[0416]

Table 19-174

[0417]

Table 19-175

[0418]

Table 19-176

[0419]

Table 19-177

[0420]

Table 19-178

[0421]

Table 19-179

[0422]

Table 19-180

[0423]

Table 19-181

[0424]

Table 19-182

[0425]

Table 19-183

[0426]

Table 19-184

[0427]

Table 19-185

[0428]

Table 19-186

[0429]

Table 19-187

[0430]

Table 19-188

[0431]

Table 19-189

[0432]

Table 19-190

[0433]

Table 19-191

[0434]

Table 19-192

[0435]

Table 19-193

[0436]

Table 19-194

[0437]

Table 19-195

[0438]

Table 19-196

[0439]

Table 19-197

[0440]

Table 19-198

[0441]

Table 19-199

[0442]

Table 19-200

[0443]

Table 19-201

[0444]

Table 19-202

[0445]

Table 19-203

[0446]

Table 19-204

[0447]

Table 19-205

[0448]

Table 19-206

[0449]

Table 19-207

[0450]

Table 19-208

[0451]

Table 19-209

[0452]

Table 19-210

[0453]

Table 19-211

[0454]

Table 19-212

[0455]

Table 19-213

[0456]

Table 19-214

[0457]

Table 19-215

[0458]

Table 19-216

[0459]

Table 19-217

[0460]

Table 19-218

[0461]

Table 19-219

[0462]

Table 19-220

[0463]

Table 19-221

[0464]

Table 19-222

[0465]

Table 19-223

[0466]

Table 19-224

[0467]

Table 19-225

[0468]

Table 19-226

[0469]

Table 19-227

[0470]

Table 19-228

[0471]

Table 19-229

[0472]

Table 19-230

[0473]

Table 19-231

[0474]

Table 19-232

[0475]

Table 19-233

[0476]

Table 19-234

[0477]

Table 19-235

[0478]

Table 19-236

[0479]

Table 19-237

[0480]

Table 19-238

[0481]

Table 19-239

[0482]

Table 19-240

[0483]

Table 19-241

[0484]

Table 19-242

[0485]

Table 19-243

[0486]

Table 19-244

[0487]

Table 19-245

[0488]

Table 19-246

[0489]

Table 19-247

[0490]

Table 19-248

[0491]

Table 19-249

[0492]

Table 19-250

[0493]

Table 19-251

[0494]

Table 19-252

[0495] [Table 19-253]

[0496] [Table 19-254]

[0497] [Table 19-255]

[0498] [Table 19-256]

[0499] [Table 19-257]

[0500] [Table 19-258]

[0501] [Table 19-259]

[0502] definition

[0185] In some embodiments of the present disclosure, the term "subject" refers to any mammal, including but not limited to humans.

[0503]

[0186] The terms "a," "an," and "the" include their plural forms unless the context clearly dictates otherwise.

[0187] The term "and" is used interchangeably with "or" unless the context clearly dictates otherwise.

[0504]

[0188] The term "and / or" should be interpreted as a specific disclosure of each of two particular features or components with or without the other. Thus, when "and / or" is used in phrases such as "A and / or B," it includes "A and B," "A or B," "A" (single), and "B" (single). Similarly, when "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0505]

[0189] Numeric ranges include the numerical values ​​that define the range. When a range of values ​​is described, each intervening integer value between the recited upper and lower limits of the range, and each fraction thereof, is also specifically disclosed as are each subrange between such values. The upper and lower limits of any range can be independently included or excluded from the range, and each range in which either limit is included, neither is included, or both are included is also encompassed by the present disclosure. Thus, a range is understood to be concise for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0506]

[0190] When a value is explicitly stated, it will be understood that values ​​that are approximately the same quantity or amount as the stated value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, their combinations are also disclosed. Where any element of the disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded alone or in any combination with other alternatives are also disclosed in the specification, and more than one element of the disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed in the specification.

[0507]

[0191] Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise", "comprising", and the like, are to be construed in their inclusive sense, that is, in the sense of "including, but not limited to," as opposed to their exclusive or exhaustive sense.

[0508]

[0192] Singular or plural words also include plural and singular, respectively. Thus, for example, if the specification describes a gene of interest, the disclosure includes a polynucleotide having a single gene of interest or multiple genes of interest.

[0509]

[0193] Units, prefixes, and symbols are expressed in their Systeme International de Unites (SI) accepted form.

[0194] Headings are included herein for reference and to aid in locating the various sections. These headings are not intended to limit the scope of the concepts described therein. Such concepts may have applicability throughout the entire specification. EXAMPLES

[0510] Example 1: General Method ELISA experimental details material

[0511] [Table 20]

[0512]

[0195] 50 micrograms (mg) of CD25-Fc (R&D 1020-RL-050) is dissolved in 500 microliters (mL) of phosphate buffered saline (PBS). This 100 mg / mL solution is frozen in 20 mL aliquots at -80°C.

[0513]

[0196] Dissolve 50 mg of CD25-His (R&D 10305-RL-050) in 100 mL of PBS. Freeze this 500 mg / mL solution in 10 mL aliquots at -80°C.

[0514]

[0197] STII-tagged designed cytokines from undiluted supernatants (100 mL / well) were captured on streptactin plates for 1 h at room temperature. Plates were washed 4 x 200 mL with PBS-T.

[0515] Primary antibodies were prepared and added to the plate: Dilution buffer contains casein block and PBS-T (1:1). CD25-Fc at 2 mg / mL was added in casein block + PBS-T (1:1), and 100 mL of CD25-Fc at 100 mg / mL was diluted in 5 milliliters (mL) of dilution buffer. CD25-Fc at 0.5 mg / mL was added in casein block + PBS-T (1:1), followed by dilution of 25 mL of CD25-Fc at 100 mg / mL in 5 mL of dilution buffer. scIL12-anti-PDL1-scFv-STII + IL12Rb2-Fc was mixed at the indicated concentration in casein block + PBS-T (1:1). Incubated at room temperature for 1 hour.

[0516]

[0199] The plate was washed with 4 x 200 mL of PBS-T.

[0200] Secondary antibody was prepared and added to the plate: Make secondary antibody composition by mixing Protein G and HRP (1:5000) in casein block + PBS-T (1:1). Incubate at room temperature for 45 minutes.

[0517]

[0201] The plate was washed with 4 x 250 mL of PBS-T.

[0202] 75 mL of TMB-Ultra was added and incubated for approximately 5 minutes.

[0203] 75 mL of 2M HCl was added.

[0518]

[0204] The absorbance was measured at 450 nm (A450) on a plate reader using an ELISA program. HEK Blue - Experimental details

[0519] [Table 21]

[0520] [Table 22]

[0521]

[0205] Detach cells using 10 mL PBS / flask, count cells, centrifuge at 300 x g for 5 minutes, seed 1e6 cells into a new flask, resuspend cell pellet in DMEM + 10% FBS to a density of 280K / mL, and plate 180 mL cells / well into a flat-bottom 96-well plate.

[0522]

[0206] Dilutions of the supernatants were prepared in 96-well PCR plates.

[0207] Dilutions of the supernatants were prepared for these plates: 120 mL / well of undiluted supernatant was added to the PCR plate.

[0523]

[0208] 20 mL of this was transferred to 180 mL of Expi293 medium for a 1:10 dilution.

[0209] 40 mL of 1:10 was transferred to 160 mL of Expi293 medium for a final dilution of 1:50.

[0524]

[0210] 100 mL of 1:50 was transferred to 100 mL of Expi293 for a 1:100 dilution.

[0211] 100 mL of 1:100 was transferred to 100 mL of Expi293 for a 1:200 dilution.

[0525]

[0212] 100 mL of 1:200 was transferred to 100 mL of Expi293 for a 1:400 dilution.

[0213] Dilutions of the designed cytokines were prepared: 100 mM acetic acid was prepared from glacial acetic acid (17.4M). 57.5 μL of glacial acetic acid was diluted in 10 mL of ultrapure water. Sterile filtered. 100 mg of IL-2 (R&D 10453-IL-100) was dissolved in 200 mL of 100 mM acetic acid to make a 500 mg / mL solution.

[0526]

[0214] Designed cytokines were used over the range of 1,000 nanograms per milliliter (ng / mL) to 0.01 ng / mL. IL2 formulation at 500mg / mL. Diluted 1:50 in Expi293 medium followed by 5-fold serial dilutions (transfer 20mL to 80mL Expi293). After diluting this 20mL to a final volume of 200mL for the assay, the concentration range is 1000ng / mL>200ng / mL>40ng / mL>8ng / mL>1.6ng / mL>0.32ng / mL>0.06ng / mL>0.012ng / mL.

[0527] Plates were incubated overnight at 37°C with serial dilutions of the designated cytokines or WT IL-2. 180 mL of Quantiblue reagent was plated per well of a 96-well flat-bottom plate. 20 mL of culture supernatant was added to each well. Incubated for 30 minutes at 37°C. A640 was measured on a plate reader using the Quantiblue program.

[0528] Octet: Binding to IL2Ra

[0217] Octet settings: ProA chip, Octet buffer (HBS-EP + 0.25% BSA), 2.6 mg / mL IL2Ra-Fc used for capture; Zeba column (0.5 mL, 7 kDa) used to buffer exchange protein into HBS-EP (no BSA).

[0529]

[0218] Protein Octet dilutions were prepared in octet buffer (HBS-EP+0.25% BSA).

[0219] Chips were hydrated in 200 mL / well of buffer for 10 min excess during the experimental setup (one row of chips required).

[0530]

[0220] Set up assay plates (black 96 well plates) with protein dilutions and buffer. Use 200 mL of sample / buffer per well (add Octet buffer for baseline step).

[0531] [Table 23]

[0532]

[0221] Two 20 mL aliquots of 100 mg / mL IL2Ra-Fc were thawed. 1.5 mL of loading solution was made up in Octet buffer resulting in an IL2Ra-Fc concentration of 2.6 mg / mL.

[0533]

[0222] For the designed cytokine columns, 5-fold serial dilutions were performed according to the layout table above.

[0223] Prepare 300 mL of the highest concentration in the plate, transfer 50 mL down into 200 mL of buffer, and remove 50 mL at the final concentration so the final volumes are equal between samples.

[0534] For the 300 nM samples in row G, 20 mL of the highest concentration sample (3 mM, row A) was transferred to 180 mL of Octet buffer. An additional 30 mL of the 3 mM, row A sample was then removed so the final volumes were equal between samples.

[0535] [Table 24]

[0536] [Table 25]

[0537] Octet: Binds to IL2Rb / g (beta / gamma)

[0225] Octet setup information: ProA chip, Octet buffer (HBS-EP + 0.25% BSA), used 12 μg / mL IL2Rb / g-Fc for capture, used a Zeba column (0.5 mL, 7 kDa) to buffer exchange the protein into HBS-EP (no BSA).

[0538]

[0226] Protein Octet dilutions were prepared in octet buffer (HBS-EP+0.25% BSA).

[0227] Chips were hydrated in 200 mL / well of buffer for 10 min in excess during the experimental setup (a new chip set was required for each construct, 5 rows of chips).

[0539]

[0228] Set up assay plates (black 96 well plates) with protein dilutions and buffer. Use 200 mL of sample / buffer per well (add Octet buffer for baseline step).

[0540]

[0229] An Evap cover was used for this run.

[0541] [Table 26]

[0542]

[0230] Two 15 mL aliquots of IL2Rb / g-Fc at 600 mg / mL were thawed. 1.5 mL of loading solution was made up in Octet buffer resulting in an IL2Rb / g-Fc concentration of 12 mg / mL.

[0543]

[0231] For the designed cytokine column, 3-fold serial dilutions were performed according to the layout table above.

[0232] After preparing 1 mL of the highest concentration in an Eppendorf, 300 mL was transferred to the top well in the plate. For 3-fold dilutions, 100 mL was transferred down to 200 mL of buffer and 100 mL was removed at the final concentration so the final volumes were equal between samples.

[0544] [Table 27]

[0545] [Table 28]

[0546]

[0233] We encountered problems with the Evap covers of the last two constructions above that caused them to fail, so we need to set up the experiments again for these.

[0234] Chips were hydrated in 200 mL / well of buffer for 10 min in excess during the experimental setup (a new set of chips is required for each construct, 2 rows of chips).

[0547]

[0235] Buffer exchanged designed cytokine proteins (in HBS-EP) were used.

[0236] The same setup as in the replicates was used, except that the Evap cover was not used.

[0548] [Table 29]

[0549] Intrinsic tryptophan fluorescence

[0237] Material:

[0550] [Table 30]

[0551] Experimental Details: Designed cytokine proteins were diluted to 0.2 mg / mL in Gibco PBS pH 7.2 (with one exception in grey)

[0552] [Table 31]

[0553]

[0240] 25 μL of diluted protein was added to each well of a round-bottom black 96-well plate.

[0241] 75 μL of GndHCl diluted from 8 M GndHCl was added as shown below, with pre-mix and post-mix concentrations listed below.

[0554] [Table 32]

[0555]

[0242] Cover and incubate the plate at 37°C for 45 minutes.

[0243] Fluorescence is measured on a BioTek Synergy H1 plate reader as follows: 37℃ Excitation 280nm Orbital mixing 30 seconds Scans from 310 to 450 nm in 5 nm width Example 2: Engineered Cytokine Signaling

[0244] Designed cytokines were engineered to not bind CD25 (IL2Ra) and activate IL2R via IL2Rb and the common gamma chain receptor. This study demonstrates the ability of the designed cytokines to be expressed in transiently transfected Expi293 cells, to bind CD25 (IL2Ra) by ELISA, and to activate HEK-Blue reporter lines expressing [IL2Ra, IL2Rb, and the gamma subunit common to IL-2 and IL-15] or [IL2Rb, and the gamma subunit common to IL-2 and IL-15].

[0556]

[0245] Referring to Figure 5A and the designed cytokines having SEQ ID NO:1 through SEQ ID NO:38, respectively, the data demonstrate that, when compared to WT IL-2, the exemplary designed cytokines of the present disclosure do not bind to or signal through the alpha subunit of the IL-2 receptor (IL-2R).

[0557]

[0246] Referring to Figure 5A and the designed cytokines having SEQ ID NO:1 through SEQ ID NO:38, respectively, the data demonstrate that 33 of the 38 exemplary designed cytokines of the present disclosure retain IL-2 / 15Rβγ signaling when compared to WT IL-2.

[0558] Based on this initial screen, a number of designed cytokines were identified that 1) expressed in Expi293 cells, 2) activated only IL2R and CD122 / 132 in the HEK-Blue assay even at relatively high dilutions of transfection supernatants, and 3) were unable to bind CD25 (despite relatively good expression). These designs were marked for further development. Among these, designed cytokine No. 39 and designed cytokine No. 40 appear to be of particular interest since these were among the most highly expressed proteins by anti-STII jess. Designed cytokine No. 39 and designed cytokine No. 40 showed good activation in the HEK-Blue assay and they did not bind CD25. These proteins are also closely related, and therefore the fact that their behavior is similar supports a structure-function relationship.

[0559]

[0248] For binding to IL2Rb / g, we compared the designed cytokines to WT IL2 (both "endogenous" IL-2 and commercially available IL2).

[0249] Referring to Figure 5B, the data demonstrate that the designed cytokines bind to IL2Rb / g with nanomolar affinity.

[0560] IL2Ra binding: No detectable binding of the designed cytokines to IL2Ra was observed even up to 3 μM. WT IL2, in contrast, binds to IL2Ra with a Kd of approximately 15 nM. Thus, this experiment confirms that our WT IL2 binds to IL2Ra as expected, whereas the designed cytokines show no binding.

[0561] IL2R b / g binding: The designed cytokines bind to IL2Rb / g with nanomolar affinities similar to WT IL2 binding. Designed cytokine No. 169 has a Kd of approximately 1.6 nM, whereas WT IL2 has a Kd of approximately 0.4 nM. Designed cytokine No. 153 has a lower affinity of 3.9 nM, which is consistent with its lower activity in functional assays.

[0562] Example 3: Engineered cytokines have increased stability compared to WT IL-2 To test whether the designed cytokines were stabilized relative to WT IL2, the change in autologous tryptophan fluorescence was measured with increasing concentrations of the denaturant GndHCl. Tryptophan fluorescence is highly sensitive to its environment, and solvent exposure causes a "red shift" in the emission spectrum of tryptophan upon excitation at 280 nm.

[0563]

[0253] With reference to Figure 12, the designed cytokines exhibit lower autofluorescence when compared to that of WT IL-12 and therefore greater stability upon contact with denaturing agents.

[0564] Other embodiments

[0254] Although the present disclosure has been described in some detail as illustrations and examples for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be implemented. References such as "the present disclosure" are intended to refer to any of the various embodiments or aspects of the present disclosure and are not intended to limit the present disclosure to a single embodiment or aspect. When used throughout this disclosure, the terms "aspect" and "embodiment" are interchangeable. Features discussed in the context of "certain," "some," or "other" aspects or embodiments of the present disclosure may be found in any embodiment of the present disclosure, but in these cases, the features may be considered preferred features in those highlighted embodiments.

[0565]

[0255] The descriptions and examples should not be construed as limiting the scope of the disclosure to the embodiments and examples described herein, but rather as including all modifications and alternatives that fall within the true scope and spirit of the disclosure.

Claims

1. A designed cytokine comprising alpha-helix H1, H2, H3, and H4, with a structure from the amino terminus to the carboxyl terminus. The first loop (L1) connects H1 and H4, The second loop (L2) connects H4 and H2, The third loop (L3) connects H2 and H3, The aforementioned designed cytokine is a cytokine that binds to the IL-2 receptor βγ (IL-2Rβγ).

2. The designed cytokine according to claim 1, wherein the designed cytokine does not bind to IL-2 receptor alpha (IL-2Rα).

3. The cytokine designed above (a) The sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (Sequence ID 9791); (b) Sequence of sequence numbers 1 to 350; (c) A sequence having at least 70% identity with the sequence of (a); or (d) Sequences of sequence numbers 1 to 38 or sequence numbers 150 to 350 The designed cytokine according to claim 1, comprising one or more of the following.

4. The cytokine designed above (i) APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK; (ii) APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK; (iii) APTSSSSTKKTQLQLEHLLLDLQMILNGINNMNADPPELVEFFLNRWITFCQSIISTLTAGGSLSGDLKHLQNLSEELKPLEEVLNLAQSKNFHLRPRDLIISNINVIVLELK, or (iv) APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPE The designed cytokine according to claim 1, comprising the sequence LVEFLNRWITFCQSIISTGSVDPEELAKELQKLEEELKPLEEVLNLAQSKNFHLRPRDLISINNIVLELK.

5. The designed cytokine according to claim 1, wherein the designed cytokine is operably linked to a targeting portion; or the designed cytokine includes a targeting portion.

6. The designed cytokine according to claim 1, wherein the fusion protein comprises the designed cytokine and a targeting portion.

7. The designed cytokine according to claim 5, wherein the targeting portion binds to a component of the tumor microenvironment (TME), or the targeting portion binds to one or more of the following: T cell surface glycoprotein CD8 (also known as differentiation antigen group 8), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1; also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immune receptor (TIGIT) having Ig and ITIM domains, cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), or T cell immunoglobulin mucin receptor 3 (TIM3).

8. The designed cytokine according to claim 5, wherein the targeting portion comprises an antibody, an antibody mimetic, or a functional fragment thereof, or the targeting portion comprises one or more monoclonal antibodies, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or genes encoding them, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and one or more nanobodies.

9. The designed cytokine according to claim 1, wherein the designed cytokine is operably linked to a tether; or the designed cytokine comprises a tether.

10. The designed cytokine according to claim 1, wherein the fusion protein comprises the designed cytokine and a tether.

11. The designed cytokine according to claim 9, wherein the tether comprises one or more nucleic acid sequences, amino acid sequences, and small molecules, optionally comprising a sequence isolated or derived from a transmembrane sequence, and optionally comprising the sequence PLFIPVAVMVTAFSGLAFIIWLARRLKKGKK.

12. The designed cytokine according to claim 1, wherein the designed cytokine is operably linked to a second cytokine or a second designed cytokine, optionally the designed cytokine comprises a second cytokine or a second designed cytokine, or the fusion protein comprises the designed cytokine and a second cytokine or a second designed cytokine, optionally the second designed cytokine comprises one or more sequences of SEQ ID NOs: 1 to 38 or SEQ ID NOs: 150 to 350.

13. The second cytokine is one of the following: IL-2 polypeptide, IL-12 polypeptide, IL-15 polypeptide, IL-18 polypeptide, IL-21 polypeptide, IL-23 polypeptide, interferon alpha polypeptide, interferon beta polypeptide, interferon gamma polypeptide, and interferon omega polypeptide. The designed cytokine according to claim 12, comprising one or more sequences isolated or derived from

14. The designed cytokine according to claim 13, wherein the designed cytokine includes a first targeting portion and the second cytokine includes a second targeting portion, and optionally, the first targeting portion and the second targeting portion are identical, or the first targeting portion and the second targeting portion are not identical.

15. The designed cytokine according to claim 12, wherein the designed cytokine includes a first targeting portion and the second designed cytokine includes a second targeting portion, and optionally, the first targeting portion and the second targeting portion are the same, or the first targeting portion and the second targeting portion are not the same.

16. The designed cytokine according to claim 13, wherein the designed cytokine comprises a first tether, and the second cytokine or the second designed cytokine comprises a second tether, optionally, the first tether and the second tether are identical, or the first tether and the second tether are not identical.

17. A nucleic acid encoding a designed cytokine according to any one of claims 1 to 16 or a fusion protein comprising a designed cytokine according to any one of claims 1 to 16.

18. The invention further comprises a regulatory element capable of driving the expression of a designed cytokine, and optionally the regulatory element comprises a promoter, optionally the promoter is inducible, optionally the promoter comprises a minimal promoter, optionally the minimal promoter comprises a sequence isolated or derived from one or more minimal promoters-1 ("minP1"), YB-TATA, and human betaglobin, optionally minP1 comprises the sequence AGAGGGTATATAAAAAGCTCGACTTCCAG, optionally the minimal promoter comprises TAGAGGTATATAATGGGGGCCAACTAGTCTACTACCAGAAAAAGCTTGGTACCGAGCTCCGATCCCAGCCCAC, or CTAGAGGGTATATAATGGGGGCCAACTAGTCTA The nucleic acid according to claim 17, comprising the sequence CTACCAGAAAGCTTGGGTACCGAGCTCCGGATCCAGCCCACC, and optionally, the regulatory element comprises (i) a response element, and optionally, the response element comprises a repeat sequence, or (ii) a non-coding or untranslated sequence isolated or derived from one or more of NFAT, NFκB, REL, RELA, IRF2, GATA3, and ATF3, optionally, the regulatory element comprises a non-coding or untranslated sequence isolated or derived from the GATA3 gene, optionally, the GATA3 sequence comprises GTTATTCTCCACGAGATCT, and optionally, the regulatory element comprises a non-coding or untranslated sequence isolated or derived from RELA, and optionally, the RELA sequence comprises GGGGATTTCCA.

19. A vector comprising the nucleic acid described in claim 17, wherein the vector optionally comprises an expression vector or a delivery vector.

20. The vector according to claim 19, further comprising a sequence encoding an exogenous receptor, optionally comprising an antigen-binding moiety, a T-cell receptor (TCR), or a chimeric antigen receptor (CAR); and optionally, the antigen being expressed in or secreted within tumor cells, cancer cells, components of TMEs, and one or more TMEs. —.

21. A cell comprising a cytokine designed according to any one of claims 1 to 16, or a vector comprising a nucleic acid encoding the cytokine or a fusion protein containing the cytokine.

22. The cell according to claim 21, wherein the cell is a mammalian cell, a human cell, a primary cell, a cultured cell, an immortalized cell, an immune cell, a stem cell or progenitor cell capable of producing immune cells; optionally, the stem cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), or a dedifferentiated immune cell; optionally, the immune cell is a T lymphocyte (T cell), an alpha-beta T cell, a gamma-delta T cell, a B lymphocyte (B cell), a macrophage, or a natural killer (NK) cell; and optionally, the cell exists ex vivo, in vivo, or in vitro.

23. A composition comprising a cytokine designed according to any one of claims 1 to 16, a nucleic acid encoding the cytokine or a fusion protein containing the cytokine, a vector containing the nucleic acid, or a cell containing the cytokine, nucleic acid, or vector.

24. (1) a cytokine designed according to any one of claims 1 to 16, a nucleic acid encoding the cytokine or a fusion protein containing the cytokine, a vector containing the nucleic acid, or a cell containing the cytokine, nucleic acid, or vector, and (2) one or more pharmaceutically acceptable carriers.

25. A pharmaceutical composition according to claim 24 for use in the treatment of a disease or condition.

26. A pharmaceutical composition for use according to claim 25, wherein the disease or disorder comprises cancer or a subtype thereof, optionally comprising liquid cancer, blood cancer, or solid cancer.