WO2025043177A1

WO2025043177A1 - Akt1 fusion proteins and methods of use

Info

Publication number: WO2025043177A1
Application number: PCT/US2024/043631
Authority: WO
Inventors: Brian Curtis Turner; Yosef Refaeli
Original assignee: Ascensus Therapeutics Inc
Current assignee: Ascensus Therapeutics Inc
Priority date: 2023-08-23
Filing date: 2024-08-23
Publication date: 2025-02-27
Anticipated expiration: 2026-02-23

Abstract

Provided herein are fusion proteins having (i) a signaling activator comprising an AKT1 polypeptide and (ii) a protein transduction domain. The fusion proteins can activate cytokine pathway signaling in a cell, independently of cytokine-binding to a cytokine receptor. Also provided herein are nucleic acids and vector encoding the fusion proteins, as well as cells and compositions having the fusion protein. In addition, provided herein are methods of using the fusion proteins to activate cytokine signaling in a cell, prepare therapeutic cells, and treat a disease or disorder in a subject.

Description

AKT1 FUSION PROTEINS AND METHODS OF USE

CROSS REFERENCE TO REEATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application No. 63/578,244, filed August 23, 2023, U.S. Provisional Application No. 63/609,934, filed December 14, 2023, and U.S. Provisional Application No. 63/665,555, filed June 28, 2024, the entire disclosure of each of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on August 14, 2024, is named ASE-003WO_SL.xml and is 86.2 kilobytes in size.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates generally to fusion proteins having a cytokine pathway activator comprising an AKT1 fusion polypeptide or a functional fragment thereof and a protein transduction domain, as well as methods of using the fusion proteins.

BACKGROUND

[0004] Cytokines are involved in immune cell function, and dysregulation of cytokine signaling can severely impair immune cell function. For instance, solid tumors generate a microenvironment that can weaken the immune system’s ability to control infection and tumor growth and metastasis. Moreover, after an infection, cytokine signaling is necessary for the generation of immune memory, and dysregulation of cytokine signaling can lead to failure of leukocytes to clear the infection or differentiate into memory cells. Immunosenescence can also result in exhaustion of leukocytes. Leukocytes can also fail to mount a complete immune response as a result of immune cell anergy. For example, in T cells, anergy is a hyporesponsive state that can be induced by TCR-antigen engagement in the absence of appropriate costimulation, which can impact the ability of a subject’s immune system to mount a complete response, e.g., against a cancer. Increasing cytokine signaling in these contexts can decrease or reverse immune cell exhaustion and impairment, thereby restoring effector function after or during a chronic infection, or during the treatment of cancer.

[0005] Cytokine pathway signaling is also involved in the function of immunosuppressive regulatory T cells (Tregs). Disruption of cytokine signaling (e.g., IL-2 pathway signaling) in Tregs can impair Treg development, maintenance, and function, which can contribute to immune system dysregulation and the development of autoimmune disease. Increasing cytokine signaling in this context can restore the function of immunosuppressive Tregs, thereby supporting the treatment of autoimmune disease.

[0006] Cytokine pathway activation is also involved in the reversal of damage and restoration of function following ischemic injury of an organ. Tissues deprived of blood and oxygen undergo ischemic necrosis or infarction with possible irreversible organ damage. Once the flow of blood and oxygen is restored to the organ or tissue (reperfusion), the organ does not immediately return to the normal preischemic state. Although reperfusion restores oxygen and reverses ischemia, repletion of high energy nucleotides, such as adenosine triphosphate (ATP), and reversal of ischemic membrane damage is slow, and tissue function may be decreased for a long period of time. Stimulation of cytokine signaling in cells after ischemic reperfusion may prevent or lessen the damage to the tissue.

[0007] Several approaches for improving cytokine signaling and function during cancer, infection, and ischemic injury have been explored, including treatment of patients with either high doses of cytokines, or with vectors capable of driving expression of effector genes in cells. However, these approaches have been hindered by both the short half-life of cytokines, as well as toxicity associated with high levels of expression of effector genes. Additionally, configuring cells to express effector genes may lead to neoplasia. Thus, alternative approaches for activating the signaling pathway downstream of cytokine receptors are of significant interest. The present disclosure satisfies this need and provides related advantages.

SUMMARY OF THE INVENTION

[0008] In one aspect, the present disclosure provides a fusion protein comprising: (a) a signaling activator comprising a constitutively active AKT1 polypeptide or a functional fragment or variant thereof, and (b) a protein transduction domain (PTD). In some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof is phosphatase resistant.

[0009] In some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution and/or an amino acid sequence that facilitates sequestration of the fusion protein at the plasma membrane. For example, in some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a Src myristoylation sequence, e.g., a Src myristoylation sequence comprising the amino acid sequence of SEQ ID NO: 5 or 6. In some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a Gag myristoylation sequence, e.g., a Gag myristoylation sequence comprising the amino acid sequence of SEQ ID NO: 7.

[0010] In some embodiments of any of the foregoing fusion proteins, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of: (i) a glutamate residue at a position corresponding to position 17 of wild-type human AKT1 (E 17), e.g., wherein the glutamate residue is substituted by lysine (E17K); (ii) a leucine residue at a position corresponding to position 52 of wild-type human AKT1 (L52), e.g., wherein the leucine residue is substituted by arginine (L52R); (iii) a cysteine residue at a position corresponding to position 77 of wild-type human AKT1 (C77), e.g., wherein the cysteine residue is substituted by phenylalanine (C77F); (iv) a glutamine residue at a position corresponding to position 79 of wild-type human AKT1 (Q79), e.g., wherein the glutamine residue is substituted by lysine (Q79K); and/or (v) a glycine residue at a position corresponding to position 171 of wild-type human AKT1 (G171), e.g., wherein the glycine residue is substituted by arginine (G171R).

[0011] In some embodiments of any of the foregoing fusion proteins, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a deletion of the pleckstrin homology (PH) domain of AKT1. For example, in some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a deletion of the residues corresponding to residues 4 through 129 of wild-type AKT1.

[0012] In some embodiments of any of the foregoing fusion proteins, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution that prevents AKT-induced neoplasia. In some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a threonine residue at a position corresponding to position 308 of wild-type human AKT1 (T308). For example, in some embodiments, the threonine residue at a position corresponding to position 308 of wild-type human AKT1 is substituted by aspartic acid (T308D). In some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a serine residue at a position corresponding to position 473 of wild-type human AKT1 (S473). For example, in some embodiments, the serine residue at a position corresponding to position 473 of wild-type human AKT1 is substituted by aspartic acid (S473D). [0013] In some embodiments of any of the foregoing fusion proteins, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises the amino acid sequence of any one of SEQ ID NOs: 2-4 or 8. In some embodiments, the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises the amino acid sequence of any one of SEQ ID NOs: 9-10 or 61.

[0014] In some embodiments of any of the foregoing fusion proteins, the PTD comprises a cationic PTD, a hydrophobic PTD, or a cell-type specific PTD.

[0015] In some embodiments, the PTD comprises a cationic PTD, e.g., a VP- 16 peptide, an antennapedia peptide, a PTD-5 peptide, a polylysine peptide, a polyarginine peptide, an HIV VPR peptide, an HIV Tat peptide, or a functional variant of any of the foregoing. In some embodiments, the PTD comprises an HIV-1 Tat peptide or a functional variant thereof. In some embodiments, the PTD comprises the amino acid sequence of SEQ ID NO: 11 or 12.

[0016] In some embodiments, the PTD comprises a hydrophobic PTD, e.g., atransportan peptide, a MAP peptide, a TP 10 peptide, or a functional variant of any of the foregoing.

[0017] In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 24-40 and 64.

[0018] In some embodiments of any of the foregoing fusion proteins, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 24-29 and 64, e.g., the amino acid sequence of SEQ ID NO: 26. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 29.

[0019] In a related aspect, the present disclosure provides a nucleic acid encoding the fusion protein of any of the foregoing embodiments, a vector comprising said nucleic acid, or a cell comprising said vector. In some embodiments, the cell is a bacterial cell.

[0020] In a related aspect, the present disclosure provides a pharmaceutical composition comprising the fusion protein of any one of the foregoing embodiments and a pharmaceutically acceptable carrier or excipient.

[0021] In a related aspect, the present disclosure provides a composition comprising the fusion protein of any of the foregoing embodiments and an immune cell. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0022] In another aspect, the present disclosure provides a method of preparing a cell therapeutic composition, the method comprising a step of contacting an immune cell with the fusion protein of any of the foregoing embodiments. In some embodiments, the method further comprises cryopreserving the cell therapeutic composition and, optionally, thawing the cell therapeutic composition. In some embodiments, the step of contacting the immune cell with the fusion protein occurs prior to cryopreservation. In other embodiments, the step of contacting the immune cell with the fusion protein occurs after thawing the cell therapeutic composition. In some embodiments, the thawed immune cell exhibits increased surface expression of CD25, CD44, and/or CD69, as compared to a frozen and thawed immune cell that was not contacted with the fusion protein. In some embodiments, the contacting step comprises contacting the immune cell with a medium comprising 0.05-500 pg/mL of the fusion protein.

[0023] In a related aspect, the disclosure provides a cell therapeutic composition generated by any one of the foregoing methods.

[0024] In another aspect, the present disclosure provides a method of genetically modifying an immune cell, the method comprising: (a) contacting an immune cell with the fusion protein of any of the foregoing embodiments, thereby generating an activated immune cell; and (b) contacting the activated immune cell with a vector encoding a gene of interest. In some embodiments, the immune cell is in a resting state prior to step (a). In some embodiments, the step of contacting the immune cell with the fusion protein induces the immune cell to enter the G1 phase of the cell cycle.

[0025] In some embodiments of the method of genetically modifying an immune cell, the vector encoding a gene of interest is a viral vector, e.g., an adenoviral vector or a retroviral vector, e.g., a type-C retroviral vector. In some embodiments, the vector is RNA. In some embodiments, step (b) of the method comprises contacting the cell with a liposome encapsulating the vector.

[0026] In a related aspect, the disclosure provides a method of expanding an immune cell in a culture, the method comprising: (a) contacting the immune cell with a growth medium comprising a mitogenic stimulus, and (b) contacting the immune cell with the fusion protein of any of the foregoing embodiments. In some embodiments, the mitogenic stimulus is an anti- CD3 antibody and/or an anti-CD28 antibody. In some embodiments, the growth medium further comprises one or more cytokines, e.g., IL-2, IL-4, IL-7, and/or IL-15.

[0027] In some embodiments, the immune cell is incubated in the growth medium for at least 3 days, e.g., 3 to 5 days. In some embodiments, additional copies of the fusion protein and/or the one or more cytokines are added to the culture every 72-120 hours. [0028] In some embodiments, steps (a) and (b) are carried out simultaneously. In some embodiments, the growth medium comprises the fusion protein and the mitogenic stimulus.

[0029] In some embodiments, step (a) is carried out prior to step (b). In some embodiments, step (b) comprises incubating the immune cell in a medium comprising the fusion protein for at least 5 minutes, e.g., at least 5, 15, 30, 45, or 60 minutes. In some embodiments, following step (b), the immune cell is removed from the medium comprising the fusion protein, washed, and incubated in a second growth medium comprising the mitogenic stimulus. In some embodiments, the second growth medium is the same growth medium used in step (a).

[0030] In some embodiments, following steps (a) and (b), the immune cell expresses a higher level of CD25, CD44, and/or CD69 relative to an immune cell which was contacted with the growth medium comprising the mitogenic stimulus without being contacted with the fusion protein. In some embodiments, following steps (a) and (b), the immune cell exhibits increased survival and/or proliferation relative to an immune cell which was contacted with the growth medium comprising the mitogenic stimulus without being contacted with the fusion protein.

[0031] In a related aspect, the present disclosure provides a method of activating a cytokine signaling pathway in an immune cell, the method comprising contacting the immune cell with the fusion protein of any of the foregoing embodiments. In some embodiments, the cytokine is IL-2. In some embodiments, the activation of signaling through the IL-2 signaling pathway occurs independently of IL-2 -mediated activation of the signaling pathway.

[0032] In some embodiments of the foregoing methods, the step of contacting the immune cell occurs in vivo or ex vivo.

[0033] In some embodiments of the foregoing methods, the immune cell is selected from a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an NKT cell, a myeloid cell, hematopoietic stem cell, and a red blood cell. In some embodiments, the immune cell is a T cell, e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, and an activated T cell. In some embodiments, the immune cell is a genetically modified immune cell. In some embodiments, the immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), e.g., wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell. The target cell can be, for example, a cancer cell or an infected cell. [0034] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a fusion protein of the foregoing embodiments, or a pharmaceutical composition comprising said fusion protein. In a related aspect, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising a fusion protein of the foregoing embodiments and an immune cell, or an immune cell that was contacted ex vivo with the fusion protein of the foregoing embodiments or a pharmaceutical composition comprising said fusion protein. In some embodiments, the cancer is selected from breast cancer (e.g., triple negative breast cancer), colorectal cancer, and lung cancer (e.g, NSCLC).

[0035] In another aspect, the disclosure provides a method of treating or preventing ischemia reperfusion injury in a subject in need thereof, the method comprising administering to the subject the fusion protein of the foregoing embodiments, or a pharmaceutical composition comprising the fusion protein. In a related aspect, the disclosure provides a method of treating or preventing ischemia reperfusion injury in a subject in need thereof, the method comprising administering to the subject a composition (e.g, a pharmaceutical composition) comprising a fusion protein of the foregoing embodiments and an immune cell, or an immune cell that was contacted ex vivo with the fusion protein of one of the foregoing embodiments or a pharmaceutical composition comprising said fusion protein.

[0036] In another aspect, the disclosure provides a method of treating an infection in a subject in need thereof, the method comprising administering to the subject the fusion protein of any of the foregoing embodiments, or a pharmaceutical composition comprising said fusion protein. In a related aspect, the disclosure provides a method of treating an infection in a subject in need thereof, the method comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising a fusion protein of any of the foregoing embodiments and an immune cell, or an immune cell that was contacted ex vivo with the fusion protein of one of the foregoing embodiments or a pharmaceutical composition comprising said fusion protein. In some embodiments, infection is a bacterial infection, e.g, an infection of Staphylococcus aureus, Streptococcus pnuemoniae, Heamophila influenzae, Neisseria meningitidis, Klebsiella pneumoniae , Mycobacterium tuberculosis, Escherichia coli, and group B Streptococci). In some embodiments, the infection is a viral infection, such as a chronic viral infection (e.g., an infection of a virus selected from Hepatitis A Virus Hepatitis B Virus, Hepatitis C Virus, LCMV, herpes virus (e.g., HSV, Epstein Barr Virus (EBV), or Kaposi’s sarcoma-associated herpesvirus (KSHV)), Human Immunodeficiency Virus (HIV), or Human Papilloma Virus (HPV)) or an acute viral infection (e.g., an infection of a virus selected from an influenza virus, West Nile Virus, Respiratory syncytial virus (RSV), a coronavirus, measles, Dengue virus, Ebola virus, Japanese encephalitis virus (JEV), or a rhinovirus). In some embodiments, the infection is a fungal infection, e.g., an infection from a fungal pathogen selected from Candida albicans, Aspergillus, Candida auris, Pneumocystis jirovecii, Cryptococcus neoformans, or Sporothrix. In some embodiments, the infection is a protozoan infection. In some embodiments, the infection is a parasitic infection, e.g., an infection from a parasite selected from Taenia, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichomoniasis, Babesiosis, Blastocytosis, Cryptospridium, Trypanosomes, Trichonomas, Sarcocystis, Rhinosporodium, Malaria, Leishmania, Giardia, or an amoeban parasite.

[0037] In some embodiments of the foregoing therapeutic methods utilizing an immune cell, the immune is selected from a T cell, a B cell, a natural killer (NK) cell, an NKT cell, a dendritic cell, and a mast cell. In some embodiments, the immune cell is a T cell, e.g. a CD4+ T cell, a CD8+ T cell, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, and an activated T cell. In some embodiments, the immune cell is a genetically modified immune cell. In some embodiments, the immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), e.g., wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell. The target cell can be, for example, a cancer cell or an infected cell.

[0038] In another aspect, the disclosure provides a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a fusion protein of any of the foregoing embodiments, or a pharmaceutical composition comprising said fusion protein. In a related aspect, the disclosure provides a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising a fusion protein of the disclosure and an immune cell, or an immune cell that was contacted ex vivo with a fusion protein of any of the foregoing embodiments or a pharmaceutical composition comprising the fusion protein. In some embodiments, the immune is selected from a T cell, a B cell, a natural killer (NK) cell, an NKT cell, a dendritic cell, and a mast cell. In some embodiments, the immune cell is a T cell, e.g., a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, or an activated T cell. In some embodiments, the T cell is a CD25+ CD4+ Treg. In some embodiments, the immune cell is a genetically modified immune cell. In some embodiments, the immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), e.g., wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell. The autoimmune disease to be treated can be a T-cell dependent autoimmune disease, e.g., an autoimmune disease selected from Type 1 diabetes, rheumatoid arthritis, LADA, multiple sclerosis, lupus, scleroderma pigmentosa, Myasthenia Gravis, Guillain Barre Syndrome, amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, and a chronic inflammatory disorder of the central nervous system. In some embodiments, the autoimmune disease is Type 1 diabetes.

[0039] In another aspect, the disclosure provides a use of a fusion protein of any of the foregoing embodiments in the manufacture of a medicament for the treatment of cancer in a subject in need thereof. In a related aspect, the disclosure provides a use of a fusion protein of any of the foregoing embodiments in the manufacture of a medicament for the treatment of an autoimmune disease in a subject in need thereof. In a related aspect, the disclosure provides a use of a fusion protein of any of the foregoing embodiments in genetically modifying an immune cell. In a related aspect, the disclosure provides a use of a fusion protein of any of the foregoing embodiments in expanding an immune cell in a culture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The disclosure can be more completely understood with reference to the following drawings.

[0041] FIG. 1 is a schematic representation of fusion proteins of the disclosure, and the method by which they can activate a cytokine signaling pathway in a cell to stimulate survival and proliferation, while bypassing the need for the cytokine and the cytokine’s receptor.

[0042] FIGs. 2A-2B depict exemplary protocols for manufacturing immune cells for use in cell therapy. FIG. 2A depicts an exemplary process for isolating, stimulating, and administering immune cells to a subject. FIG. 2B depicts an exemplary process for manufacturing and administering genetically modified immune cells to a subject (FIG. 2B).

[0043] FIGs. 3A-3C depict the results of ex vivo experiments to test the role of AKT1 signaling in cytokine-mediated activation of T cells. Activated murine CD4+ T cells were cultured ex vivo in the presence of the indicated cytokine, and T-cell survival and proliferation were measured post-treatment by monitoring the number of viable T cells (FIG 3A). AKT1 activation (FIG. 3B and FIG. 3C) and Bcl-2 production (FIG. 3B) were measured 30-minutes post-treatment via western blotting with anti-phospho-AKTl and anti -Bel -2 antibodies.

[0044] FIG. 4 is a bar graph depicting the results of an ex vivo experiment testing the ability of a dominant-negative form of AKT1 (“DN AKT”) to block cytokine-mediated T-cell survival. Activated murine CD4+ T cells were retrovirally transduced ex vivo with a vector encoding DN AKT or an empty vector control (“MIG”), and cells were cultured in the presence of the indicated cytokine. T-cell survival and proliferation were measured post-treatment by monitoring the number of viable T cells.

[0045] FIGs. 5A-5C depict the results of ex vivo experiments testing whether constitutively active and conditionally active forms of AKT1 can stimulate T cell proliferation and survival in the absence of supplemental cytokines. Activated murine CD4+ T cells were retrovirally transduced ex vivo with a vector encoding constitutively active AKT (FIG. 5A; “Myr-AKT”) or conditionally active AKT that is active in the presence of TMX (FIG. 5B; “AKTER”). T cells transduced with an empty vector were used as controls (“MIG”). T-cell survival and proliferation were measured every 24 h post-infection by monitoring the number of viable T cells. AKT1 activation was measured post-infection via western blotting with an anti-phospho- AKTl antibody (FIG 5C).

[0046] FIG. 6 is a bar graph depicting the results of an in vivo experiment testing the ability of antigen specific and/or anergic T cells to inhibit tumor formation in a syngeneic murine lymphoma model. Mice were inoculated with HEL-expressing lymphoma cells (“EpMYC/MD4/ML5”) in combination with either wild-type T cells (non-antigen-specific; “+ WT”), antigen-specific T-cells (“+ 3A9”), or anergic, antigen-specific T-cells (“+ 3A9/ML5”). Prior to transplantation, T cells were retrovirally transduced with an empty vector (“pMIG”), a vector encoding a constitutively active Myr-Akt (“pMIG-Akt*), or a vector encoding Bcl2 (“pMIG-Bcl2”). Non-transduced T cells were used as a control (“None”).

[0047] FIG. 7 depicts the results of an ex vivo experiment testing the ability of a PTD-fusion protein to promote the survival of an activated T cell. Activated murine CD4+ T cells were treated ex vivo with a fusion protein comprising a Tat PTD and Bcl-2 at the indicated concentrations, and the percentage of remaining viable T cells was measured 48 hours posttreatment. Untreated T cells were used as a control (“NT”). [0048] FIGs. 8A-8B depict the results of a Coomassie stain (FIG. 8A) and an anti-His6 western blot (FIG. 8B) to detect PTD-MyrAkt ectopically expressed in E. coli transformed with a plasmid encoding a 6His-tagged form of the protein. T indicates total protein; S indicates soluble protein, and “531315” indicates the E. coli strain transformed with the plasmid encoding the PTD-MyrAkt-6His construct. E. coli transformed with a plasmid comprising a non-coding gene sequence was used as a negative control (“Neg Cntrl”). E. coli transformed with a plasmid encoding a known-molecular-weight 6His-tagged protein was used as a positive control (“Pos Cntrl”).

[0049] FIG. 9 depicts the results of a non-reducing SDS-PAGE for the detection of purified 6His-tagged PTD4-MyrAkt.

[0050] FIG. 10 depicts the results of an ex vivo experiment testing the ability of a PTD4- MyrAkt fusion protein to promote cell survival during T cell activation. Primary murine lymphocyte cells were activated for 72 hours with ionomycin and PMA in the presence of the indicated concentration of the PTD4-MyrAkt fusion protein, and the percentage of apoptotic cells was measured via 7AAD staining post-treatment. Treatment with PMA and ionomycin in the presence of denatured fusion protein was used as a control.

[0051] FIG. 11 depicts the results of an ex vivo experiment testing the ability of a PTD4- MyrAkt fusion protein to promote expansion of activated T cells in the absence of added cytokines. Primary activated murine T cells were treated for an hour at seeding with the indicated concentration of a fusion protein comprising MyrAkt and a Tat4 PTD. The cells were then washed and incubated for 48 hours in media alone. The percentage of viable cells was measured post-treatment via FACS. Treatment with medium without the fusion protein (“Media”) or with denatured fusion protein (“Denatured Protein”) were used as controls.

[0052] FIG. 12 is a bar graph summarizing the results of an ex vivo experiment testing the ability of a PTD-MyrAkt fusion protein to promote survival and proliferation of primary T cells. Primary murine CD4+ T cells were treated ex vivo with either a PTD-MyrAkt fusion protein or with IL-2 (“hIL-2”) at the indicated concentrations, and the percentage of viable T cells was measured post-treatment via FACS. T cells treated with non-supplemented medium (“Media alone”), with denatured PTD-MyrAkt (“Denatured Protein”), or with heat-inactivated IL-2 (“hIL-2 (heat inactivated)”) were used as controls.

[0053] FIG. 13 is a survival plot depicting the result of an in vivo experiment testing the anticancer effect of immune cells treated with two different cytokine-pathway fusion proteins in a syngeneic murine colorectal cancer model. Immune cells were harvested from mice 7 days postinjection with MC38 tumor cells and treated with either PTD-MyrAkt fusion protein (“PTD- MyrAkt;” 2.5 pg/mL) or with a fusion protein comprising MYC and a Tat PTD (“Tat-MYC;” 25 pg/mL). Fusion-protein-treated immune cells were administered to MC38 -tumor-bearing mice, and survival of each treatment cohort was monitored over time. Untreated, non-tumor-bearing mice (“WT”) and tumor-bearing mice that were not administered immune cells (“No-Tx”) were used as controls. X axis represents days post-MC38-injection; “Cell Rx” indicates time at which immune cells were administered.

[0054] FIG. 14 is a bar graph summarizing the results of an ex vivo experiment testing the ability of a PTD-MyrAkt fusion protein to promote survival of primary human regulatory T cells (Tregs). Approximately 100,000 affinity-purified human Tregs isolated from the peripheral blood of one of two healthy volunteers (“Nl”) or one of four rheumatoid arthritis patients (“utf- 10,” “utf-11,” “utf-14,” and “utf-16”) were cultured ex vivo for five days with either PTD- MyrAkt fusion protein, with Tat-MY C fusion protein, or with IL-2 at the indicated concentrations. The number of live Tregs was determined by incubating the culture with CCK8 reagent for 4 hours and analyzing UV optical density at 450 nm. T cells cultured in nonsupplemented medium were used as controls (“None”).

DETAILED DESCRIPTION OF THE INVENTION

[0055] Provided herein are fusion proteins having a signaling activator comprising an AKT1 polypeptide or a functional variant thereof and a protein transduction domain. Also provided herein are compositions including said proteins, as well as methods for using the proteins to modulate cytokine signaling in cells to treat a disease or disorder in a subject, and/or to prepare cell therapeutic compositions.

[0056] The function of cytokine receptors is to enable communication between cells. Cells modulate signaling through the cytokine receptors by controlling availability of a ligand and cytokine receptor expression. Once a cytokine binds to its receptor on a target cell, signaling is initiated and can result in proliferation, survival, and differentiation. Signaling from the receptor is transduced though a common set of mediator molecules. When the target cell cannot respond properly, triggering the signal by circumventing the receptor may be the key to triggering the desired activity. The fusion proteins of the disclosure allow for transient activation of cytokine signaling in cells independently of cytokine availability, receptor surface expression or function, stage of cell cycle for the target cell, or cell permissiveness to cytokine signaling. I. Definitions

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred embodiments of the compositions, methods, and materials are described herein. For the present invention, the following terms are defined below.

[0058] The singular terms “a,” “an,” and “the” include plural referents unless context makes clear otherwise. Similarly, the word “or” is intended to include “and” unless the context makes clear otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods, and materials are described below. The abbreviation, “e.g.” stems from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0059] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0060] The term “constitutively active,” as used herein in reference to a polypeptide, is understood to mean that the polypeptide is modified (e.g., comprises a truncation; comprises one or more substitutions, insertions, and/or deletions; and/or is fused to an ectopic amino acid sequence) such that the polypeptide has increased activity (e.g., increased enzymatic activity) relative to a wild-type form of the polypeptide lacking said modification(s). In some embodiments, a constitutively active polypeptide remains active or is increasingly likely to remain active regardless of inhibitory mechanisms that may be present in the surrounding environment. For example, a phosphatase-resistant form of Aktl is considered to be a constitutively active form of Aktl . For the avoidance of doubt, a wild-type polypeptide that is phosphorylated (e.g., phosphorylated Aktl) is not considered to be a “constitutively active” form of the polypeptide for the purposes of this disclosure.

[0061] “Activator,” as used herein, generally refers to the ability of a polypeptide to induce, enhance or promote the function of a given target or signaling pathway. Accordingly, a “cytokine pathway activator” refers to a polypeptide (e.g., a cytokine) that can induce, enhance, or promote signaling through cytokine pathway signaling and cause a cell to exhibit one or more properties associated with cytokine signaling.

[0062] As used herein, the term “administering,” refers to the placement of a fusion polypeptide, cell, or population of cells as described herein into a subject by a method or route that results in at least partial delivery of the agent at a desired site. For example, pharmaceutical compositions including the fusion polypeptide or population of cells described herein can be administered by any appropriate route that results in an effective treatment in the subject.

[0063] As used herein, a cell or population of cells is “autologous” to the subject from which the cell or population of cells was derived.

[0064] As used herein, a cell or population of cells is “allogeneic” to a subject that is genetically distinct from the subject from which the cell or population of cells was derived.

[0065] The term “cancer,” as used herein, generally relates to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade adjacent tissues. A “cancer cell” or “tumor cell” refers to an individual cell that is a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by abnormal growth of cells, which may be benign, premalignant, or malignant. Most cancers form tumors, but some cancers (e.g., leukemias) do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cancer cells) and tumor (tumor cells) can be used interchangeably.

[0066] As used herein, the term “cell” also refers to individual cells, cell lines, or cultures derived from such cells. A “cell type” refers to cells having a particular set of identifying characteristics. A “culture,” when use in reference to cells, refers to a composition including isolated cells of the same cell type or different cell types in a medium (e.g., liquid medium).

[0067] A “population of cells” or “cell population,” as used herein, can be and are used interchangeably and its meaning will be clear depending on the context. For example, the term “population” can be a cell culture of more than one cell having the same identifying characteristics or it can be a culture of multiple one cell types having different identifying characteristics, e.g., a population in one context may be a sub-population in another context. The term “sub-population” or “portion” of cells refers to a subset of a cell culture or population when used to describe certain cell types within the cell culture or cell population. [0068] As used herein, “contacting” refers to combining two or more agents (e.g., fusion polypeptides, combining agents and cells), or combining two populations of different cells, which can be achieved in many ways. Contacting can occur in vitro, e.g. , mixing a fusion polypeptide with a population of cells in a test tube or growth medium. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by co-expression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate. Contacting may also occur ex vivo e.g. , contacting a fusion polypeptide with a tissue or organ. A population of cells may be contacted with a fusion polypeptide by culturing the population of cells in the presence of the fusion polypeptide for a period of time, such as for two or more days.

[0069] An “engineered,” “modified,” and “genetically modified” cell or cells, as used herein, refers to a cell or cell that includes added, deleted or altered genetic material (e.g., DNA or RNA) as compared to a non-engineered or modified cell or cells.

[0070] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a nucleic acid (e.g., a gene, a DNA molecule, or a mRNA) to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g. , a rRNA, tRNA, or mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleic acid encoding a fusion polypeptide” includes nucleic acids having nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0071] As used herein, “enriching” or “isolating” a population of cells refers to producing a population in which the relative proportion of cells of a particular type has increased in comparison with a previous population of cells (e.g., cells exhibiting one or more properties associated with cytokine signaling).

[0072] To “expand,” “specifically expand” or “preferentially expand” a cell or population of cells means to culture the cell(s) so that the cell(s) proliferate to greater numbers. The term can also refer to culturing a sub-population or portion of cells so that a particular cell type(s) proliferates to numbers greater than other cell types in the population.

[0073] The terms “express” and “expression” mean allowing or causing the information in a gene or polynucleotide sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. The expression product itself, e.g. the resulting protein, may also be said to be “expressed.” In the context of a cell, expression may be characterized as intracellular, extracellular or membrane. The term “intracellular” means inside a cell. The term “extracellular” means outside a cell. The term “membrane” means at least a portion of a polypeptide is contacting or embedded in a cell membrane. The term “cytoplasmic” means residing within the cell membrane, outside the nucleus.

[0074] As used herein, the term “functional fragment or variant,” when used in reference to a peptide, polypeptide or protein, is intended to refer to a portion and/or a derivative of the peptide, polypeptide, or protein that retains some or all of the activity (e.g., kinase activity) of the original peptide, polypeptide, or protein from which the fragment or variant was derived. These functional fragments or variants can, for example, be truncations (e.g., C-terminal or N- terminal truncations) of a peptide, polypeptide, or protein. Functional fragments or variants can also include one or more amino acid substitutions, such as an amino acid substitution described herein, and/or a deletion of one or more amino acid residues.

[0075] A “fusion” protein or polypeptide refers to a polypeptide having at least two heterologous polypeptides and optionally a linking sequence or a linkage to operatively link the two heterologous polypeptides into one continuous polypeptide. The two heterologous polypeptides linked in a fusion protein are typically derived from two independent sources, and therefore a fusion polypeptide includes two linked polypeptides not normally found linked in nature. A fusion protein of the disclosure may include, e.g., an AKT1 polypeptide and a protein transduction domain.

[0076] The terms “increased,” “increase,” or “enhance” are all used herein to mean an increase by a measurable amount as compared to a reference level. In some embodiments, the terms “increased,” “increase,” or “enhance,” when used to refer to cytokine pathway signaling, can mean an increase of at least 10% signaling as compared to a reference level. [0077] An “infection” or “infectious disease,” as used herein, refers to a disease (e.g., the common cold) that can be transmitted between people or between organisms and is caused by microbial matter (microbial agents).

[0078] As used herein, the term “isolated,” when used in reference to a molecule (e.g., peptide, polypeptide, protein, nucleic acid, polynucleotide, vector), a cell or a population of cells, refers to a molecule, cell or population of cells that is substantially free of at least one component with which the referenced molecule, cell or population of cells is found in nature. The term includes a molecule, cell, or population of cells that is removed from some or all components with which it is found in its natural environment. Therefore, an isolated molecule, cell or population of cells can be partly or completely separated from other substances with which it is found in nature or with which it is grown, stored or subsisted in non-naturally occurring environments.

[0079] A “ligand” or “cytokine,” as used herein, refers to a molecule that is recognized by a cytokine receptor. Exemplary ligands include, but are not limited to, IL-1, IL-2, IL-4, IL-5, IL- 6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-17, IL-21, IL-22, IL-23, IL-27, IL-35, a Toll-like receptor (TLR) ligand, TNL-a, IFNa, IFN[3, IFNy, G-CSF, GM-CSL, M-CSL, erythropoietin (EPO), oncostatin, MCP-1, nitrogen oxide (NO), growth hormone (GH), leukemia inhibitory factor (LIP), leptin, granzyme B (GZMB), macrophage inflammatory protein (MIP-la), vascular endothelial growth factor (VEGF), stem cell factor (SCF), and ciliary neurotrophic factor (CNTF). Binding of a cytokine or ligand to the receptor results in activation of a signaling cascade associated with the receptor (e.g., ligand-mediated activation).

[0080] ‘ ‘Kinase activity” as used herein, refers to the ability of an enzyme to add a phosphate group to a target protein at a tyrosine residue, serine residue, and/or threonine residue.

[0081] The term “operatively linked,” when used in reference to a nucleic acid encoding a fusion polypeptide described herein, refers to connection of a nucleotide sequence encoding a fusion polypeptide described herein to another nucleotide sequence (e.g. , a promoter) is such a way as to allow for the connected nucleotide sequences to function (e.g., express the fusion polypeptide in a cell).

[0082] A “pharmaceutical composition,” as used herein, refers to a mixture of a fusion protein or cell or population of cells described herein, with other pharmaceutically acceptable chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. In certain instances, the pharmaceutical composition facilitates administration of the fusion polypeptide or cell or population of cells to an individual. In certain embodiments of practicing the methods of treatment or use provided herein, therapeutically effective amounts of a fusion polypeptide, cell, or population of cells described herein are administered in a pharmaceutical composition to a subject having a disorder, disease, or condition to be treated. In specific embodiments, the subject is a human.

[0083] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0084] As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms also refer to proteins or polypeptides that include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. Proteins and polypeptides are often relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.

[0085] As used herein, the term “promoter,” when used in reference to a nucleic acid encoding a fusion polypeptide described herein, refers to a nucleotide sequence where transcription of a linked open reading frame (e.g., a nucleotide sequence encoding a fusion polypeptide) by an RNA polymerase begins. A promoter sequence can be located directly upstream or at the 5’ end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to a promoter sequence and initiate transcription. Promoter sequences define the direction of transcription and indicate which DNA strand will be transcribed, e.g., the sense strand.

[0086] A “progenitor cell” as used herein, in general, can be any cell in a cell differentiation pathway that is capable of differentiating into a more mature cell. Thus, a progenitor cell can be a pluripotent cell, or it can be a partially differentiated multipotent cell, or reversibly differentiated cell.

[0087] As used herein, a “peptide transduction domain (PTD)” (also termed Cell Penetrating peptide or “CPP”), is a peptide that can be used to facilitate uptake of a macromolecule (e.g. , a fusion polypeptide) that is attached to the peptide. PTDs can be used to deliver a wide variety of macromolecular cargo, including peptides, proteins, PNAs, and DNA vectors. In some embodiments, a PTD can be used to deliver macromolecular cargo into 100% of primary and transformed cells. In some embodiments, a PTD can be used to deliver macromolecular cargo into most, if not all, tissues. The PTD may be a cationic PTD or a hydrophobic PTD, and may be cell-specific or a general PTD.

[0088] As used herein, the term “recombinant,” with respect to a nucleic acid, such as a nucleic acid having a gene that encodes a protein or polypeptide (e.g., a fusion polypeptide described herein), refers to: a nucleic acid that has been artificially supplied to a biological system; a nucleic acid that has been modified within a biological system, or a nucleic acid whose expression or regulation has been manipulated within a biological system. The recombinant nucleic acid can be supplied to the biological system, for example, by introduction of the nucleic acid into genetic material of a host cell, such as by integration into a chromosome, or as non- chromosomal genetic material such as a plasmid. A recombinant nucleic acid that is introduced into or expressed in a host cell may be a nucleic acid that comes from a different organism or species than the cell, or may be a synthetic nucleic acid, or may be a nucleic acid that is also endogenously expressed in the same organism or species as the cell. A recombinant nucleic acid that is also endogenously expressed in the same organism or species as the cell can be considered heterologous if: the sequence of the recombinant nucleic acid is modified relative to the endogenously expressed sequence, the sequence of a regulatory region such as a promoter that controls expression of the nucleic acid is modified relative to the regulatory region of the endogenously expressed sequence, the nucleic acid is expressed in an alternate location in the genome of the cell relative to the endogenously expressed sequence, the nucleic acid is expressed in a different copy number in the cell relative to the endogenously expressed sequence, and/or the nucleic acid is expressed as non-chromosomal genetic material such as a plasmid in the cell.

[0089] As used herein, the term “self-renewal” refers to the ability of a stem cell to produce daughter stem cells with the same phenotype, characteristics, and functional potential as the original stem cell. In particular, self-renewal, as used herein, is defined as the ability to continue proliferation while maintaining an undifferentiated multi -potent stem cell state.

[0090] As used herein, the term “substitution” refers to a replacement of an amino acid occupying a position with a different amino acid. A “conservative substitution” refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Alternatively, the term “non-conservative substitution” refers to the replacement of one amino acid residue for another such that the replaced residue is going from one family of amino acids to a different family of residues. Genetically encoded amino acids can be divided into four families: (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg (R), His (H); (3) nonpolar (hydrophobic) = Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic = Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic = Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar = Asn (N), Gin (Q), Ser (S), Thr (T). In alternative fashion, the amino acid repertoire can be grouped as (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg (R), His (H), and (3) aliphatic = Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl; (4) aromatic = Phe (F), Tyr (Y), Trp (W); (5) amide = Asn (N), Glu (Q); and (6) sulfur-containing = Cys (C) and Met (M) (see, for example, Biochemistry, 4th ed., Ed. by L. Stryer, WH Freeman and Co., 1995, which is incorporated by reference herein in its entirety).

[0091] As used herein, the term “sufficient amount of time” or “sufficient period of time” refers to the time required by a cell to exhibit a cellular response (e.g. , activation of cytokine pathway signaling) following a cell stimulus. The amount of time required for eliciting the cellular response may depend on the particular cell type, culture conditions and the stimulating agent (e.g., a fusion polypeptide described herein). For instance, the amount of time may correspond to minutes, hours, or days depending on the particular stimuli, cell type, culture condition and desired cellular response.

[0092] The terms “treating” or “treatment,” as used herein, refer to a therapeutic intervention that results in any observable beneficial effect on a sign or symptom of a disease or pathological condition after it has begun to develop. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. [0093] As used herein, the term “variant,” when used in reference to any peptide, polypeptide, protein, nucleic acid, or polynucleotide described herein, refers to a sequence of amino acids or nucleotides having at least one substitution, deletion or insertion as compared to a parent sequence. With regards to a polypeptide, a substitution means replacement of the amino acid occupying a position with a different amino acid, a deletion means removal of an amino acid occupying a position and an insertion means addition of amino acids adjacent to an amino acid occupying a position. A variant sequence of amino acids or nucleotides is not naturally occurring. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a modified variant of a wild-type sequence or homolog thereof.

[0094] As used herein, the term “vector” refers to a compound and/or composition that transduces, transforms, or infects a host cell, thereby causing the host cell to express nucleic acids and/or proteins other than those native to the host cell, or in a manner not native to the host cell. Vectors can be constructed to include a fusion polypeptide described herein, encoded by a nucleotide sequence operably linked to expression control sequences (e.g., promoter) that are functional in the host cell (“expression vector”). Expression vectors applicable for use in the host cells described herein include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more recombinant or exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of a recombinant or exogenous nucleic acid encoding an enzyme or protein involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid or its corresponding gene product (e.g. , enzyme or protein). It is understood by those skilled in the art that the recombinant or exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as described herein.

[0095] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0096] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0097] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of the disclosure unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

II. Fusion Polypeptides

[0098] In some aspects of the disclosure, provided herein are fusion proteins having a signaling activator comprising an AKT1 polypeptide or a functional fragment or variant thereof, and a protein transduction domain (PTD).

[0099] The term “AKT1” is used herein to refer to an AKT1 polypeptide. AKT1 (UniProt # P31749 for human AKT1) is also known as protein kinase B, PKB, and AKT oncogene. AKT1 belongs to the AKT subfamily of serine/threonine kinases that contain SH2 (Src homology 2- like) domains, and has a role in cell growth, survival, and metabolism. AKT1 contains an N- terminal pleckstrin homology (PH) domain, a kinase domain, and a C-terminal regulatory domain. Growth factor signaling via receptor tyrosine kinases activates class I phosphoinositide 3-kinases (PI3Ks), which phosphorylate the lipid second messenger phosphatidylinositol-4, 5- bisphosphate (PIP2), thereby converting it into phosphatidylinositol-3,4,5-trisphosphate (PIP3). Plasma-membrane-associated PIP3 binds to the PH domain of AKT1, which alleviates PH- domain-mediated autoinhibition of the protein. AKT1 is activated by phosphorylation at two key residues: T308 and S473. Localization of Akt to PIP3 in the plasma membrane promotes phosphorylation at its T308 residue by phosphoinositide-dependent kinase 1 (PDK1) and at its S473 residue by mammalian target of rapamycin complex 2 (mT0RC2), which leads to activation and downstream signaling. Activated AKT1 phosphorylates a diverse array of over 100 substrates. AKT1 can also be dephosphorylated, e.g., by the activity of phosphatases such as Protein Phosphatase 1 (PPI).

[00100] In some embodiments, the AKT polypeptide is a human AKT1 polypeptide. In some embodiments, the AKT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, or a functional fragment or variant thereof.

[00101] In some embodiments, the AKT1 polypeptide is a variant of a naturally occurring AKT1 polypeptide (e.g., a variant of human AKT1). In some embodiments, the variant AKT1 polypeptide has an amino acid sequence is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant AKT1 polypeptide is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a portion of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant AKT1 polypeptide, or the functional fragment thereof, has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more activity relative to the wildtype AKT1 polypeptide. In some embodiments, the variant AKT1 polypeptide, or the functional fragment thereof, has at least 10% activity relative to the wild-type AKT1 polypeptide. In some embodiments, the functional fragment has at least 25% activity relative to the wild-type polypeptide. In some embodiments, the variant AKT1 polypeptide, or the functional fragment thereof, has at least 50% activity relative to the wild-type AKT1 polypeptide.

[00102] In some embodiments, the AKT1 polypeptide is a fragment of a naturally occurring polypeptide, or a variant thereof. For example, in some embodiments, the AKT1 polypeptide comprises at least 100, 150, 200, 250, 300, 350, 400, or 450 consecutive amino acids present in a naturally occurring AKT1 polypeptide. For example, in some embodiments, the AKT1 polypeptide comprises the residues 131 through 477 ofhuman AKT1 (z.e., SEQ ID NO: 2). In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 2.

[00103] In some embodiments, the AKT1 polypeptide comprises the residues 130 through 477 ofhuman AKT1 (z.e., SEQ ID NO: 3). In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 3.

[00104] In some embodiments, the AKT1 polypeptide comprises the residues 1 through 477 ofhuman AKT1. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to residues 1 through 477 of human AKT1. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of residues 1 through 477 ofhuman AKT1. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of residues 1 through 477 ofhuman AKT1. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 4.

[00105] In some embodiments, the AKT1 polypeptide is constitutively active. For example, in some embodiments, the AKT1 polypeptide has at least 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more activity relative to wild-type AKT1. In some embodiments, the constitutively active AKT1 polypeptide is phosphatase resistant. Examples of constitutively active AKT1 polypeptides include, for example, AKT1 polypeptides comprising a myristoylation sequence, AKT1 polypeptides comprising certain amino acid substitutions that increase the protein’s activity, AKT1 polypeptides comprising a deletion of the PH domain of the protein, as well as AKT1 polypeptides having any combination of the foregoing features.

[00106] In some embodiments, the AKT1 polypeptide comprises a substitution and/or an amino acid sequence that facilitates sequestration of the fusion protein at the plasma membrane. For example, in some embodiments, the AKT1 polypeptide comprises an amino acid sequence having a myristoylation sequence, e.g., a Src myristoylation sequence or a Gag myristoylation sequence. In some embodiments, the Src myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 5 (MGSSKSKPKDPSQRSE), or a functional fragment or variant thereof. In some embodiments, the Src myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 6 (MGSSKSKPKSR), or a functional fragment or variant thereof. In some embodiments, the Gag myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 7 (

MGQTVTTPLSLTLDHWSEVRTRAHNQGVEVRKKKWVTLCEAEWVIFTMNVGWPREG TFSLDNISQVEKKIFAPGPYGHPDQVPYITTWRSLATDPPSWVRPFLPPPTKPPTPLPQPLS PQPSAPPTSSLYPVLPKSDPPKPPVLPPDP), or a functional fragment or variant thereof. In some embodiments, the myristoylation sequence (e.g., the Src myristoylation sequence) is N- terminally linked to the AKT1 polypeptide. Myristoylated forms of AKT1 are described, for example, in Kohn et al. (1998) Cell Biology and Metabolism 273(19): 11937-11943 and in Ahmed et al. (1993) Oncogene 8: 1957-1963, which are herein incorporated by reference in their entireties. In some embodiments, the myristoylation sequence is fused to full-length AKT1, e.g., an AKT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the myristoylation sequence is fused to a fragment of AKT1, e.g., a fragment lacking the PH domain of the protein as described herein, e.g., a fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3. In some embodiments, the myristoylation sequence is fused to an AKT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[00107] In some embodiments, the myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the myristoylation sequence comprises an amino acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 5. In some embodiments, the myristoylation sequence comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the myristoylation sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the myristoylation sequence comprises an amino acid sequence at least 80% or 90% identical to SEQ ID NO: 6. In some embodiments, the myristoylation sequence comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the myristoylation sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the myristoylation sequence comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the myristoylation sequence comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the myristoylation sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 7.

[00108] In some embodiments, the AKT1 polypeptide comprises an amino acid substitution that renders the protein constitutively active. For example, in some embodiments, the AKT1 polypeptide comprises a substitution associated with an oncogenic or cancer- associated form of the protein. In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a glutamate residue at a position corresponding to position 17 of wild-type human AKT1 (E 17), e.g., wherein the glutamate residue is substituted by lysine (E17K). In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a leucine residue at a position corresponding to position 52 of wild-type human AKT1 (L52), e.g., wherein the leucine residue is substituted by arginine (L52R). In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a cysteine residue at a position corresponding to position 77 of wild-type human AKT1 (C77), e.g., wherein the cysteine residue is substituted by phenylalanine (C77F). In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a glutamine residue at a position corresponding to position 79 of wild-type human AKT1 (Q79), e.g., wherein glutamine residue is substituted by lysine (Q79K). In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a glycine residue at a position corresponding to position 171 of wild-type human AKT1 (G171), e.g., wherein the glycine residue is substituted by arginine (G171R). In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises any combination of the foregoing substitutions. Point mutations that increase the activity of AKT1 are known in the art and are described, for example, in the following references, which are incorporated herein by reference in their entirety: Kumar et al. (2013) PLoS One 8: e64364; Parikh et al. (2012) Proc. Natl. Acad. Sci 109: 19368-19373; and Yi et al. (2013) Oncotarget 4: 29-34.

[00109] In some embodiments, the AKT1 polypeptide does not comprise the PH domain of wild-type AKT1, or comprises a deletion of part or all of the PH domain of AKT1. For example, in some embodiments, the AKT1 polypeptide comprises a deletion of the consecutive series of amino acids corresponding to residues 4 through 129 or 4 through 130 of SEQ ID NO: 1. In some embodiments, the AKT1 polypeptide is a fragment of a naturally occurring AKT1 polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2 or 3. AKT1 polypeptides comprising deletions of the PH domain are described, for example, in Parikh et al. (2012).

[00110] Constitutively active AKT1 polypeptides can have one or more of the foregoing features that increase the activity of the polypeptide relative to the wild-type counterpart. For example, in some embodiments, the AKT1 polypeptide comprises both a myristoylation sequence (e.g., a Src myristoylation sequence) and a substitution associated with increased AKT1 activity (e.g., E17K, L52R, C77F, Q79K, G171R, or any combination thereof). In some embodiments, the AKT1 polypeptide comprises both a myristoylation sequence (e.g., a Src myristoylation sequence) and a deletion of the PH domain. In some embodiments, the AKT1 polypeptide comprises both a substitution associated with increased AKT1 activity (e.g., E17K, L52R, C77F, Q79K, G171R, or any combination thereof) and a deletion of the PH domain. In some embodiments, the AKT1 polypeptide comprises (1) a myristoylation sequence (e.g., a Src myristoylation sequence), (2) a substitution associated with increased AKT1 activity (e.g., E17K, L52R, C77F, Q79K, G171R, or any combination thereof), and (3) a deletion of the PH domain.

[00111] In some embodiments, the variant AKT1 polypeptide, or the functional fragment thereof, further comprises one or more substitutions that prevent AKT-induced neoplasia in a subject. For example, in some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a threonine residue at a position corresponding to position 308 of wild-type human AKT1 (T308). In some embodiments, the threonine at position 308 comprises a non-conservative amino acid substitution, e.g., a substitution with a negatively charged amino acid, e.g., aspartate (T308D). In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a serine residue at a position corresponding to position 473 of wild-type human AKT1 (S473). In some embodiments, the serine at position 473 comprises a non-conservative amino acid substitution, e.g., a substitution with a negatively charged amino acid, e.g., aspartate (S473D). In some embodiments, the AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of both T308 and S473, e.g, wherein both residues are substituted by aspartate. In some embodiments, the AKT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 8.

[00112] In some embodiments, the AKT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 9. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 9.

[00113] In some embodiments, the AKT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 10. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 10.

[00114] In some embodiments, the AKT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 53. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 53.

[00115] In some embodiments, the AKT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 61. In some embodiments, the AKT1 polypeptide comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the AKT1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 61. Table 1: Exemplary AKT1 Polypeptides

[00116] In some embodiments, the fusion protein comprising an AKT1 polypeptide or functional fragment or variant thereof as described herein (e.g., as in Table 1) is capable of penetrating a plasma membrane of a cell. In some embodiments, the ability of the fusion polypeptide to penetrate the plasma membrane may be conferred by the PTD.

[00117] The fusion protein comprising an AKT1 polypeptide or functional fragment or variant thereof as described herein (e.g. , as in Table 1) can include any PTD known in the art and that has been demonstrated to facilitate uptake by a cell of heterologous molecules linked to the PTD. The PTD may facilitate uptake through a process referred to a micropinocytosis, a form of endocytosis performed by all cells.

[00118] The discovery of several proteins which could efficiently pass through the plasma membrane of eukaryotic cells has led to the identification of a class of proteins from which PTDs have been derived. Some of the best characterized of these proteins are the Drosophila homeoprotein antennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3: 1121-34, 1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-8, 1991; Le Roux et al., Proc. Natl.

Acad. Sci. USA, 90:9120-4, 1993), the herpes simplex virus structural protein VP22 (Elliott and O’Hare, Cell 88:223-33, 1997), the HIV-1 transcriptional activator TAT protein (Green and Loewenstein, Cell 55: 1179-1188, 1988; Frankel and Pabo, Cell 55: 1189-1193, 1988), and more recently the cationic N-terminal domain of prion proteins. Not only can these proteins pass through the plasma membrane but the attachment of other proteins, such as the enzyme [3- galactosidase, can stimulate the cellular uptake of these complexes. Such chimeric proteins are present in a biologically active form within the cytoplasm and nucleus. Characterization of this process has shown that the uptake of these fusion polypeptides is rapid, often occurring within minutes, in a receptor independent fashion. Moreover, the transduction of these proteins does not appear to be affected by cell type and can efficiently transduce approximately 100% of cells in culture with no apparent toxicity (Nagahara et al., Nat. Med. 4: 1449-52, 1998). In addition to full-length proteins, PTDs have also been used successfully to induce the intracellular uptake of DNA (Abu-Amer, supra), antisense oligonucleotides (Astriab-Fisher et a/., Pharm. Res., 19:744- 54, 2002), and small molecules (Polyakov et al., Bioconjug. Chem. 11:762-71, 2000). Thus, fusion of a PTD with a heterologous molecule (e.g., a cytokine pathway activator) is sufficient to cause their transduction into a variety of different cells in a concentration-dependent manner. Exemplary PTDs are summarized in Table 2 below.

Table 2: Exemplary Protein Transduction Domains

[00119] In some embodiments, the fusion protein comprises an AKT1 polypeptide or functional fragment or variant thereof (e.g. , Table 1) and a cationic PTD. Cationic PTDs track into lipid raft endosomes carrying with them their linked cargo and release their cargo into the cytoplasm by disruption of the endosomal vesicle. Examples of PTDs include AntHD, TAT, VP22, cationic prion protein domains and functional fragments thereof. In some embodiments, the cationic PTD is derived from a VP- 16 peptide, an antennapedia peptide, a PTD-5 peptide, a polylysine peptide, a polyarginine peptide, an HIV VPR peptide, or an HIV Tat peptide, or a variant thereof. In some embodiments, the cationic PTD has an amino acid sequence set forth in any one of SEQ ID NOs: 11-15 or 19-20. In some embodiments, the AKT1 polypeptide is a constitutively active form of AKT1, as described herein. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-10, 53, or 61, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 24-26.

[00120] In some embodiments, the fusion protein comprises an AKT1 polypeptide or functional fragment or variant thereof described herein (e.g., Table 1) and a hydrophobic PTD. In some embodiments, the hydrophobic PTD is derived from a transportan peptide, a MAP peptide, a TP 10 peptide, or a variant thereof. In some embodiments, the cationic PTD has an amino acid sequence set forth in any one of SEQ ID NOS: 16-18. In some embodiments, the AKT1 polypeptide is a constitutively active form of AKT1, as described herein. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-10, 53, or 61, or a functional fragment or variant thereof.

[00121] In some embodiments, the fusion protein comprises an AKT1 polypeptide or functional fragment or variant thereof described herein (e.g. , Table 1) and a cell-type specific PTD. A cell-type specific PTD may direct uptake of the fusion polypeptide by a particular cell type(s). The cell-type specific PTD may also be combined with any additional domain or modification that increases cell-type specificity of the uptake. In some embodiments, the AKT1 polypeptide is a constitutively active form of AKT1, as described herein. In some embodiments, the AKT1 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-10, 53, or 61, or a functional fragment or variant thereof.

[00122] In some embodiments, the fusion protein further has a peptide linker linking the AKT1 polypeptide and the PTD. Peptide linkers that can be used in the fusion proteins of the disclosure may be from about 1 to 20 amino acids in length, e.g., 1 to 10, 1 to 15, 1 to 10, 1 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 amino acids in length. The linker sequence is generally flexible so as not to hold the fusion protein in a single rigid conformation. The linker sequence can be used, e.g, to space the PTD domain from the AKT1 polypeptide. For example, the peptide linker sequence can be positioned between the PTD and AKT1 polypeptide to provide molecular flexibility. The length of the linker moiety is chosen to optimize the biological activity of the fusion protein and can be determined empirically without undue experimentation. The linker can, additionally or alternatively, be interposed between the AKT1 polypeptide and one or more protein tags, e.g., a tag described hereinbelow. Exemplary linkers include Gly-Gly-, GGGGS (SEQ ID NO: 21), GKSSGSGSESKS (SEQ ID NO: 22), GSTSGSGKSSEGKG (SEQ ID NO: 23), KGELNSKLE (SEQ ID NO: 52), and STAMA (SEQ ID NO: 63). Linking moieties are described, for example, in Huston et al. , Proc. Nat ’I Acad. Sci. 85:5879, 1988; Whitlow et al., Protein Engineering 6:989, 1993; and Newton et al., Biochemistry 35:545, 1996. Other suitable peptide linkers include those described in, e.g., U.S. Pat. Nos. 4,751,180 and 4,935,233.

[00123] In some embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 24-26.

[00124] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 24, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 24. In some embodiments, the fusion protein comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO:

24.

[00125] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 25, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 25. In some embodiments, the fusion protein comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the fusion protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO:

25.

[00126] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 26, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26. In some embodiments, the fusion protein comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the fusion protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO:

26.

[00127] Additionally, in some embodiments, the fusion protein may include a tag. For example, the fusion polypeptide may further include a tag that facilitates identification and/or purification of the fusion polypeptide, e.g., a V5 epitope tag (GKPIPNPLLGLDST; SEQ ID NO: 55) and/or a 6-His tag (HHHHHH; SEQ ID NO: 56).

[00128] The AKT1 polypeptide and the PTD, as well as any additional linkers or tags, can be organized in nearly any fashion provided that the fusion protein described herein retains its intended function (e.g., activation of cytokine pathway signaling). In some embodiments, the PTD is N-terminally fused to the AKT1 polypeptide. In some embodiments wherein the fusion protein comprises a myristoylation sequence, the fusion protein comprises, in the N-to-C- terminal direction: the PTD, the myristoylation sequence, and the AKT1 polypeptide. For example, the fusion protein may comprise, in the N-to-C-terminal direction: a PTD of any one of SEQ ID NOs: 11-20, a myristoylation sequence of any one of SEQ ID NOs: 5-7, and an AKT1 polypeptide sequence of any one of SEQ ID NOs: 1-4 and 8. In particular embodiments, the fusion protein may comprise, in the N-to-C-terminal direction: a PTD of SEQ ID NO: 12, a myristoylation sequence of SEQ ID NO: 5 or 6, and an AKT1 polypeptide sequence of SEQ ID NO: 2. In particular embodiments, the fusion protein may comprise, in the N-to-C-terminal direction: a PTD of SEQ ID NO: 12 and an AKT1 polypeptide sequence of SEQ ID NO: 9. In particular embodiments, the fusion protein may comprise, in the N-to-C-terminal direction: a PTD of SEQ ID NO: 12, a myristoylation sequence of SEQ ID NO: 5 or 6, and an AKT1 polypeptide sequence of SEQ ID NO: 4 or 8. In particular embodiments, the fusion protein may comprise, in the N-to-C-terminal direction: a PTD of SEQ ID NO: 12 and an AKT1 polypeptide sequence of SEQ ID NO: 10.

[00129] In some embodiments, the PTD is C-terminally fused to the AKT1 polypeptide. In some embodiments, the fusion protein may comprise, in the N-to-C-terminal direction, the myristoylation sequence, the AKT1 polypeptide, and the PTD. For example, the fusion protein may comprise, in the N-to-C-terminal direction: a myristoylation sequence of any one of SEQ ID NOs: 5-7, an AKT1 polypeptide sequence of any one of SEQ ID NOs: 1-4 and 8, and a PTD of any one of SEQ ID NOs: 11-20. In particular embodiments, the fusion protein may comprise, in the N-to-C-terminal direction, the myristoylation sequence of SEQ ID NO: 6, the AKT1 polypeptide sequence of SEQ ID NO: 8, and the PTD of SEQ ID NO: 12. [00130] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 27, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 27. In some embodiments, the fusion protein comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the fusion protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO:

27.

[00131] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 28, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28. In some embodiments, the fusion protein comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the fusion protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO:

28.

[00132] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 29, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 29. In some embodiments, the fusion protein comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO:

29.

[00133] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 64, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 64. In some embodiments, the fusion protein comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the fusion protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 conservative substitutions relative to the amino acid sequence of SEQ ID NO: 64.

[00134] In some embodiments, the fusion protein comprising an AKT1 polypeptide (e.g., Table 1) and a PTD described herein (e.g., Table 2) is capable of inducing activation of cytokine pathway signaling. Without wishing to be bound by theory, upon entry into the cytoplasm of a cell, the fusion polypeptide is understood to be able to modulate effectors of a cytokine pathway. By bypassing the plasma membrane and directly activating cytokine pathway signaling, the fusion protein can activate signaling in a cell downstream of a cytokine receptor, thereby bypassing the need for the signaling through cytokine receptor. Activation of cytokine pathway signaling would lead to a cell exhibiting one or more properties associated with cytokine receptor stimulation, even in the absence of a cytokine. In some embodiments, the activation of the cytokine pathway in a cell occurs independently of ligand-mediated activation of the cytokine pathway. In some embodiments, the ligand is selected from IL-2, IL-4, IL-7, and IL-15. Cytokine pathway activation by the fusion protein would subside upon turnover of the fusion protein in the cell.

[00135] Exemplary fusion proteins of the disclosure are shown in Table 3 below. In some embodiments, the fusion protein having an AKT1 polypeptide and a PTD has an amino acid sequence corresponding to any of SEQ ID NOs: 24-40 and 64.

Table 3: Exemplary Fusion Proteins

a. Recombinant Nucleic Acids

[00136] In some embodiments, provided herein is a recombinant nucleic acid that includes a nucleotide sequence encoding a fusion protein of the disclosure. Exemplary nucleic acids encoding exemplary fusion proteins described herein (e.g., Table 3) are summarized in Table 4 below. Table 4: Exemplary Nucleic Acids Encoding a Fusion Protein

[00137] In some embodiments, the recombinant nucleic acid includes a promoter operatively linked to the nucleic acid sequence encoding the fusion protein. Such a promoter can express the fusion protein in a host cell. The promoter may be a constitutive promoter. Alternatively, the promoter may be an inducible promoter. Moreover, the promoter may be a weak or a strong promoter, depending on the level of expression to be achieved. Suitable prokaryotic promoters are, for example, the tetracycline responsive promoter, the lacUV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter. In addition, the recombinant nucleic acid may further include one or more regulatory elements. For instance, the recombinant nucleic acid may include, without limitation, an enhancer element, a transcription termination signal, a translation start signal, a polyadenylation signal, or any other signal required for expression of the encoded fusion protein in a given host cell.

[00138] A recombinant nucleic acid encoding a fusion protein described herein also have at least a certain sequence identity to a nucleotide sequence described herein. Accordingly, in some aspects, a recombinant nucleic acid encoding a fusion protein has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, or is identical, to a nucleic acid described herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence described herein. b. Vectors

[00139] In some embodiments, provided herein is a vector comprising a nucleic acid (e.g. , a recombinant nucleic acid) described herein. In some embodiments, the vector comprises a recombinant nucleic acid encoding a fusion protein described herein (e.g., as set forth in Table 4). In some embodiments, the vector is an expression vector. In some embodiments, the vector is a prokaryotic expression vector. In some embodiments, the vector is a eukaryotic expression vector. In some embodiments, the vector is a mammalian expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector has double stranded DNA. In some embodiments, the vectors provided herein may be used to construct a host strain. In some embodiments, constructing a host cell can include, among other steps, introducing a vector described herein into a cell, for example, that is capable of expressing an amino acid sequence encoded by the vector. Vectors described herein can be introduced stably or transiently into a cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. Additional methods are described herein, any one of which can be used in the method described herein. For exogenous expression in E. coli or other prokaryotic cells, some recombinant nucleic acid can further encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, a fusion polypeptide can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into a recombinant nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the fusion polypeptide.

[00140] An expression vector or vectors can be constructed to include one or more recombinant nucleic acids encoding a fusion protein described herein operably linked to an expression control sequence functional in the host cell. Expression vectors applicable for use in the host cells provided herein include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more recombinant nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of recombinant nucleic acid sequences encoding a fusion polypeptide provided herein can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the recombinant nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as described herein.

[00141] A vector or expression vector can also be used to express an encoded nucleic acid to produce an encoded polypeptide by in vitro transcription and translation. Such a vector or expression vector will comprise at least a promoter, and includes the vectors described herein above. Such a vector for in vitro transcription and translation generally is double stranded DNA. Methods of in vitro transcription and translation are well known to those skilled in the art (see Sambrook et al. , Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New Y ork (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). Kits for in vitro transcription and translation are also commercially available (see, for example, Promega, Madison, WI; New England Biolabs, Ipswich, MA; Thermo Fisher Scientific, Carlsbad, CA). c. Cells

[00142] In some embodiments, provided herein is a cell (e.g., a host cell) having a vector described herein having a recombinant nucleic acid described herein. Also provided is a cell (e.g., a host cell) having a recombinant nucleic acid described herein (e.g., Table 4). In some embodiments, the nucleic acid is integrated into a chromosome of the host cell. In some embodiments, the integration is site-specific. In other embodiments, the recombinant nucleic acid is present in the cell, but not integrated into a chromosome of the host cell. In some embodiments, the recombinant nucleic acid is expressed. In some embodiments, provided herein is a host cell having a fusion protein as described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2).

[00143] In some embodiments, the cell may be a prokaryotic cell. For instance, the prokaryotic cell may be a bacterial cell. Exemplary bacterial cells include, but are not limited to cells from Escherichia (e.g., E. coli). Enterohacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans). Shigella, as well as Bacilli such as B. subtilis and B. lichenifarmis. and, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[00144] In some embodiments, the cell may be a eukaryotic cell. For instance, the eukaryotic cell may be a yeast or fungal cell, an insect cell, a plant cell, an algae, or a mammalian cell.

[00145] In some embodiments, the eukaryotic cell is a yeast or fungal cell. Exemplary yeast or fungi include Saccharomyces cerevisiae. Schizosaccharomyces pombe, Kluyveromyces (e.g., K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, and K. marxianus)' , Pichia pastoris, Candida; Trichoderma, Neurospora, Schwanniomyces such as Schwanniomyces occideniaUs. Penicillium, Tolypocladium, and Aspergillus such as A. nidulans and A. niger.

[00146] In some embodiments, the eukaryotic cell is an insect cell. Exemplary insect cells include those from Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),), Aedes albopictus (mosquito),), Drosophila melanogaster (fruit fly), and Bombyx mori.

[00147] In some embodiments, the eukaryotic cell is a mammalian cell. Without limitation, examples of mammalian cells include those from cell lines corresponding to, monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. , J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells

(TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; human hepatoma cells (Hep G2); Chinese hamster ovary (CHO) cells, including DHFR" CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cells such as NSO and Sp2/0. In addition, the cell may correspond to a cell from a commercially available cell line (see, for example, the American Type Culture Collection (ATCC; Manassas VA); Life Technologies, Carlsbad CA).

[00148] In some embodiments, the cell is a primary cell from a subject, e.g., a human. In some embodiments, the primary cell is an immune cell, e.g., a T cell.

[00149] Conventional gene transfer methods can be used to introduce vectors and recombinant nucleic acids described herein into cells (e.g. , immune cells) or tissues. In some embodiments, the vectors or recombinant nucleic acids are introduced into cells in vivo or ex vivo. Methods of nucleic acid delivery include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Lipofection is described in e.g., U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355 and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art.

[00150] Viral vector delivery systems, such as RNA or DNA viral based systems, may also be used to introduce vectors and/or recombinant nucleic acids of the disclosure into cells. Viral vectors can be administered directly to subjects (in vivo) or they can be used to treat cells in vitro (ex vivo) and the modified cells then are administered to a subject. The subject can be human or non-human. Conventional viral based systems include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene or recombinant nucleic acid. d. Production

[00151] In some embodiments, provided herein is a method for producing a fusion protein as described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2).

[00152] In some embodiments, the method for producing the fusion protein includes culturing a host cell transformed with a nucleic acid, preferably an expression vector, containing a recombinant nucleic acid encoding the fusion protein construct under the appropriate conditions and for a sufficient amount of time to induce or cause expression of the fusion polypeptide. In some embodiments, the conditions appropriate for expression varies with the expression vector and the host cell chosen (e.g., a cell described herein).

[00153] In some embodiments, host cells used to produce fusion proteins of the disclosure are grown in media suitable for culturing of the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Any of these media may be supplemented as necessary with hormones and/or other growth factors, salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression of the fusion polypeptide, and will be apparent to the ordinarily skilled artisan.

[00154] In some embodiments, the method for producing a fusion protein having an AKT1 polypeptide described herein (e.g., Table 1) and a PTD described herein (e.g., Table 2) includes recovering the fusion protein. In some embodiments, recovery involves disrupting the host cell, for example by osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris is removed by centrifugation or filtration. The proteins can then be further purified. In some embodiments, a fusion protein of the disclosure is purified by various methods of protein purification, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, and size -exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, in some embodiments, the fusion protein is isolated and purified by appropriately selecting and combining affinity columns such as Protein A column (e.g., POROS Protein A chromatography) with chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultra-filtration, de-salting and dialysis procedures. In some embodiments, a polypeptide is conjugated to a tag to facilitate purification. Exemplary tags include, but are not limited to, a hexa-histidine peptide (His6-tag; SEQ ID NO: 56, HHHHHH), a FLAG tag (SEQ ID NO: 57, DYKDDDDK), a V5 epitope tag (SEQ ID NO: 55, GKPIPNPLLGLDST), and a hemagglutinin (HA) tag (SEQ ID NO: 58, YPYDVPDYA). In some embodiments, the fusion polypeptide has a V5 epitope tag and/or a His6-tag (SEQ ID NO: 56).

[00155] In some embodiments, the method for producing the fusion protein includes chemical synthesis. For instance, the fusion protein may be produced by solid phase or solution phase polypeptide synthesis. Methods for the solid phase and/or solution phase synthesis of proteins are well known in the art (see e.g., Bruckdorfer, T. et al. Curr. Pharm. Biotechnol. [2004] 5, 29-43; Stewart et al., In Solid Phase Peptide Synthesis, 2^nd Edition [1984] Pierce Chemical Company, Rockford, IL; Bodanszky and Bodanszky, The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York; and Barany and Merrifield, Solid-Phase Peptide Synthesis,' pp. 3284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield). The fusion protein may also be produced by in vitro transcription and translation from an encoding nucleic acid employing well-established methods known to those skilled in the art. The fusion protein may be synthesized as a single continuous polypeptide, or as separate fragments. In the latter case, the separate fragments may be fused to each other by condensation of the amino terminal end of one molecule with the carboxyl end of the other molecule, thereby forming a peptide bond, thus forming a continuous polypeptide.

III. Compositions

[00156] In another aspect of the disclosure, provided herein are compositions comprising a fusion protein as described herein, wherein the fusion protein comprises an AKT1 polypeptide as (e.g., as in Table 1) and a PTD (e.g., as in Table 2). [00157] In some embodiments, the composition (e.g., pharmaceutical composition) comprises a fusion protein comprising as described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2). In some embodiments, the AKT1 polypeptide is constitutively active. In some embodiments, the AKT1 polypeptide is phosphatase resistant. In some embodiments, the fusion polypeptide has an AKT1 polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-4, 8-10, 53, and 61 or a functional fragment or variant thereof.

[00158] In some embodiments, the composition (e.g., pharmaceutical composition) comprises a fusion polypeptide comprising a cationic PTD, a hydrophobic PTD, or a cell-type specific PTD. In some embodiments, the cationic PTD is derived from a VP- 16 peptide, an antennapedia peptide, a PTD-5 peptide, a polylysine peptide, a polyarginine peptide, an HIV VPR peptide, or an HIV Tat peptide, or a variant thereof. In some embodiments, the hydrophobic PTD is derived from a transportan peptide, a MAP peptide, a TP 10 peptide, or a variant thereof. In some embodiments, the PTD has a sequence set forth in any one of SEQ ID NOs: 11-20. In some embodiments, the fusion protein further has a linker and/or a tag.

[00159] In some embodiments, the fusion polypeptide present in the composition (e.g., pharmaceutical composition) is capable of penetrating a plasma membrane of a cell. In some embodiments, the fusion polypeptide is capable of inducing activation of cytokine pathway signaling independently of the presence of a ligand. In some embodiments, the ligand is selected from a ligand selected from the group consisting of IL2, IL-4, IL-7, and IL-15.

[00160] In some embodiments, the composition is a pharmaceutical composition having a fusion protein as described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g, as in Table 2) and a pharmaceutically suitable excipient, diluent or carrier. The pharmaceutical composition may be formulated in a conventional manner using one or more physiologically acceptable carriers, excipient and/or auxiliaries which facilitate processing of the active compounds into preparations which are suitable for pharmaceutical use. The pharmaceutical composition can also optionally include one or more preservatives, for example, antibacterial agents, pharmaceutically acceptable carriers, excipients, or stabilizers described elsewhere herein provided they do not adversely affect the physicochemical stability of the fusion polypeptide. Examples of acceptable carriers, excipients, and stabilizers include, but are not limited to, additional buffering agents, co-solvents, surfactants, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (for example, Zn-protein complexes), and biodegradable polymers such as polyesters. A thorough discussion of formulation and selection of pharmaceutically acceptable carriers, stabilizers, and isomolytes can be found in Remington’s Pharmaceutical Sciences (18^th ed.; Mack Publishing Company, Eaton, Pa., 1990). In some embodiments, the pharmaceutical composition is formulated as an aqueous composition. In some embodiments, the pharmaceutical composition is formulated as a gelatinous composition. In some embodiments, the pharmaceutical composition is formulated in the form of a nanoparticle, a microbead, a microsphere, or a virus like particle composition.

[00161] In certain embodiments, proper formulation is dependent upon the route of administration chosen. A summary of components for generating pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). In some embodiments, the pharmaceutical formulation is formulated for administration to a subject in any manner, including one or more of multiple administration routes, such as, by way of non-limiting example, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, oral, topical, rectal, or transdermal administration routes. In some embodiments, the pharmaceutical compositions is formulated for topical, transdermal, intranasal, oral, intravenous, intratracheal, intraperitoneal, subcutaneous, intracranial, intramuscular, transdermal, intraorbital, intratracheal, or intratumoral administration to a subject.

[00162] The pharmaceutical formulations comprising a fusion protein described herein comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2) include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[00163] The pharmaceutical composition comprising a fusion protein described herein comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2) may also be manufactured by conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or compression processes. It would be understood by one of ordinary skill in the art that the method of manufacture will depend on the type of formulation and delivery method of the pharmaceutical composition.

[00164] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2) includes a carrier. The carrier may be selected on the basis of compatibility with the fusion polypeptide, and the properties of the desired formulation. Exemplary carrier materials include, e.g, binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa. : Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[00165] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated as a dosage form. As such, in some embodiments, provided herein is a dosage form having the fusion protein, suitable for administration to an individual. In certain embodiments, suitable dosage forms include, by way of non-limiting example, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multi-particulate formulations, and mixed immediate release and controlled release formulations. The dosage form may optionally include a pharmaceutically acceptable additive, such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof. In some aspects, using standard coating procedures, such as those described in Remington’s Pharmaceutical Sciences, 20^th Edition (2000), a film coating is provided around the formulation of the fusion polypeptide. [00166] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of the fusion polypeptide. In some embodiments, the unit dosage is in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions are optionally packaged in single-dose non-reclosable containers. In some embodiments, multipledose re-closeable containers are used. In certain instances, multiple dose containers comprise a preservative in the composition. By way of example only, formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multidose containers, with an added preservative.

[00167] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated for topical administration (e.g., the formulation is administered to the surface of the skin). For topical administration, the pharmaceutical composition may be formulated as a solution, a cream, a paste, a lotion, a gel, an ointment, a foam, a microemulsion, or a combination thereof. The pharmaceutical composition may be formulated to delivery by any suitable method of topical administration, such as skin abrasion, chemical delivery (e.g, a chemical substance (e.g., PEG, ethanol, glycerol monolaurate, sodium dodecyl sulfate, phosphatidyl choline, or urea) is used to facilitate penetration of an active agent across the skin) electroporation (e.g., the permeabilization of a barrier (e.g., the skin) via application of an electric current), or sonophoresis (e.g., the permeabilization of a barrier (e.g., the skin) via application of ultrasound).

[00168] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated as a cream. In certain instances, creams are semisolid (e.g., soft solid or thick liquid) formulations that include a fusion polypeptide having an active form of a cytokine pathway activator described herein (e.g., Table 1) and a PTD described herein (e.g., Table 2) dispersed in an oil-in-water emulsion or a water-in-oil emulsion.

[00169] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated as a lotion. In certain instances, lotions are fluid emulsions (e.g., oil-in- water emulsions or a water-in-oil emulsions). In some embodiments, the hydrophobic component of a lotion and/or cream is derived from an animal (e.g., lanolin, cod liver oil, and ambergris), plant (e.g., safflower oil, castor oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, pine oil, sesame oil, or sunflower seed oil), petroleum (e.g. , mineral oil, or petroleum jelly), or a combination thereof. In some embodiments, the pharmaceutical composition is formulated as an ointment. In certain instances, ointments are semisolid preparations that soften or melt at body temperature.

[00170] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated as a paste. In certain instances, pastes contain at least 20% solids. In certain instances, pastes are ointments that do not flow at body temperature.

[00171] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated as a gel. In certain instances, gels are semisolid (or semi-rigid) systems consisting of dispersions of large organic molecules dispersed in a liquid. In certain instances, gels are water-soluble and are removed using warm water or saline.

[00172] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated as a stick. In certain instances, sticks are solid dosage forms that melt at body temperature. In some embodiments, a stick has a wax, a polymer, a resin, dry solids fused into a firm mass, and/or fused crystals.

[00173] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated is in the form of a styptic pencil (e.g. a stick prepared by (1) heating crystals until they lose their water of crystallization and become molten, and (2) pouring the molten crystals into molds and allowing them to harden). In some embodiments, the pharmaceutical composition is formulated in the form of a stick that includes a wax (e.g., the wax is melted and poured into appropriate molds in which they solidify in stick form). In some embodiments, the pharmaceutical composition is formulated in the form of a stick that includes a melting base (e.g., a base that softens at body temperature). Examples of melting bases include, but are not limited to, waxes, oils, polymers and gels. In some embodiments, the pharmaceutical composition is formulated in the form of a stick that includes a moisten base (e.g., a base that is activated by the addition of moisture). In some embodiments, the pharmaceutical composition is formulated for administration via a transdermal patch. In some embodiments, a topical formulation described herein is dissolved and/or dispersed in a polymer or an adhesive. In some embodiments, a patch described herein is constructed for continuous, pulsatile, or on demand delivery of a fusion polypeptide described herein.

[00174] In some embodiments, the pharmaceutical composition is formulated for administration via a wound dressing. Wound dressings include, but are not limited to gauzes, transparent film dressings, hydro-gels, polyurethane foam dressings, hydrocolloids and alginates.

[00175] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), includes a penetration enhancer. Penetration enhancers include, but are not limited to, sodium lauryl sulfate, sodium laurate, polyoxyethylene-20-cetyl ether, laureth-9, sodium dodecylsulfate, dioctyl sodium sulfosuccinate, polyoxyethylene-9-lauryl ether (PLE), Tween 80, nonylphenoxypolyethylene (NP- POE), polysorbates, sodium glycocholate, sodium deoxycholate , sodium taurocholate, sodium taurodihydrofusidate, sodium glycodihydrofusidate, oleic acid, caprylic acid, mono- and di-glycerides, lauric acids, acylcholines, caprylic acids, acylcamitines, sodium caprates, EDTA, citric acid, salicylates, DMSO, decylmethyl sulfoxide, ethanol, isopropanol, propylene glycol, polyethylene glycol, glycerol, propanediol, and diethylene glycolmonoethyl ether.

[00176] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), includes a gelling (or thickening) agent. In some embodiments, atopical formulation described herein further has from about 0. 1% to about 5%, from about 0.1% to about 3%, or from about 0.25% to about 2% of a gelling agent. In certain embodiments, the viscosity of atopical formulation described herein is in the range from about 100 to about 500,000 cP, about 100 cP to about 1,000 cP, about 500 cP to about 1500 cP, about 1000 cP to about 3000 cP, about 2000 cP to about 8,000 cP, about 4,000 cP to about 10,000 cP, about

10,000 cP to about 50,000 cP. Suitable gelling agents for use in preparation of the gel topical formulation include, but are not limited to, celluloses, cellulose derivatives, cellulose ethers (e.g., carboxym-ethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose), guar gum, xanthan gum, locust bean gum, alginates (e.g, alginic acid), silicates, starch, tragacanth, carboxyvinyl polymers, carrageenan, paraffin, petrolatum, acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium 25 stearate, bladderwrack, bentonite, carbomer, carrageenan, carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chondrus, dextrose, furcellaran, gelatin, ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, polyethylene glycol (e.g. PEG 200-4500), gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxy-ethyl methacrylate ), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PYM/MA), poly(methoxyethyl meth- acrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), or combinations thereof.

[00177] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), includes an emollient. Emollients include, but are not limited to, castor oil esters, cocoa butter esters, safflower oil esters, cottonseed oil esters, com oil esters, olive oil esters, cod liver oil esters, almond oil esters, avocado oil esters, palm oil esters, sesame oil esters, squalene esters, kikui oil esters, soybean oil esters, acetylated monoglycerides, ethoxylated glyceryl monostearate, hexyl laurate, isohexyl laurate, isohexyl palmitate, isopropyl palmitate, methyl palmitate, decyloleate, isodecyl oleate, hexadecyl stearate decyl stearate, isopropyl isostear ate, methyl isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl adipate, diisopropyl sebacate, lauryl lactate, myristyl lactate, and cetyl lactate, oleyl myristate, oleyl stearate, and oleyl oleate, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, hydrox- ystearic acid, oleic acid, linoleic acid, ricinoleic acid, arachidic acid, behenic acid, erucic acid, lauryl alcohol, myristyl alcohol, cetyl alcohol, hexadecyl alcohol, stearyl alcohol, isostearyl alcohol, hydroxystearyl alcohol, oleyl alcohol, ricinoleyl alcohol, behenyl alcohol, erucyl alcohol, 2-octyl dodecanyl alcohol, lanolin and lanolin derivatives, beeswax, spermaceti, myristyl myristate, stearyl stearate, carnauba wax, candelilla wax, lecithin, and cholesterol. [00178] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated for parenteral, intradermal, or subcutaneous administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00179] In some embodiments, the pharmaceutical composition comprising a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), is formulated for intravenous administration. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [00180] Sterile injectable solutions can be prepared by incorporating the fusion protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the fusion polypeptide into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

IV. Methods of Use

[00181] In another aspect of the disclosure, provided herein are methods of using a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), to activate a cytokine pathway signaling in a cell, and/or to prepare a population of cells for therapeutic use.

[00182] In some embodiments, the method comprises contacting a cell, or a population of cells, with a fusion protein described herein comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g, as in Table 2). In some embodiments, the AKT1 polypeptide is constitutively active. In some embodiments, the AKT1 polypeptide is phosphatase resistant. In some embodiments, the fusion polypeptide comprises an AKT1 polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-10, 53, or 61.

[00183] In some embodiments, the fusion protein used in the methods of the disclosure comprises a cationic PTD, a hydrophobic PTD, or a cell-type specific PTD. In some embodiments, the cationic PTD is derived from a VP- 16 peptide, an antennapedia peptide, a PTD-5 peptide, a polylysine peptide, a polyarginine peptide, an HIV VPR peptide, or an HIV Tat peptide, or a variant thereof. In some embodiments, the hydrophobic PTD is derived from a transportan peptide, a MAP peptide, a TP 10 peptide, or a variant thereof. In some embodiments, the PTD has a sequence set forth in any one of SEQ ID NOs: 11-20. In some embodiments, the fusion protein further comprises a linker and/or a tag. In some embodiments, the fusion protein comprises a sequence of any one of SEQ ID NOs: 24-40 and 64 (shown in Table 3).

[00184] In some embodiments, the fusion protein used in a method is capable of penetrating a plasma membrane of a cell. In some embodiments, the fusion polypeptide is capable of inducing activation of cytokine pathway signaling independently of the presence of a ligand. In some embodiments, the ligand is selected from IL-2, IL-4, IL-7, and IL- 15. a. Activation of Cytokine Pathway Signaling

[00185] In another aspect of the disclosure, provided herein are methods of activating cytokine pathway signaling in a cell. In some embodiments, the method of activating cytokine pathway signaling in a cell comprises contacting the cell with a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), for a sufficient amount of time to induce activation of the signaling pathway. In some embodiments, the contacting occurs in vivo. In some embodiments, the contacting occurs ex vivo.

[00186] In some embodiments, the contacting is performed for about 1 to 24 hours. In some embodiments, the contacting is performed for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours or at least 24 hours. In some embodiments, the contacting is performed for 1 to 6 hours. In some embodiments, the contacting is performed for 4 to 8 hours.

[00187] In some embodiments, activation of cytokine pathway signaling after the contacting occurs independently of ligand-mediated activation of the cytokine pathway. In some embodiments, the ligand is selected from the group consisting of IL-2, IL-4, IL-7, and IL-15.

[00188] In some embodiments, the cell exhibits of one or more properties associated with cytokine pathway signaling after the contacting. In some embodiments, the one or more properties associated with cytokine pathway signaling are selected from: (a) cell division and proliferation; (b) cell migration; (c) stem or progenitor cell differentiation; (d) cytokine and/or growth factor production; (e) increased expression of pro-inflammatory genes; (f) degranulation; (g) survival; (h) differentiation; (i) self-renewal; (j) cell activation; (k) increased expression of cell surface markers; and (1) any combination of (a)-(k). In some embodiments, the cytokine pathway signaling is active for hours or days after contacting the population of cells with the fusion protein. In some embodiments, the activation of cytokine pathway signaling has a duration of at least about 48 hours after contacting the population of cells with the fusion protein. In some embodiments, the activation of cytokine pathway signaling has a duration of at least about 120 hours after contacting the population cells with the fusion protein. In some embodiments, the activation of cytokine pathway signaling has a duration of at least about 168 hours after contacting the population of cells with the fusion protein.

[00189] In some embodiments, the cell is an immune cell, e.g. , a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an eosinophil, a microglia, a monocyte, a neutrophil, an astrocyte, a basophil, a plasma cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, a red blood cell, or any progenitor cell thereof. In some embodiments, the cell is a T cell, e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, an activated T cell, a genetically modified T cell, and/or a CAR T cell (e.g., a T cell comprising a nucleic encoding a CAR comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell, such as a cancer cell or an infected cell). In some embodiments, the immune cell is a cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor. For example, in some embodiments, the immune cell expresses a TCR. In some embodiments, the immune cell expresses CD3. b. Therapeutic Cells

[00190] In yet another aspect of the disclosure, provided herein are methods for preparing a cell therapeutic composition, e.g. , comprising a cell or a population of cells for therapeutic use. In some embodiments, the method for preparing a cell or a population of cells for therapeutic use comprises contacting cells with the fusion comprises contacting the cell with a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2). In some embodiments, the contacting is performed in vivo. In some embodiments, the contacting is performed ex vivo.

[00191] In some embodiments, the cell to be contacted with the fusion protein is an immune cell, e.g., a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an eosinophil, a microglia, a monocyte, a neutrophil, an astrocyte, a basophil, a plasma cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, a red blood cell, or any progenitor cell thereof. In some embodiments, the cell is a T cell, e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, an activated T cell, a genetically modified T cell, and/or a CAR T cell. In some embodiments, the immune cell is a cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor (e.g., CD3). In some embodiments, the cell or population of cells is autologous to a subject to be treated with the immune cell composition. In some embodiments, the cell to be contacted with the fusion protein is not genetically modified. In some embodiments, the cell or population of cells is allogeneic to a subject to be treated with the immune cell composition.

[00192] In some embodiments, the cell therapeutic composition comprises a genetically modified T cell, e.g, a T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR), e.g., a CAR comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell. In some embodiments, the target cell is a cell intended to be targeted to be killed in accordance with a therapeutic method of the disclosure. For example, in some embodiments, the target cell is a cancer cell or an infected cell.

[00193] In some embodiments, the contacting is performed for a sufficient amount of time to induce activation of cytokine pathway signaling in at least a portion of the cells. In some embodiments, the contacting is performed for about 1 to 24 hours. In some embodiments, the contacting is performed for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours or at least 24 hours. In some embodiments, the contacting is performed for 1 to 6 hours. In some embodiments, the contacting is performed for 4 to 8 hours.

[00194] In some embodiments, the contacting is performed shortly prior to administering the population of cells to a subject in need thereof. In some embodiments, this can improve the therapeutic efficacy of the population of cells by improving proliferation and survival in vivo for those cells which have taken up the fusion protein. For example, in some embodiments, the contacting step is performed not more than 30 minutes, not more than 1 hour, not more than 2 hours, not more than 3 hours, not more than 4 hours, not more than 5 hours, not more than 6 hours, not more than 8 hours, not more than 10 hours, not more than 12 hours, not more than 16 hours, not more than 20 hours, not more than 24 hours, not more than 36 hours, not more than 48 hours, or not more than 72 hours prior to administering the population of cells (e.g., CAR-T cells) to a subject in need thereof.

[00195] In some embodiments, cytokine pathway signaling is active for hours or days after hours after contacting the cell or the population of cells with the fusion protein. In some embodiments, the activation of cytokine pathway signaling has a duration of at least about 48 hours after contacting the cell or the population of cells with the fusion protein. In some embodiments, the activation of cytokine pathway signaling has a duration of at least about 120 hours after contacting the cell or the population cells with the fusion protein. In some embodiments, the activation of cytokine pathway signaling has a duration of at least about 168 hours after contacting the cell or the population of cells with the fusion protein.

[00196] In some embodiments, the cell, the population of cells, or at least a portion of the contacted population of cells exhibits of one or more properties associated with cytokine pathway signaling after the contacting. In some embodiments, the one or more properties associated with cytokine pathway signaling are selected from: (a) cell division and proliferation; (b) cell migration; (c) stem or progenitor cell differentiation; (d) cytokine and/or growth factor production; (e) increased expression of pro-inflammatory genes; (f) degranulation; (g) survival; (h) differentiation; (i) self-renewal; (j) cell activation; (k) increased expression of cell surface markers; and (1) any combination of (a)-(k).

[00197] In some embodiments, the method of preparing a cell therapeutic composition comprises a step of cryopreserving the cell therapeutic composition (e.g., as in step 6 in the exemplary T cell therapy manufacturing schematic depicted in FIG. 2B). Accordingly, the method can also further comprise a step of subsequently thawing the cryopreserved cell therapeutic composition. In some embodiments, the step of contacting the cell or the population of cells with the fusion protein occurs prior to cry opreservation, e.g. , immediately prior to cryopreservation. In other embodiments, the step of contacting the cell or population of cells occurs after cryopreservation. In some embodiments, the contacting step occurs after thawing the cell therapeutic composition. In some embodiments, the thawed immune cell exhibits increased surface expression of CD25, CD44, and/or CD69, as compared to a frozen and thawed immune cell that was not contacted with the fusion protein. In some embodiments, the thawed immune cell exhibits improved functional recovery after thawing, as compared to a frozen and thawed immune cell that was not contacted with the fusion protein.

[00198] In some embodiments, the method comprises contacting the cell or population of cells with a medium comprising 0.05-500 pg/mL of the fusion protein of the disclosure. The medium can comprise, for example, 0.05-500 pg/mL, 0.05-400 pg/mL, 0.05-300 pg/mL, 0.05- 250 pg/mL, 0.05-200 pg/mL, 0.05-150 pg/mL, 0.05-100 pg/mL, 0.05-50 pg/mL, 0.05-25 pg/mL, 0.05-10 pg/mL, 0.05-5 pg/mL, 0.05-1 pg/mL, 0.05-0.5 pg/mL, 0.05-0.1 pg/mL, 0.1-500 pg/mL, 0.1-400 pg/mL, 0.1-300 pg/mL, 0.1-250 pg/mL, 0.1-200 pg/mL, 0.1-150 pg/mL, 0.1- 100 pg/mL, 0.1-50 pg/mL, 0.1-25 pg/mL, 0.1-10 pg/mL, 0.1-5 pg/mL, 0.1-1 pg/mL, 0. 1-0.5 pg/mL, 0.5-500 pg/mL, 0.5-400 pg/mL, 0.5-300 pg/mL, 0.5-250 pg/mL, 0.5-200 pg/mL, 0.5- 150 pg/mL, 0.5-100 pg/mL, 0.5-50 pg/mL, 0.5-25 pg/mL, 0.5-10 pg/mL, 0.5-5 pg/mL, 0.5-1 pg/mL, 1-500 pg/mL, 1-400 pg/mL, 1-300 pg/mL, 1-250 pg/mL, 1-200 pg/mL, 1-150 pg/mL, 1-100 pg/mL, 1-50 pg/mL, 1-25 pg/mL, 1-10 pg/mL, 1-5 pg/mL, 5-500 pg/mL, 5-400 pg/mL, 5-300 pg/mL, 5-250 pg/mL, 5-200 pg/mL, 5-150 pg/mL, 5-100 pg/mL, 5-50 pg/mL, 5-25 pg/mL, 5-10 pg/mL, 10-500 pg/mL, 10-400 pg/mL, 10-300 pg/mL, 10-250 pg/mL, 10-200 pg/mL, 10-150 pg/mL, 10-100 pg/mL, 10-50 pg/mL, 10-25 pg/mL, 25-500 pg/mL, 25-400 pg/mL, 25-300 pg/mL, 25-250 pg/mL, 25-200 pg/mL, 25-150 pg/mL, 25-100 pg/mL, 25-50 pg/mL, 50-500 pg/mL, 50-400 pg/mL, 50-300 pg/mL, 50-250 pg/mL, 50-200 pg/mL, 50-150 pg/mL, 50-100 pg/mL, 100-500 pg/mL, 100-400 pg/mL, 100-300 pg/mL, 100-250 pg/mL, 100- 200 pg/mL, 100-150 pg/mL, 150-500 pg/mL, 150-400 pg/mL, 150-300 pg/mL, 150-250 pg/mL, 150-200 pg/mL, 200-500 pg/mL, 200-400 pg/mL, 200-300 pg/mL, 200-250 pg/mL, 250-500 pg/mL, 250-400 pg/mL, 250-300 pg/mL, 300-500 pg/mL, 300-400 pg/mL, or 400-500 pg/mL of the fusion protein.

[00199] In some embodiments, the method further includes isolating the cell or population of cells that exhibit activation of cytokine pathway signaling after the contacting. Isolation of the population of cells may be used to produce a sub-population or portion of cells with active cytokine pathway signaling. Isolation may comprise detection of one or more biomarker (e.g. , a cell surface protein) associated with active cytokine pathway signaling. For instance, cells exhibiting active cytokine pathway signaling may express a cell surface biomarker associated with the active cytokine pathway. The cells could be cultured in the presence of a binding agent (e.g., an antibody) that binds the cell surface marker and has a detectable label attached thereto to and use flow cytometry (e.g., fluorescence activated cell sorting) to separate cells that express the marker (indicative of active cytokine pathway signaling) from cells that do not. In other embodiments, an affinity-based separation method is used to separate cells with active cytokine pathway signaling from cells that do not. Useful methods to separate cells based on affinity include the use of agarose or agarose-based matrices (e.g., agarose or sepharose beads), particles that consist at least in part of a magnetic material (e.g., magnetic beads), particles having polymers such as styrene or latex, tissue culture vessels or plates, tubes (e.g., microfuge tubes), membranes, etc. Isolation of the cells may also be based on expression of a selectable marker, where activation of cytokine signaling leads to expression of a gene that confers resistance or increased survival in a given condition (e.g., lack of a particular nutrient in the media). The isolation may also include selection for morphological features associated with activation of cytokine signaling. In some embodiments, the method further includes expanding the isolated population of cells. The method may also comprise immortalizing, or preserving (e.g., by cry opreservation) the isolated population of cells.

[00200] In some embodiments, the method of preparing a population of cells for therapeutic use further includes genetically modifying the population of cells. Any method known in the art for genetic modification of cells may be used. The population of cells may be modified to insert exogenous genetic material, such as nucleic acids encoding fluorescent markers or a desired enzyme, correct genetic errors, or to regulate expression of one or more genes.

[00201] In some embodiments, the use of a fusion protein of the disclosure in one of the foregoing methods of preparing a population of cells (e.g. , CAR-T cells) for therapeutic use reduces the amount of time needed to prepare a sufficiently large number of cells (e.g., CAR-T cells) for cell therapy. Accordingly, in some embodiments, the method of preparing a population of cells is completed in less than 30 days, less than 29 days, less than 28 days, less than 27 days, less than 26 days, less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days. In some embodiments, the cell therapeutic composition (e.g, the cryopreserved cell therapeutic composition) is generated within 30 days, within 29 days, within 28 days, within 27 days, within 26 days, within 25 days, within 24 days, within 23 days, within 22 days, within 21 days, within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, within 15 days, within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within

5 days, within 4 days, or within 3 days of obtaining the initial immune cells from the subject by leukapheresis. In some embodiments, the cell therapeutic composition (e.g., a composition of CAR-T cells) is administered to the subject within 30 days, within 29 days, within 28 days, within 27 days, within 26 days, within 25 days, within 24 days, within 23 days, within 22 days, within 21 days, within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, within 15 days, within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, or within 3 days of obtaining the initial immune cells from the subject by leukapheresis.

[00202] In some embodiments, the population of cells prepared by the method described herein includes one or more cell types. In some embodiments, the population of cells includes two or more, three or more, four or more, five or more, six or more, or seven or more cell types. In some embodiments, the population of cells includes two or more cell types. In some embodiments, each of the two or more cells exhibits the same level of activation of cytokine pathway signaling. In some embodiments, each of the two or more cell types exhibits complete cessation of cytokine pathway signaling. In some embodiments, each of the two or more cell types exhibit different levels of activation of cytokine pathway signaling. In some embodiments, the method further includes separating each of the two or more cell types after contacting.

[00203] Also provided herein is a cell, a population of cells, or a cell therapeutic composition prepared by any of the methods described herein.

[00204] Disclosed herein are methods of using the disclosed fusion proteins e.g., PTD- MyrAkt fusion protein, to improve the outcomes of cryopreservation of CART cells. In some embodiments, the fusion protein is used to treat established CART cell culture prior to cryopreservation to provide for a higher frequency of viable cells that are functional (e.g., as defined by responsiveness to CD3 stimulation). In some embodiments, disclosed herein are methods for using the disclosed fusion protein, e.g. , PTD-MyrAkt fusion protein to treat freshly thawed CAR-T cells. In this instance, the pro-survival activities of the PTD-MyrAkt fusion protein may achieve a higher percentage of the starting population of thawed CAR-T cells to survive the initial hours.

[00205] Additionally disclosed herein is the use of the disclosed fusion proteins e.g., the PTD-MyrAkt fusion protein, to treat CAR-T cells prior to administration to a recipient patient. CAR-T cells are currently administered to patients following lymphodepletion. In addition, in order to promote the proliferation and survival of CAR-T cells in vivo following infusion to the patient, these individuals are given up to 6 injections of systemic high dose IL-2 (maximum tolerated doses). The presence of the PTD-MyrAkt fusion protein within the CART cells themselves will promote the proliferation and survival of these cells in vivo in a direct manner without adversely affecting the patient. In addition, the cell intrinsic presence of this fusion protein will be more effective at promoting the proliferation and survival of the cells in vivo, hence leading to increased therapeutic efficacy. Lastly, the presence of the PTD-MyrAkt fusion protein within the CART cells will bridge the cells from their reliance on super physiological levels of survival signals they experienced in culture to the levels of cytokines normally present in vivo. c. Genetically Modifying a Cell

[00206] In yet another aspect of the disclosure, provided herein are methods of genetically modifying an immune cell, e.g., an immune cell for use in cell therapy (e.g., as in step 4 in the exemplary T cell therapy manufacturing schematic depicted in FIG. 2B). In some embodiments, the method of genetically modifying an immune cell comprises a step of (a) contacting the cell with a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), thereby generating an activated immune cell, and a step of (b) contacting the activated immune cell with a vector encoding a gene of interest. In some embodiments, step (a) and/or step (b) is performed ex vivo.

[00207] The vector can, for example, be any vector suitable for stably or transiently introducing an ectopic nucleic acid into the cell. In some embodiments, the vector is a viral vector, such as an adenoviral vector or a retroviral vector (e.g., a type-C retroviral vector). In some embodiments, the vector is RNA. In some embodiments, step (b) comprises contacting the cell with a liposome encapsulating the vector.

[00208] In some embodiments of the methods, the immune cell is in a resting state prior to the step of being contacted with the fusion protein. In some embodiments, contacting the cell enhances the efficiency of genetically modifying the immune cell or population of immune cells, e.g., as compared to an immune cell or population of immune cells that was not treated with the fusion protein.

[00209] In some embodiments, the immune cell is a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an eosinophil, a microglia, a monocyte, a neutrophil, an astrocyte, a basophil, a plasma cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, a red blood cell, or any progenitor cell thereof. In some embodiments, the immune cell is a T cell, e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, an activated T cell, a genetically modified T cell, and/or a CAR T cell. In some embodiments, the cell is autologous to a subject to be treated with the immune cell. In some embodiments, the cell is allogeneic to a subject to be treated with the immune cell. In some embodiments, the cell is an immune cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor. For example, in some embodiments, the immune cell expresses a TCR. In some embodiments, the immune cell expresses CD3.

[00210] In some embodiments, the vector comprises a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell. In some embodiments, the target cell is a cell intended to be targeted to be killed in accordance with a therapeutic method of the disclosure. For example, in some embodiments, the target cell is a cancer cell or an infected cell.

[00211] In yet another aspect of the disclosure, provided herein are methods of genetically modifying a cell, e.g. , an immune cell for use in cell therapy and/or a primary cell. In some embodiments, the method of genetically modifying a cell comprises a step of (a) contacting the cell with a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g, as in Table 2), and a step of (b) subsequently modifying the cell with a genomic editing system. In some embodiments, step (a) and/or step (b) is performed ex vivo.

[00212] Certain genomic editing systems may be used to introduce mutations into a cell genome (e.g., by introducing one or more substitutions, insertions, or deletions, into one or more copies of a target gene or an associated regulatory region, and/or by partially or completely deleting one or more copies of a gene). Certain genomic editing systems may also be used to introduce heterologous nucleic acids into the genome of a modified cell. The introduction of heterologous nucleic acids into the genome can be used to disrupt gene or protein expression, e.g., via the introduction of a nucleic acid that disrupts the transcription, translation, or function of a target gene. Additionally or alternatively, the introduction of heterologous DNA via a genomic editing system may be used to introduce a nucleic acid encoding one or more genes or proteins of interest (e.g. , a nucleic acid encoding a CAR). The introduction of heterologous regulatory elements into certain genomic sites (or, conversely, the disruption of native regulatory elements) may likewise be used to alter expression of a gene or protein. Genomic editing systems include, but are not limited to, transposon systems (e.g. retrotransposon systems or DNA transposon systems) and nuclease genomic editing systems (e.g., rare-cutting endonucleases, e.g., CRISPR-Cas systems). Nuclease genomic editing systems can be used, for example, to introduce mutations into a desired genomic locus by non homologous end joining, or can be used to introduce a heterologous nucleic acid (e.g., a nucleic acid encoding a CAR) into the genome via homology-directed repair. A nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activator-like effector nuclease (TALEN) or derivative thereof, a zinc -finger nuclease (ZFN) or derivative thereof, or a homing endonuclease (HE) or derivative thereof.

[00213] In some embodiments, the cell that is modified by a genomic editing system is an immune cell, e.g., a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an eosinophil, a microglia, a monocyte, a neutrophil, an astrocyte, a basophil, a plasma cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, a red blood cell, or any progenitor cell thereof. In some embodiments, the immune cell is a T cell, e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, an activated T cell, a genetically modified T cell, and/or a CAR T cell. In some embodiments, the cell is autologous to a subject to be treated with the cell. In some embodiments, the cell is allogeneic to a subject to be treated with the cell. In some embodiments, the cell is an immune cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor. For example, in some embodiments, the immune cell expresses a TCR. In some embodiments, the immune cell expresses CD3. In some embodiments, the cell is modified by the genomic editing system to express a CAR.

[00214] In some embodiments, contacting the cell with a fusion protein of the disclosure enhances the efficiency of genetically modifying the cell or population of cells with the genomic editing system, e.g., as compared to a cell or population of cells that was not treated with the fusion protein. d. Expanding an Immune Cell

[00215] In yet another aspect of the disclosure, provided herein are methods of expanding an immune cell, e.g. , an immune cell for use in cell therapy. These methods can occur in the context of preparing a cell therapeutic composition (e.g., as in step 3 and/or step 5 in the exemplary T cell therapy manufacturing schematic depicted in FIG. 2B). In some embodiments, the method of expanding an immune cell comprises a step of (a) contacting the immune cell with a growth medium comprising a mitogenic stimulus, and a step of (b) contacting the immune cell a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g, as in Table 2). In some embodiments, step (a) and/or step (b) is performed ex vivo. Steps (a) and (b) can be carried out separately (e.g., sequentially), or the steps can be carried out simultaneously.

[00216] The mitogenic stimulus in the growth medium of step (a) can be, for example, an anti-CD3 antibody and/or an anti-CD28 antibody. The growth medium can also further comprise one or more growth factors, e.g. a cytokine, e.g., a cytokine selected from IL-2, IL-4, IL-7, and IL- 15, or any combination thereof. In some embodiments, the method comprises incubating the immune cell in the growth medium for at least 3 days, e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the method comprises incubating the immune cell in the growth medium for 3-10 days, e.g, 3-10, 3-7, 3-6, 3-5, 3-4, 4-10, 4-7, 4-5, 5-10, 5-7, or 7-10 days. In some embodiments, the method comprises incubating the immune cell in the growth medium for 3-5 days.

[00217] In embodiments wherein steps (a) and (b) are carried out simultaneously, the growth medium can further comprise the fusion protein. The growth medium can comprise, for example, 0.05-500 pg/mL, 0.05-400 pg/mL, 0.05-300 pg/mL, 0.05-250 pg/mL, 0.05-200 pg/mL, 0.05-150 pg/mL, 0.05-100 pg/mL, 0.05-50 pg/mL, 0.05-25 pg/mL, 0.05-10 pg/mL, 0.05-5 pg/mL, 0.05-1 pg/mL, 0.05-0.5 pg/mL, 0.05-0.1 pg/mL, 0.1-500 pg/mL, 0.1-400 pg/mL, 0.1-300 pg/mL, 0.1-250 pg/mL, 0.1-200 pg/mL, 0.1-150 pg/mL, 0.1-100 pg/mL, 0.1-50 pg/mL, 0.1-25 pg/mL, 0.1-10 pg/mL, 0.1-5 pg/mL, 0.1-1 pg/mL, 0. 1-0.5 pg/mL, 0.5-500 pg/mL, 0.5- 400 pg/mL, 0.5-300 pg/mL, 0.5-250 pg/mL, 0.5-200 pg/mL, 0.5-150 pg/mL, 0.5-100 pg/mL, 0.5-50 pg/mL, 0.5-25 pg/mL, 0.5-10 pg/mL, 0.5-5 pg/mL, 0.5-1 pg/mL, 1-500 pg/mL, 1-400 pg/mL, 1-300 pg/mL, 1-250 pg/mL, 1-200 pg/mL, 1-150 pg/mL, 1-100 pg/mL, 1-50 pg/mL, 1- 25 pg/mL, 1-10 pg/mL, 1-5 pg/mL, 5-500 pg/mL, 5-400 pg/mL, 5-300 pg/mL, 5-250 pg/mL, 5- 200 pg/mL, 5-150 pg/mL, 5-100 pg/mL, 5-50 pg/mL, 5-25 pg/mL, 5-10 pg/mL, 10-500 pg/mL, 10-400 pg/mL, 10-300 pg/mL, 10-250 pg/mL, 10-200 pg/mL, 10-150 pg/mL, 10-100 pg/mL, 10-50 pg/mL, 10-25 pg/mL, 25-500 pg/mL, 25-400 pg/mL, 25-300 pg/mL, 25-250 pg/mL, 25- 200 pg/mL, 25-150 pg/mL, 25-100 pg/mL, 25-50 pg/mL, 50-500 pg/mL, 50-400 pg/mL, SO- SOO pg/mL, 50-250 pg/mL, 50-200 pg/mL, 50-150 pg/mL, 50-100 pg/mL, 100-500 pg/mL, 100-400 pg/mL, 100-300 pg/mL, 100-250 pg/mL, 100-200 pg/mL, 100-150 pg/mL, 150-500 pg/mL, 150-400 pg/mL, 150-300 pg/mL, 150-250 pg/mL, 150-200 pg/mL, 200-500 pg/mL, 200-400 pg/mL, 200-300 pg/mL, 200-250 pg/mL, 250-500 pg/mL, 250-400 pg/mL, 250-300 pg/mL, 300-500 pg/mL, 300-400 pg/mL, or 400-500 pg/mL of the fusion protein.

[00218] In some embodiments wherein the steps are not carried out simultaneously, step (a) is carried out before step (b). In some embodiments, step (b) comprises incubating the immune cell in medium comprising the fusion protein, e.g., at a concentration of 0.05-500 pg/mL, 0.05-400 pg/mL, 0.05-300 pg/mL, 0.05-250 pg/mL, 0.05-200 pg/mL, 0.05-150 pg/mL, 0.05-100 pg/mL, 0.05-50 pg/mL, 0.05-25 pg/mL, 0.05-10 pg/mL, 0.05-5 pg/mL, 0.05-1 pg/mL, 0.05-0.5 pg/mL, 0.05-0.1 pg/mL, 0.1-500 pg/mL, 0.1-400 pg/mL, 0.1-300 pg/mL, 0.1-250 pg/mL, 0.1-200 pg/mL, 0.1-150 pg/mL, 0.1-100 pg/mL, 0.1-50 pg/mL, 0.1-25 pg/mL, 0.1-10 pg/mL, 0.1-5 pg/mL, 0.1-1 pg/mL, 0.1-0.5 pg/mL, 0.5-500 pg/mL, 0.5-400 pg/mL, 0.5-300 pg/mL, 0.5-250 pg/mL, 0.5-200 pg/mL, 0.5-150 pg/mL, 0.5-100 pg/mL, 0.5-50 pg/mL, 0.5-25 pg/mL, 0.5-10 pg/mL, 0.5-5 pg/mL, 0.5-1 pg/mL, 1-500 pg/mL, 1-400 pg/mL, 1-300 pg/mL, 1- 250 pg/mL, 1-200 pg/mL, 1-150 pg/mL, 1-100 pg/mL, 1-50 pg/mL, 1-25 pg/mL, 1-10 pg/mL, 1-5 pg/mL, 5-500 pg/mL, 5-400 pg/mL, 5-300 pg/mL, 5-250 pg/mL, 5-200 pg/mL, 5-150 pg/mL, 5-100 pg/mL, 5-50 pg/mL, 5-25 pg/mL, 5-10 pg/mL, 10-500 pg/mL, 10-400 pg/mL, 10-300 pg/mL, 10-250 pg/mL, 10-200 pg/mL, 10-150 pg/mL, 10-100 pg/mL, 10-50 pg/mL, 10- 25 pg/mL, 25-500 pg/mL, 25-400 pg/mL, 25-300 pg/mL, 25-250 pg/mL, 25-200 pg/mL, 25- 150 pg/mL, 25-100 pg/mL, 25-50 pg/mL, 50-500 pg/mL, 50-400 pg/mL, 50-300 pg/mL, 50- 250 pg/mL, 50-200 pg/mL, 50-150 pg/mL, 50-100 pg/mL, 100-500 pg/mL, 100-400 pg/mL, 100-300 pg/mL, 100-250 pg/mL, 100-200 pg/mL, 100-150 pg/mL, 150-500 pg/mL, 150-400 pg/mL, 150-300 pg/mL, 150-250 pg/mL, 150-200 pg/mL, 200-500 pg/mL, 200-400 pg/mL, 200-300 pg/mL, 200-250 pg/mL, 250-500 pg/mL, 250-400 pg/mL, 250-300 pg/mL, 300-500 pg/mL, 300-400 pg/mL, or 400-500 pg/mL of the fusion protein. In some embodiments, the immune cell is incubated in the medium comprising the fusion protein for at least 5 minutes, e.g., at least 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, or 180 minutes. In some embodiments, the immune cell is incubated in the medium comprising the fusion protein for at least 60 minutes. In some embodiments, the immune cell is incubated in the medium for 5 to 180, 5 to 150, 5 to 120, 5 to 90, 5 to 60, 5 to 45, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 180, 10 to 150, 10 to 120, 10 to 90, 10 to 60, 10 to 45, 10 to 30, 10 to 20, 10 to 15, 15 to 180, 15 to 150, 15 to 120, 15 to 90, 15 to 60, 15 to 45, 15 to 30, 15 to 20, 20 to 180, 20 to 150, 20 to 120, 20 to 90, 20 to 60, 20 to 45, 20 to 30, 30 to 180, 30 to 150, 30 to 120, 30 to 90, 30 to 60, 30 to 45, 45 to 180, 45 to 150, 45 to 120, 45 to 90, 45 to 60, 60 to 180, 60 to 150, 60 to 120, 60 to 90, 90 to 180, 90 to 150, 90 to 120, 120 to 180, 120 to 150, or 150 to 180 minutes. In some embodiments, following step (b), the immune cell is removed from the medium comprising the fusion protein, washed, and incubated in a second growth medium comprising the mitogenic stimulus. The second growth medium can be, for example, the same growth medium used in step (a).

[00219] In some embodiments of the foregoing methods, following steps (a) and (b), the immune cell expresses a higher level of CD25, CD44, and/or CD69 relative to an immune cell which was contacted with the growth medium comprising the mitogenic stimulus without being contacted with the fusion protein. In some embodiments, following steps (a) and (b), the immune cell exhibits increased survival and/or proliferation relative to an immune cell which was contacted with the growth medium comprising the mitogenic stimulus without being contacted with the fusion protein. In some embodiments, following steps (a) and (b), in addition to exhibiting improved expansion, the immune cell simultaneously becomes more susceptible to viral transduction relative to an immune cell which was contacted with the growth medium comprising the mitogenic stimulus without being contacted with the fusion protein.

[00220] In some embodiments of the foregoing methods, carrying out steps (a) and (b) (optionally concurrently) results in an enhanced expansion efficiency, as compared to the expansion of immune cells that are not contacted with the fusion protein. In some embodiments, the enhanced expansion efficiency can reduce the time that the immune cells need to be expanded in order to generate a sufficient number of immune cells to prepare a cell therapeutic composition. For example, in some embodiments, contacting the immune cell with a fusion protein of the disclosure (e.g., a PTD-MyrAkt fusion protein) reduces the amount of time needed to prepare the sufficient number of immune cells by at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 3 days, or at least 4 days, as compared to the amount of time to prepare the sufficient number of immune cells using an immune cell that was not contacted with the fusion protein.

[00221] In some embodiments, the immune cell is a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an eosinophil, a microglia, a monocyte, a neutrophil, an astrocyte, a basophil, a plasma cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, a red blood cell, or any progenitor cell thereof. In some embodiments, the immune cell is a T cell, e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, an activated T cell, a genetically modified T cell, and/or a CAR T cell. In some embodiments, the immune cell is a cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor (e.g., CD3). In some embodiments, the cell is autologous to a subject to be treated with the immune cell. In some embodiments, the cell is allogeneic to a subject to be treated with the immune cell.

[00222] Further disclosed herein are methods of using the disclosed fusion proteins (e.g., PTD-MyrAkt fusion protein) to improve CART cell production and therapeutic efficacy. In some embodiments, the levels or viral gene transduction may be improved by the addition of the disclosed fusion proteins to the viral transduction culture. In this instance, the ability of the fusion protein to activate signals that would normally be triggered by cytokine receptors, in a naive T cell population, is able to promote entry into the cell cycle in those cells regardless of surface expression of the cytokine receptor. Furthermore, the presence of the PTD-MyrAkt fusion protein in the priming culture will also facilitate cell expansion during the first days of culture by augmenting the proliferation and survival activities of the cytokines produced by the newly activated T-cells.

[00223] Further disclosed herein are methods of using the disclosed fusion proteins (e.g., PTD-MyrAkt fusion protein) to improve the expansion of established CAR-T cells to generate a sufficiently large number of cells for treating patients. The ability of the fusion protein to enhance the proliferation and survival signals normally induced by cytokine receptors that utilize the common gamma chain will promote the generation of a larger number of cells in a shorter period of time. For example, in some embodiments, contacting the immune cell with a fusion protein of the disclosure (e.g., a PTD-MyrAkt fusion protein) reduces the amount of time needed to prepare the sufficiently large number of CAR-T cells by at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days, as compared to the amount of time to prepare the sufficiently large number of CAR-T cells using cells that were not contacted with the fusion protein.

[00224] Further disclosed herein are methods of using the disclosed fusion proteins (e.g., PTD-MyrAkt fusion protein) to treat Tregs ex vivo prior to transfusion to improve their survival and expansion in vivo in the absence of systemically administered IL-2. In some embodiments, the target Treg cell population may be naturally occurring CD25+CD4+ Treg cells. In this instance, the Tregs may be treated ex vivo hour with a disclosed fusion protein, abrogating the need for ex vivo expansion and lengthy production campaigns under cGMP conditions. The fusion protein may also be used to treat “induced” Tregs ex vivo in a similar manner, prior to infusion in order to extend their proliferation, survival and regulatory activity in vivo.

[00225] Further disclosed herein are methods of using the disclosed fusion proteins (e.g., PTD-MyrAkt fusion protein) to improve the expansion during the initial priming ex vivo to generate a large number of cells for therapeutic applications. The fusion protein may also be used before the final step of production to improve the survivability of expanded Treg populations after cryopreservation. The disclosed fusion proteins may be used to treat the ex vivo expanded Treg cell populations immediately prior to infusion in order to mimic the signal derived from the IL-2 receptor in vivo, without the need for systemic IL-2 administration to the patient.

[00226] Further disclosed herein are methods for using the disclosed fusion proteins (e.g., PTD-MyrAkt fusion protein) for enhancing the viral transduction of CART constructs into ex vivo activated T cells (e.g., Tregs). The fusion protein may enhance the T cell (e.g., Treg) activation and initial expansion required for viral gene transduction and the expansion of transduced cells ex vivo. In some embodiments of the foregoing methods, the enhanced viral transduction efficiency and/or the reduced expansion efficiency can reduce the time required to generate a sufficiently large number of T cells (e.g., Tregs) for a cell therapeutic composition. For example, in some embodiments, contacting the T cells (e.g., Tregs) with a fusion protein of the disclosure (e.g., a PTD-MyrAkt fusion protein) reduces the amount of time needed to prepare the sufficiently large number of T cells by at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 3 days, or at least 4 days, as compared to the amount of time to prepare the sufficiently large number of T cells when the fusion protein is not used. e. Cell Types [00227] The cell or population of cells used with the methods described herein may include any desired cell type or combination of cell types. For instance, for a particular downstream application, one or more particular cell types may be desired.

[00228] In some embodiments, the cell or population of cells includes one or more immune cells. In some embodiments, the one or more immune cells comprise one or more of a T cell (e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, an activated T cell, a genetically modified T cell, and/or a CAR T cell), a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an eosinophil, a microglia, a monocyte, a neutrophil, an astrocyte, a basophil, a plasma cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, a red blood cell, or any progenitor cell thereof. In some embodiments, the immune cell is a cell (e.g, a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor (e.g., CD3). In some embodiments, the one or more immune cells are autologous to a subject to be treated with the one or more immune cells. In some embodiments, the one or more immune cells are allogeneic to a subject to be treated with the one or more immune cells.

[00229] In some embodiments, the cell or population of cells includes one or more of a hematopoietic stem cell, an induced pluripotent stem cell, a trophoblast cell, a placenta-derived cell, or a progenitor cell.

[00230] In some embodiments, the cell or population of cells is one or more of a cardiomyocyte, a fibroblast, a hepatocyte, an adipocyte, an endothelial cell, a bone marrow stromal cell, or an epithelial cell, or any progenitor cell thereof.

[00231] In some embodiments, the cell or population of cells are human. In other embodiments, the cell or population of cells from a non-human mammal. The non-human mammal may be, for example, a dog, cat, horse, cattle, dairy cow, swine, sheep, lamb, goat, primate, mouse, or rat.

V. Methods of Treatment

[00232] In another aspect of the disclosure, provided herein are methods of treating or preventing a disease or disorder in a subject. In some embodiments, the disease or disorder is an ischemia reperfusion injury, a cancer, an infection, or an autoimmune disease or disorder.

[00233] In some embodiments, the method of treating or preventing a disease or disorder in a subject includes administering to the subject a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2). In some embodiments, the AKT1 polypeptide is constitutively active. In some embodiments, the AKT1 polypeptide is phosphatase resistant. In some embodiments, the fusion polypeptide comprises an AKT1 polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-10, 53, or 61. [00234] In some embodiments, the fusion protein used in the methods of treating or preventing a disease or disorder in a subject described herein has a cationic PTD, a hydrophobic PTD, or a cell-type specific PTD. In some embodiments, the cationic PTD is derived from a VP- 16 peptide, an antennapedia peptide, a PTD-5 peptide, a polylysine peptide, a polyarginine peptide, an HIV VPR peptide, or an HIV Tat peptide, or a variant thereof. In some embodiments, the hydrophobic PTD is derived from a transportan peptide, a MAP peptide, a TP 10 peptide, or a variant thereof. In some embodiments, the PTD has a sequence set forth in SEQ ID NOs: 11-20. In some embodiments, the fusion protein further comprises a linker and/or a tag.

[00235] In some embodiments, the fusion polypeptide used in the methods of treating or preventing a disease or disorder in a subject described herein is capable of penetrating a plasma membrane of a cell. In some embodiments, the fusion polypeptide is capable of inducing activation of cytokine pathway signaling independently of the presence of a ligand. In some embodiments, the ligand is selected from a ligand selected from the group consisting of IL-2, IL- 4, IL-7, and IL- 15.

[00236] In some embodiments, the method of treating or preventing a disease or disorder in a subject comprises administering to the subject a cell or a population of cells that has been contacted ex vivo with the fusion polypeptide (e.g. , in accordance with one of the foregoing methods). In some embodiments, the population of cells exhibits activation of cytokine pathway signaling after the contacting. In some embodiments, the method of treating or preventing a disease or disorder in a subject comprises administering to the subject a genetically modified cell or a cell therapeutic composition generated via a method of the disclosure. In other embodiments, the method of treating or preventing a disease or disorder in a subject comprises administering the subject a cell therapeutic composition of the disclosure that comprises immune cells that have not been genetically modified.

[00237] It would be understood by one of skill in the art that the particular cell type(s) included in the population of cells used in the methods described herein will depend on the particular disease or disorder being treated, the degree of progression of the disease or disorder, and the route of administration. In some embodiments, the population of cells include one or more immune cells. In some embodiments, the one or more immune cells include one or more of a T cell (e.g., a T cell selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, an activated T cell, a genetically modified T cell, and/or a CAR T cell), a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an eosinophil, a microglia, a monocyte, a neutrophil, an astrocyte, a basophil, a plasma cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, a red blood cell, or any progenitor cell thereof. In some embodiments, population of cells includes one or more of a hematopoietic stem cell, a trophoblast, a placenta-derived cell, an induced pluripotent stem cell, or a progenitor cell. In some embodiments, the population of cells includes one or more of a cardiomyocyte, a fibroblast, a hepatocyte, an adipocyte, an endothelial cell, a bone marrow stromal cell, or an epithelial cell, or a progenitor cell thereof. In some embodiments, the population of cells is genetically modified. In other embodiments, the population of cells is not genetically modified. In some embodiments, the population of cells is autologous to the subject. In some embodiments, the population of cells is allogenic to the subject.

[00238] In some embodiments, the method of treating or preventing a disease or disorder in a subject comprises the steps as set forth in FIG. 2A, wherein step 3 comprises contacting the immune cells with a fusion protein of the disclosure. For example, in some embodiments, the method of treating or preventing a disease or disorder in a subject comprises the steps of (1) obtaining blood from the subject to be treated; (2) isolating immune cells (e.g., PBMCs) from the blood sample; (3) treating the immune cells with a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2); and (4) administering the treated immune cells to the subject. In some embodiments, PBMCs are obtained from the blood of the subject using leukapheresis, followed by steps (3) and (4). The disease or disorder to be treated and prevented using this method can be, e.g., ischemia reperfusion injury, infection, cancer, or an autoimmune disease or disorder. In some embodiments, the method of treating or preventing a disease or disorder does not comprise and/or does not require (i) a step of genetically modified the immune cells, (ii) lymphodepletion, and/or (iii) a step of expanding the immune cells.

[00239] In some embodiments, the immune cells are isolated, treated with a fusion protein of the disclosure, and administered to the subject within 1 week of obtaining the blood sample from the subject, e.g., within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 48 hours, within 36 hours, within 24 hours, within 18 hours, within 12 hours, or within 6 hours of obtaining the blood sample from the subject. In some embodiments, the immune cells are isolated, treated with a fusion protein of the disclosure, and administered to the subject within 12 to 24 hours of obtaining the blood sample from the subject.

[00240] In some embodiments, the immune cells are treated with the fusion protein shortly prior to administering the immune cells to the subject in need thereof. In some embodiments, this can improve the therapeutic efficacy of the immune cells by improving proliferation and survival in vivo for those cells which have taken up the fusion protein. For example, in some embodiments, the step of treating the immune cells with the fusion protein is carried out not more than 30 minutes, not more than 1 hour, not more than 2 hours, not more than 3 hours, not more than 4 hours, not more than 5 hours, not more than 6 hours, not more than 8 hours, not more than 10 hours, not more than 12 hours, not more than 16 hours, not more than

20 hours, not more than 24 hours, not more than 36 hours, not more than 48 hours, or not more than 72 hours prior to administering the population of cells (e.g., CAR-T cells) to a subject in need thereof. a. Ischemia Reperfusion Injury [00241] In some embodiments, provided herein is a method of treating or preventing ischemia reperfusion injury in a subject that includes administering to the subject a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), or a cell or population of cells that has been contacted ex vivo with the fusion protein. [00242] Ischemia reperfusion injury, sometimes called reperfusion injury or reoxygenation injury, refers to the tissue damage caused when blood supply returns to tissue (reperfusion) after a period of ischemia or lack of oxygen (anoxia or hypoxia). The lack of blood oxygen during the ischemic period results in inflammation and oxidative damage upon the restoration of circulation. In some embodiments, the ischemia reperfusion injury is a myocardial ischemia, a cerebral ischemia, a hepatic ischemia, a pulmonary ischemia, or a nephritic ischemia.

[00243] In some embodiments, the method for treating or preventing ischemia reperfusion injury in a subject includes administering to the subject the fusion protein or the cell or population of cells after occurrence of the ischemic reperfusion injury. In some embodiments, the subject is administered with the fusion protein or the cell or population of cells within 30 to 60 minutes of occurrence of the ischemic reperfusion injury. In some embodiments, the subject is administered with the fusion protein or the cell or population of cells within 30 minutes after occurrence of the ischemic reperfusion injury. In some embodiments, the subject is administered with the fusion protein or the cell or population of cells within 45 minutes after occurrence of the ischemic reperfusion injury. In some embodiments, the subject is administered with the fusion protein or the cell or population of cells after at least 1 to 6 hours of occurrence of the ischemic reperfusion injury. In some embodiments, the subject is administered with the fusion protein or the cell or population of cells after at least 1.5 hours of occurrence of the ischemic reperfusion injury. In some embodiments, the subject is administered with the fusion protein or the cell or population of cells after at least 3 hours of occurrence of the ischemic reperfusion injury.

[00244] In some embodiments, the method for treating or preventing ischemia reperfusion injury in a subject further includes ischemia pre-conditioning. Ischemic preconditioning is the exposure of the tissue (e.g. myocardium, kidney or nervous tissue) endangered by ischemia to brief, repeated periods of hypoxia, preferably ischemia (e.g. by vascular occlusion). In some embodiments, ischemic preconditioning includes exposure of the tissue by an external effect having the same result in the tissue as said repeated periods of hypoxia; this can be achieved, e.g., by treatment with pharmaceutical, physical, and/or chemical agents mimicking the preconditioning effect. Preconditioning has a cardioprotective effect, renders the tissue resistant to the deleterious effects of ischemia or reperfusion and lessens myocardial infarct size and dysfunction and arrhythmias after ischemia.

[00245] In some embodiments, the method for treating or preventing ischemia reperfusion injury in a subject further includes assessing the progression of the ischemic reperfusion injury by detecting a biomarker in the serum of the subject. Exemplary biomarkers of ischemic reperfusion injury include, but are not limited to, NGAL, KIM-1, IL- 18, RBP, FABP4, cystatin C and creatinine. In some embodiments, the biomarker is a biomarker associated with a myocardial ischemia, a cerebral ischemia, a hepatic ischemia, a pulmonary ischemia, or a nephritic ischemia. b. Infection

[00246] In some embodiments, provided herein is a method of treating a subject having an infection, the method includes administering to the subject a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), or a cell or population of cells that has been contacted ex vivo with the fusion protein. In some embodiments, the cell to be administered to the subject is a CAR T cell, wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigenbinding site specifically binds an antigen on the surface of an infected cell. In some embodiments, the cell or population of cells are autologous to the subject having the infection. In other embodiments, the cell or population of cells are allogeneic to the subject having the infection.

[00247] Infectious diseases that can be treated, protected against, and/or managed by the fusion protein or cell or population of cells may be caused by infectious agents including, but not limited to, bacteria, fungi, protozoa, and viruses. In some embodiments, the infection is a bacterial infection, a viral infection, a fungal infection, a protozoan infection, or a parasite infection.

[00248] In some embodiments, the infection treated by the methods described herein is a bacterial infection. Examples of bacterial infections include those caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecials, Proteus vulgaris, Staphylococcus viridans, Pseudomonas aeruginosa, Mycobacteria rickettsia, Mycoplasma, Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus, Streptococcus, Staphylococcus, Mycobacterium, pertissus, cholera, diphtheria, chlamydia, .S', aureus and Legionella. In some embodiments, the bacterial infection is an infection from a bacteria selected from Staphylococcus aureus, Streptococcus pnuemoniae, Heamophila influenzae, Neisseria meningitidis, Klebsiella pneumoniae, Mycobacterium tuberculosis, Escherichia coli, and group B Streptococci.

[00249] In some embodiments, the infection treated by the methods described herein is a viral infection. Exemplary viral infections include, without limitation, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza (e.g. , influenza A or influenza B), varicella, adenovirus, herpes virus (e.g. herpes simplex type I (HSV-I) or herpes simplex type II (HSV- II)), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, small pox, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and agents of viral diseases such as viral meningitis, encephalitis, dengue or small pox. In some embodiments, the viral infection is a chronic viral infection. In some embodiments, the chronic viral infection is an infection from a virus selected from Hepatitis A Virus Hepatitis B Virus, Hepatitis C Virus, Epstein Barr Virus (EBV), LCMV, HSV, Human Immunodeficiency Virus (HIV), Kaposi’s sarcoma-associated herpesvirus (KSHV), or Human Papilloma Virus (HPV). In some embodiments, the viral infection is an acute viral infection. In some embodiments, the acute viral infection is an infection from a virus selected from an influenza virus, West Nile Virus, Respiratory syncytial virus (RSV), a coronavirus, measles, Dengue virus, Ebola virus, Japanese encephalitis virus (JEV), or a rhinovirus.

[00250] In some embodiments, the infection treated by the methods described herein is a fungal infection. Exemplary pathogenic fungi that may lead to infection in a subject include, but are not limited to, Trichophyton, Epidermophyton, Candida, Micros porum. Aspergillus, Paecilomyces, Fusarium, Acremonium, Chaetomium, Phoma species, Scopulariopsis, Scytalidium, Alternaria, Epicoccum, and Curvularia. In some embodiments, the fungal infection is an infection from a fungal pathogen selected from Candida albicans, Aspergillus, Candida auris, Pneumocystis jirovecii, Cryptococcus neoformans, or Sporothrix.

[00251] In some embodiments, the infection treated by the methods described herein is a protozoan infection. In some embodiments, the protozoan infection is an infection from a protozoa selected from Giardia intestinalis, Entamoeba hystolitica, Cyclospora cayatanenensis, or cryptosporidium.

[00252] In some embodiments, the infection treated by the methods described herein is a parasitic infection. In some embodiments, the parasitic infection is an infection from a parasite selected from Taenia, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichomoniasis, Babesiosis, Blastocytosis, Cryptospridium, Trypanosomes, Trichonomas, Sarcocystis, Rhinosporodium, Malaria, Leishmania, Giardia, or an amoeban parasites. c. Cancer

[00253] In some embodiments, provided herein is a method of treating a cancer in a subject, the method includes administering to the subject a fusion protein described herein, comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), or a cell or population of cells that has been contacted ex vivo with the fusion protein. In some embodiments, the cell or population of cells are autologous to the cancer subject. In other embodiments, the cell or population of cells are allogeneic to the cancer subject.

[00254] In some embodiments, the cancer to be treated is a solid cancer. In some embodiments, the cancer to be treated is selected from brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, testicular cancer, or uterine cancer. In some embodiments, the cancer is a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., an angiosarcoma or chondrosarcoma), larynx cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, bartholin gland carcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondosarcoma, choriod plexus papilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenum cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing’s sarcoma, eye and orbit cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin’s disease, ileum cancer, insulinoma, intaepithelial neoplasia, interepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunum cancer, joint cancer, Kaposi’s sarcoma, pelvic cancer, large cell carcinoma, large intestine cancer, leiomyosarcoma, lentigo maligna melanomas, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin’s lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharynx cancer, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, urinary bladder cancer, urinary system cancer, uterine cervix cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulva cancer, well differentiated carcinoma, or Wilms tumor. In some embodiments, the cancer is non-Hodgkin’s lymphoma, such as a B-cell lymphoma or a T- cell lymphoma. In some embodiments, the non-Hodgkin’s lymphoma is a B-cell lymphoma, such as a diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In some other embodiments, the non-Hodgkin’s lymphoma is a T-cell lymphoma, such as a precursor T- lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer/T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma. In some embodiments, the cancer is a lung cancer, a kidney cancer, a bladder cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a pancreatic cancer, a stomach cancer, an esophageal cancer, a mesothelioma, a melanoma, a head and neck cancer, a thyroid cancer, a sarcoma, a prostate cancer, a glioblastoma, a cervical cancer, a leukemia, a lymphoma, a myeloma, or a hematologic malignancy. In some embodiments, the cancer is a cancer selected from breast cancer, pancreatic cancer, colorectal cancer, small cell lung cancer, a neuroendocrine tumor, rhadbomyosarcoma, hepatocellular carcinoma, ovarian cancer, prostate cancer, glioblastoma, osteosarcoma, melanoma, prostate cancer, non-small cell lung carcinoma, bladder cancer, kidney cancer, and head and neck cancer. In some embodiments, the cancer is non-metastatic. In some embodiments, the cancer is metastatic.

[00255] In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC)). In some embodiments, the cancer is breast cancer (e.g., triple negative breast cancer or HER2 -negative breast cancer). In some embodiments, the cancer is colorectal cancer.

[00256] In some embodiments, the method comprises administering an immune cell that has been contacted ex vivo with a fusion protein of the disclosure. In some embodiments, the immune cell is a T cell, e.g. , a CD4+ T cell, a CD8+ T cell, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, and/or an activated T cell. In some embodiments, the T cell is a genetically modified T cell, e.g., a CAR T cell, e.g., wherein the CAR comprises an extracellular domain comprising an antigenbinding site, wherein the antigen-binding site specifically binds an antigen on the surface of a cancer cell. d. Autoimmune Diseases and Disorders

[00257] In some embodiments, provided herein is a method of treating an autoimmune disease or disorder in a subject, the method comprising administering to the subject a fusion protein described herein comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), or a cell or population of cells that has been contacted ex vivo with the fusion protein. In some embodiments, the cell or population of cells are autologous to the subject having the autoimmune disease. In other embodiments, the cell or population of cells are allogeneic to the subject having the autoimmune disease.

[00258] In some embodiments, the autoimmune disease is a T-cell mediated autoimmune diseases, such as Type 1 diabetes, rheumatoid arthritis, LADA, multiple sclerosis, lupus, scleroderma pigmentosa, Myasthenia Gravis, Guillain Barre Syndrome, amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, or a chronic inflammatory disorder of the central nervous system. In some embodiments, the autoimmune disease is Type 1 diabetes. In some embodiments, the autoimmune disease is rheumatoid arthritis (e.g., stage 2 rheumatoid arthritis or stage 3 rheumatoid arthritis).

[00259] In some embodiments, the method comprises administering an immune cell that has been contacted ex vivo with a fusion protein of the disclosure. In some embodiments, the immune cell is a T cell, e.g., a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, and/or an activated T cell. In some embodiments, the T cell is a CD25+ CD4+ Treg. In some embodiments, the T cells (e.g., Tregs) are immunosuppressive effect, which allows for the treatment of an autoimmune disease or the prevention or alleviation of a symptom or manifestation thereof.

[00260] In some embodiments, the T cell is a genetically modified T cell, e.g., a CAR T cell, e.g., wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell. e. Methods of Administration

[00261] The site and method of administration of the fusion protein or the cell or population of cells will also depend on the disease or disorder being treated. In some embodiments, the administering is performed systemically. In some embodiments, the administering is performed locally. In some embodiments, the administering is performed intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intratracheally, intraperitoneally, intracranially, intramuscularly, intratumorally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

[00262] In some embodiments, the fusion protein or the cell or population of cells is administered before, during or after the occurrence of a disease or disorder described herein. Timing of administering the fusion protein or the cell or population of cells is optionally varied to suit the needs of the subject treated. Thus, in certain embodiments, the fusion protein or the cell or population of cells is used as a prophylactic and is administered continuously to a subject with a propensity to develop diseases or disorders in order to prevent the occurrence of the disease or disorder. In some embodiments the fusion protein or the cell or population of cells is administered to an individual during or as soon as possible after the onset of the symptoms. The administration of the fusion polypeptide or the population of cells is optionally initiated within the first 48 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration can be achieved by any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, and the like, or combination thereof. In some embodiments, the fusion protein or the cell or population of cells should be administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disorder, such as, for example, from more than 1 month to about 3 months. The length of treatment is optionally varied for each subject based on known criteria. In exemplary embodiments, the compound or a formulation containing the compound is administered for at least 2 weeks, between more than 1 month to about 5 years, or from more than 1 month to about 3 years. f. Additional Therapies

[00263] In some embodiments, the method comprises administering a fusion protein described herein comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), or a cell or population of cells as described herein in combination with an additional therapy. Administering the fusion protein or cell or population of cells in combination with another therapeutic agent (which also includes a therapeutic regimen) that may increase the therapeutic benefit to the subject. In some embodiments, regardless of the disorder, disease or condition being treated, the overall benefit experienced by the subject is additive of the combination or in other embodiments, the subject experiences a synergistic benefit.

[00264] The particular therapeutic agent to be combined with the fusion protein or the cell or population of cells will depend upon the diagnosis condition of the subject, and appropriate treatment protocol. The fusion protein or the cell or population of cells and the additional therapy are optionally administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of the patient, and the actual choice of compounds used. In certain instances, the determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is based on an evaluation of the disorder being treated and the condition of the subject.

[00265] In some embodiments, therapeutically effective dosages vary when therapies are used in treatment combinations. Methods for experimentally determining therapeutically effective dosages of therapeutic agents for use in combination treatment regimens are described in the literature. For example, the use of metronomic dosing, e.g., providing more frequent, lower doses in order to minimize toxic side effects, has been described extensively in the literature. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.

[00266] In some embodiments, a fusion protein described herein comprising an AKT1 polypeptide (e.g., as in Table 1) and a PTD (e.g., as in Table 2), or a cell or population of cells as described herein used in the methods described herein, are administered in combination with an agent that reduces cytokine pathway signaling. For instance, a fusion protein described herein may be administered sequentially with an agent that reduces cytokine pathway signaling. Without wishing to be limited by theory, the administration of the fusion polypeptide described herein would counter the effects of the agent in a subject, and vice-versa. The combination treatment may thus prevent over or under activation of cytokine pathway signaling in the subject. By administering both the fusion protein and the agent that reduces cytokine pathway signaling, cytokine pathway signaling may be fine-tuned so as to optimize the therapeutic benefit to the subject.

[00267] In some embodiments of the combination therapies described herein, dosages of the co-administered therapies vary depending on the type of therapy, on the disorder or condition being treated, and so forth. In addition, when co-administered with one or more biologically active agents, the compound provided herein is optionally administered either simultaneously with the biologically active agent(s), or sequentially. If simultaneously, the multiple therapeutic agents are optionally provided in a single, unified form, or in multiple forms. In certain instances, one of the therapeutic agents is optionally given in multiple doses. In other instances, both are optionally given as multiple doses. If not simultaneous, the timing between the multiple doses is any suitable timing, e.g. , from more than zero weeks to less than four weeks. In addition, the combination methods are not to be limited to the use of only two agents; the use of multiple therapeutic combinations are also envisioned.

[00268] In some embodiments, a dosage regimen to treat, prevent, or ameliorate the condition for which relief is sought, is modified in accordance with a variety of factors. These factors include the disorder from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, in various embodiments, the dosage regimen actually employed varies and deviates from the dosage regimens set forth herein. In some embodiments, the therapeutic agents are provided as a combined dosage form or in separate dosage forms for substantially simultaneous administration. In certain embodiments, the therapeutic agents that make up the combination therapy are administered sequentially, with either therapeutic compound being administered by a regimen calling for two-step administration. In some embodiments, two-step administration regimen calls for sequential administration of the agents or spaced-apart administration of the separate agents. In certain embodiments, the time period between the multiple administration steps varies, by way of nonlimiting example, from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the therapeutic agent. g. Subjects

[00269] In some embodiments, the subject to be treated is an animal, such as a mammal. In some embodiments, the mammal is a dog, cat, horse, cattle, dairy cow, swine, sheep, lamb, goat, primate, mouse, rat, or human. In some embodiments, the subject is a human.

[00270] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

EXAMPLES

[00271] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Example 1

[00272] This Example characterizes the role of AKT activity in the survival and proliferation of T lymphocytes.

Materials and Methods

In vitro lymphocyte cultures

[00273] Single cell suspensions were prepared from mouse spleens. T cells were purified by incubation with anti-CD4 coated magnetic beads (Dynal, Oslo, Norway) for 45 minutes at 4 °C. The non-adherent cells were discarded, and the cells bound to the beads were retrieved by incubation with a secondary antibody (mCD4 Detach, Dynal, Oslo, Norway). The T-cell suspensions were washed twice in PBS and resuspended to a final concentration of 2xl0⁷ cells/mL.

Construction and Production of Retroviruses

[00274] cDNAs encoding myristoylated AKT (Myr-AKT), dominant negative AKT (DN- AKT), or conditionally active AKT fused to the ligand binding domain of the estrogen receptor (AKT-ER) were cloned into the murine stem cell virus IRES green fluorescent protein (MIG) retroviral expression vector. The amino acid sequence of the Myr-AKT polypeptide comprised the amino acid sequence of SEQ ID NO: 9 followed by a linker comprising the amino acid sequence of SEQ ID NO: 63. The complete amino acid sequences of AKT constructs used in this example are set forth in Table 5. High titer retrovirus was obtained by transfecting 293T cells with retroviral plasmid DNA and the pCL-Eco packaging plasmid. Spin infections were performed at 2500 rpm for 1 h at 30 °C. Cells were infected twice within a 24-h period. Infection efficiency was determined by quantifying green fluorescent protein (GFP) expression by flow cytometry and was between 30 and 70% in all experiments.

[00275] Naive CD4+ T-cells were purified as described above and activated for 24 hours with antibodies to CD3 and CD28 at a final concentration of 1 pg/ml each. These activated T cells were infected with retrovirus produced in 293FT cells. Infection efficiency was monitored for 3 days after initial activation by determining the number of GFP expressing cells by flow cytometry. Retroviral vectors used are based on the bicistronic MSCV-IRES-GFP, which has been previously described. The viral constructs used include pMIG, pMIG-AKT-ER, and pMIG-DN-Akt. [00276] In some experiments, spleen and lymph node cells were first depleted of CD8 T cells by staining with CD8a microbeads and elution on an autoMACS column (Miltenyi Biotec, Auburn, CA). To obtain activated T cells, spleen and lymph node cells from wild type (C57BL/6) and IL-2" ' mice were activated with 1 pg/ml anti-CD3 Ab (BD PharMingen).

Table 5. AKT1 Construct Sequences

Survival assays

[00277] Activated C57BL6/J T cells that were left uninfected or infected with MIG, MIG- Myr AKT, MIG-DN-AKT, or MIG-AKT-ER were cultured in triplicate in a 96-well plate with or without 100 ng/ml IL-2, IL-4, IL-7, or IL-15 (BioSource International, Camarillo, CA). 4-OH tamoxifen (100 nM; “TMX;” Calbiochem, San Diego, CA) was added to some cultures of T cells infected with MIG-AKT-ER or MIG. T cell survival was assayed as follows.

[00278] Eirst, the percentage of cells that were viable at 0 h of culture was determined by flow cytometry (FACScan; BD Immunocytometry Systems, San Jose, CA) and at various subsequent time points (24, 48, and 72 h). Viable cells were distinguished from dead cells on the basis of their forward- and side-scatter characteristics. In preliminary studies, it was confirmed that this method for detecting live lymphocytes yielded results that lay within 3% of those obtained by other apoptosis assays (propidium iodide exclusion, 7AAD staining, and TUNEL assay). To determine the effect of modifying AKT activity with retroviruses on T cell survival, the percentage of viable cells that expressed GFP was also measured. Because the MIG retrovirus coordinately expresses GFP and the gene of interest (in these experiments, Myr-AKT, DN-AKT, or AKT-ER), this marker gene serves to identify retrovirally transduced cells. T cell survival at each time point (Tn) was calculated according to the following formula, which represents the ratio of viable cells at a given time point compared with the zero time point, multiplied by 100: [the percentage of viable cells (at T_n) X the percentage of GFP+ cells (at T_n)]/[the percentage of viable cells (at To) X the percentage of GFP+ cells (at To)] X 100. Western blotting

[00279] To study the activation of AKT in T cells, C57BL6/J T cells were activated for 3 days, starved in medium (RPMI supplemented with 10% FBS) overnight, and then cultured in the presence of 100 ng/ml IL-2, IL-4, IL-7, or IL-15 (BioSource International) for 30 min. T cells infected with pMIG, pMIG MyrAkt, or pMIG AKT-ER were harvested for Western blot analysis 24 or 72 h after the last infection. In experiments with DN-AKT, GFP^Wgh populations were isolated by high-speed cell sorting. In experiments with AKT-ER, T cells were cultured with or without 100 nM of TMX. All cells were lysed as previously described, run on a 12% SDS -polyacrylamide gel, and blotted on a polyvinylidine difluoride membrane. Blots were blocked overnight (TBST + 5% milk) and probed with antibodies to AKT or phospho-AKT (S473; Cell Signaling Technology, Beverly, MA), Bcl-2 (dC21; Santa Cruz Biotechnology, Santa Cruz, CA), or [3-actin (Sigma-Aldrich) and, subsequently, with a goat anti-rabbit or mouse HRP-conjugated antibody (Santa Cruz Biotechnology). All blots were developed with a luminol reagent (Santa Cruz Biotechnology) and exposed to film (Kodak, Rochester, NY).

Results

IL-2 family cytokines promote T-cell survival and activate AKT

[00280] AKT acts downstream of cytokine receptors (e.g., IL-2R) to stimulate cell proliferation and survival. To evaluate the role of AKT activity in the activation of T cells, activated C57BL6/J CD4+ T cells were cultured ex vivo with or without one of four IL-2 -family cytokines (IL-2, IL-4, IL-7, or IL- 15). T-cell survival and proliferation were measured by monitoring the number of viable T cells by flow cytometry every 24 hours. AKT activation and Bcl-2 expression were measured via western blotting with a phospho-AKT-specific antibody and a Bcl-2-specific antibody, respectively, following a 30-minute treatment with the indicated cytokine. As shown in FIG. 3A, treatment with any of the four cytokines promoted the expansion of activated T cells ex vivo. Cytokine-stimulated T-cell expansion was correlated with Akt phosphorylation and Bcl-2 expression (FIGs. 3B-3C).

A dominant negative AKT blocks cytokine -mediated T-cell survival signaling

[00281] To test whether AKT activity is necessary for cytokine-mediated T-cell expansion, activated C57BL6/J CD4+ T cells were retrovirally transduced with a vector encoding a dominant negative form of AKT (“DN AKT”) or an empty vector control (“MIG”). Activated, transduced T cells were cultured ex vivo in media supplemented with one of four IL-2 family cytokines (IL-2, IL-4, IL-7, or IL-15). Live, transduced (GFP+) cells were quantified by FACS every 24 hours to monitor T-cell viability. As summarized in FIG. 4, expression of dominant negative AKT blocked common gamma chain cytokine receptor derived survival signals.

AKT activation stimulates T-cell proliferation and survival in the absence of cytokine stimulation

[00282] To test whether AKT activity could stimulate T-cell proliferation and survival in the absence of cytokine stimulation, activated C57BL6/J IL-2 " CD4+ T cells were retrovirally transduced with a vector encoding a constitutively active form of Akt (“Myr-AKT” or “Akt*”), a conditionally active form of Akt (“AKT-ER”), or an empty vector control (“MIG”). Myr-Akt comprises an N-terminal myristoylation signal derived from Src, which promotes membrane association of the protein and leads to constitutive phosphorylation and activation. AKT-ER comprises Myr-Akt with a C-terminal fusion to a mutated portion of the estrogen receptor, which enables the AKT-ER fusion protein to be conditionally active in the presence of the synthetic steroid 4-Hydroxytamoxifen (“TMX”). The transduced T cells were cultured for 3 days ex vivo (with or without TMX, as appropriate). Live transduced (GFP+) cells were quantified by FACS every 24 hours to track T cell expansion, and AKT activation was measured via western blotting with a phospho-AKT-specific antibody after 24 and 72 hours.

[00283] As shown in FIG. 5A, expression of the constitutively active Myr-Akt fusion protein promoted T-cell survival and proliferation, even in the absence of cytokine stimulation. Similarly, expression of the conditionally active AKT-ER construct promoted T-cell survival and proliferation in the presence of TMX (FIG. 5B). AKT-ER-stimulated T-cell expansion was correlated with Akt phosphorylation (FIG. 5C).

Example 2

[00284] This example describes the ability of T cells expressing constitutively active Aktl to inhibit Non-Hodgkin’s Lymphoma (NHL) tumor formation. This example also demonstrates that constitutively active Aktl (e.g., MyrAkt) can restore antigen responsiveness in an anergic lymphoid cell population. Materials and Methods

Transgenic mice and transplantation of tumors

[00285] Mice carrying the E i -MY C transgene were obtained from the Jackson

Laboratory. These mice express MYC in a B cell-specific manner, beginning at the Pre/Pro-B cell stage. BCR^HEL mice (MD4), sHEL mice (ML5), and 3A9 mice were also obtained. MD4 mice express a pre-rearranged murine BCR from the endogenous immunoglobulin promoter, and ML5 mice ubiquitously express a transgene for the soluble form ofHEL under the control of the metallothionein promoter. 3A9 mice carry a T cell receptor transgene specific for HEL. All transgenic mouse lines were maintained on a C57/BL6 background, and were genotyped by PCR.

Adoptive transfers of cells and transplantation of tumors

[00286] Adoptive transfers and transplantation of tumors were done by injecting 106 cells intravenously (unless otherwise indicated) into syngeneic (C57/BL6) female mice ranging in age from 4-6 weeks. Primary CD4+ T cells were also used for transfer studies. These were first cultured and retrovirally transduced with the indicated retrovirus. Transfer studies were done by injecting 5xl0⁶ cells/mouse intravenously.

Assessment of tumorigenesis

[00287] The emergence of tumors was followed in two ways: (i) physical examination of living animals and necropsy of deceased animals, particularly to detect enlargement of lymphoid organs and viscera, and (ii) counting the total number of cells in organs. Three pairs of lymph nodes were collected each time (two inguinal, two axillary, and two brachial lymph nodes). These lymph nodes were pooled and processed into single-cell suspensions. Spleens were also collected and used to generate single-cell suspensions. Each spleen was individually ground on a 60-hn wire mesh screen (Sigma). The red blood cells were lysed in TAC buffer (0.017 M Tris, pH 7.65, and 0.135 M NHrCl), and the resulting pellets were resuspended in complete lymphocyte media, (RPMI1640 supplemented with 10% heat inactivated fetal calf serum, supplemented with L-glutamine, penicillin/streptomycin, nonessential amino acids, 2 mM HEPES, 2 mM sodium pyruvate, and 10 mM b-mercaptoethanol; all obtained from Invitrogen). Single-cell suspensions were counted with a Coulter counter (Coulter Diagnostics). The percentage of viable cells was determined by uptake of 7-aminoactinomycin D (7AAD) and flow cytometry. The values for total cell numbers were used to derive the number of viable cells by multiplying percentage of viable cells (obtained from the 7AAD analysis) by the total number of cells (obtained from the Coulter counter analysis) and dividing by 100. These measurements were compared with microscopic counting of trypan-blue excluding cells in a hemocytometer.

T cell purification and in vitro proliferation

[00288] Naive CD4+ T cells were purified from pooled spleen and lymph nodes harvested from c-myc mutant mice. T cell preparations were typically 96% CD4+, as determined by staining and flow cytometry. CarboxyFluorescein Succinimidyl Ester (CFSE) labelling was performed by washing the purified, naive CD4+ T cells twice in PBS. The cells were incubated with 10 pM CFSE in PBS for 7 minutes in the dark. The labelling reaction was quenched with an equal volume of FCS and washed twice in complete lymphocyte media. Proliferative responses to antigen were determined by intracellular fluorescent dye staining the cells before they were incubated in RPMI 1640 supplemented with 1 mM L-glutamine, penicillin/streptomycin, nonessential amino acids, sodium pyruvate and Hepes (Gibco/BRL, Grand Island, NY) and 10% FCS, and the indicated mitogenic stimuli. TCR induced proliferation was assayed by incubating 106 CFSE-stained CD4+ T cells with 1 pg/ml soluble anti-CD3 (clone 2C11, Pharmingen) and 10 pg/ml soluble anti-CD28 (clone 37N1, Pharmingen), for three days, and determining cell division number by flow cytometry.

[00289] Assays in which doxycycline was added to the cultures contained 100 ng/ml of doxycycline (Sigma, St. Louis MO) added to the media.

Retroviral vectors and lymphocyte infections

[00290] Naive CD4+ T cells were purified as described above, and activated for 24 hours with antibodies to CD3 and CD28. These activated T cells were infected with retrovirus containing supernatant produced in BO SC 23 cells. Infection efficiency was monitored 3 days after initial activation by flow cytometry. The retroviral vectors were based on the bicistronic MSCV-IRES-GFP. The viral constructs used included pMIG, pMIG-cMyc (generated by introducing the cDNA for human c-myc into the EcoRl site of the pMIG polylinker, or pMIG- Akt*. Experimental Design

[00291] The details of the experimental design for the study are described in Table 6 below.

Table 6: Experimental design

Results

T cells expressing constitutively active Aktl can inhibit NHL tumor development

[00292] sHEL-expressing primary tumor cells derived from an Ep-MY C/MD4/ML5 mouse line were transferred into C57BL6/J mice, either alone or in combination with (i) T cells derived from a wild-type mouse (“WT”) or antigen-specific T cells (expressing an anti-HEL TCR transgene; “3A9”). Prior to transplantation, the T cells were retrovirally transduced with a vector encoding a constitutively active Akt (“pMIG-Akt*”), a vector encoding Bcl2 (“pMIG- Bcl2”), or an empty vector as a control (“pMIG”). Non-transduced T cells were used as a further control. After 28 days, tumor cells were quantified in mouse lymph nodes and spleens. [00293] As summarized by the left four columns of FIG. 6, mice inoculated with tumor cells in combination with wild type T cells recapitulated the primary tumor, irrespective of what gene the T cells were transduced to express (FIG. 6, left four columns). Antigen-specific T cells that (i) were not retrovirally transduced or (ii) that were transduced with an empty vector control failed to inhibit tumor formation (FIG. 6, 5th and 6th columns from left). In contrast, antigenspecific T cells transduced with constitutively active Akt (but not Bcl-2) were able to inhibit NHL tumor formation (FIG. 6, 7th and 8th columns from left).

Constitutively active Aktl restores antigen responsiveness in a previously anergic T cell population

[00294] In order to test the ability of constitutively active Aktl expression to overcome T- cell anergy, C57BL6/J mice were inoculated with sHEL-expressing primary tumor cells (Ep- MYC/MD4/ML5) in combination with antigen-specific 3A9/ML5 T cells. 3A9/ML5 T cells express an anti-HEL TCR and are derived from a mouse which systemically expresses the HEL antigen, which renders the T cells anergic. Prior to transplantation, the anergic T cells were retrovirally transduced with a vector encoding a constitutively active Akt (“pMIG-Akt*”), a vector encoding Bcl2 (“pMIG-Bcl2”), or an empty vector as a control (“pMIG”). Nontransduced T cells were used as a control. After 28 days, tumor cells were quantified in mouse lymph nodes and spleens.

[00295] As summarized by the rightmost four columns of FIG. 6, the non-transduced anergic T cells failed to inhibit NHL tumor formation. By contrast, retroviral transduction with constitutively Aktl (but not Bcl-2) enabled the T cells to inhibit NHL tumor formation. These results indicate that Akt* is able to reverse the anergic phenotype and reestablish antigen reactivity.

Example 3

[00296] This example demonstrates that a PTD-cytokine fusion protein is able to confer a survival advantage to T cells in the absence of exogenous cytokines.

Materials and Methods

Cloning of pTAT-BCL2-V5-6xHis

[00297] The plasmid pTAT-BCL2-V5-6xHis was made by PCR amplification of the coding regions for human BCL2 using a forward primer that contains an in-frame N-terminal 9- amino-acid sequence of the TAT protein transduction domain of HIV- 1 (RKKRRQRRR (SEQ ID NO: 11)), and a reverse primer that removed the stop codon. The PCR product was then cloned into pETlOl/D-Topo (Invitrogen) vector, which includes a C-terminal V5 epitope and 6x-histidine (SEQ ID NO: 56) purification tag.

Protein induction and purification

[00298] BL-21 RARE cells were created by transforming BL-21 Star E. coli strain (Invitrogen) with pRARE (CamR), isolated from BL21 Rosetta cells (Novagen), that express tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons.

[00299] The plasmid pTAT-BCL2-V5-6xHis was transformed into BL21 RARE cells, and grown on TB/Amp/Cam plate at 37 °C overnight. An isolated colony was used to inoculate a 5 mb TB/Amp/Cam starter culture, and grown at 37 °C overnight. 1 liter of TB/Amp/Cam broth was inoculated with the 5 mb starter culture, grown to an OD600 of 0.5, and induced with 0.5 mM IPTG at 37 °C for 3 hrs. Bacterial cells were pelleted by centrifugation. The cell pellet was resuspended in lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris pH to 8.0) and lysed at room temperature overnight on a shaker. The lysate was cleared by centrifugation at 29,000 x g for 30 min, and the supernatant was applied to a His-TRAP nickel affinity column (GE). The column was washed with 10 volumes of lysis buffer containing 50 mM imidazole followed by elution with lysis buffer containing 200 mM imidazole. Protein was dialyzed in a stepwise fashion into dialysis buffer (500 mM NaCl, 50 mM NaH2PO4, pH 7.0 10% glycerol, pH 7.5). The dialysis went as follows: 2 hours in dialysis buffer containing 4 M urea, 2 hours in buffer with 2 M urea, then overnight in dialysis buffer alone. Purity and size of proteins were verified using SDS-PAGE electrophoresis and either Coomassie blue staining or western blot with anti- V5 (1:5000; Invitrogen) or anti-BCL2 (N-262, 1:2000; Santa Cruz Biotechnology) antibodies. Protein concentration was measured by Bradford protein assay (Sigma) compared to a standard curve of bovine serum albumin.

In vitro lymphocyte cultures and T-cell survival assays

[00300] Single cell suspensions were prepared from mouse spleens. T-cells were purified by incubation with anti-CD4 coated magnetic beads (Dynal, Oslo, Norway) for 45 minutes at 4 °C. The non-adherent cells were discarded, and the cells bound to the beads were retrieved by incubation with a secondary antibody (mCD4 Detach, Dynal, Oslo, Norway). The T-cell suspensions were washed twice in PBS, resuspended to a final concentration of 2xl0⁷ cells/mL. T-cells were activated in vitro with 1 mg/mL of anti-CD3 (Pharmingen) for 1 day. 72 hours post-activation, the live T-cells were isolated by centrifugation on a ficoll cushion. The wells were then washed and replated. The live activated T cells were either left untreated or treated with 25 pg/mL Tat-BCL2. 48 hours after Tat-fusion protein treatment, apoptosis was analyzed by 7AAD staining and flow cytometry, and the results were summarized as a dose response curve.

Results

[00301] In order to determine whether a PTD-cytokine fusion can promote T cell survival, activated C57BL6/J CD4+ T cells were treated ex vivo with a fusion protein comprising Bcl2 with an N-terminal fusion to the PTD Tat (“Tat-BCL2”) at the indicated concentrations. Untreated T cells were used as a negative control. 48 hours after Tat-fusion protein treatment, apoptosis was analyzed by 7AAD staining and flow cytometry. As summarized in FIG. 7, treatment with Tat-BCL2 conferred a survival advantage to the activated CD4+ T cells.

Example 4

[00302] This example describes the manufacture of a fusion protein comprising (i) a constitutively active form of Akt (Myr-Akt) comprising a fragment of Aktl with an N-terminal fusion of a myristoylation site derived from Src, and (ii) a Tat PTD.

[00303] A nucleic acid encoding PTD-MyrAkt was cloned into an expression vector comprising a T7 inducible promoter. The constructed plasmid also comprised an in-frame His- tag. The plasmid encoded an AKT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 26, and the complete polypeptide sequence (inclusive of tags) corresponds to the amino acid sequence of SEQ ID NO: 29. Purified plasmid was transformed into chemically competent BL21(DE3) E. colt cells via heat shock and plated on non-inducing agar with 100 mg/L kanamycin. Plates were incubated overnight at 37 °C. One colony from each transformation was picked and grown into 0.82 mb of non-inducing medium comprising 100 mg/L kanamycin and incubated overnight at 37 °C. The overnight culture was inoculated into 0.6 mb of autoinduction media (comprising 0.5% glycerol, 0.05% glucose, 0.2% lactose, 100 mg/L kanamycin). Three sets of auto induction media were inoculated from the same starter culture and incubated at three different temperatures: 20 °C cultures were grown for 27 hours, 30 °C cultures, and 37 °C cultures were grown for 6 hours. Cells were harvested by centrifugation and frozen. [00304] Cell pellets were lysed and then centrifuged to separate total and soluble factions. The total and soluble fractions were denatured and run on a polyacrylamide gel under reducing conditions. Coomassie stained SDS gels were scanned and expression levels were estimated by densitometry analysis of protein bands relative to standards of known protein concentration. Commercially available gel image software was used to quantify the intensity of observed bands. E. coli transformed with a vector comprising a non-coding gene sequence or a vector expressing a known-molecular-weight soluble 6His-tagged protein were used as negative and positive controls, respectively. As shown in FIGs. 8A-8B, the PTD-MyrAkt fusion protein was successfully produced.

Example 5

[00305] This example describes the manufacture of a fusion protein comprising (i) a constitutively active form of Akt (Myr-Akt) comprising a fragment of Aktl with an N-terminal fusion of a myristoylation site derived from Src, and (ii) a Tat4 PTD.

[00306] The amino acid sequence of a PTD4-MyrAkt construct was reverse translated and optimized for bacterial expression, and the resulting DNA sequence was synthesized and cloned into a self-inducible bacterial expression vector (pD451SR; ATUM). The constructed plasmid also comprised an in-frame His-tag. The resulting plasmid encoded an AKT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 10 (MyrAkt) and an HIV PTD comprising the amino acid sequence of SEQ ID NO: 12. The encoded fusion polypeptide comprised the amino acid sequence of SEQ ID NO: 26, and the complete polypeptide sequence (inclusive of tags) corresponds to the amino acid sequence of SEQ ID NO: 29.

[00307] BL21(DE3) E. coli cells were transformed with the plasmid by electroporation and grown overnight at 18 °C in autoinduction media. Cells were collected by centrifugation and washed in saline. The pellets were lysed using a high pressure homogenizer, and protein was purified using the following chromatography columns: Ni-His 60 column (affinity); Q-HP (ion exchange and endotoxin removal); Source 15-Q; Superdex 2000 (gel filtration). The resulting purified protein was analyzed by spectrophotometer (280 nm), SE-HPLC, mass spectrometry, and SDS PAGE (FIG. 9). As shown in FIG. 9, the PTD-MyrAkt fusion protein was successfully produced and purified. Example 6

[00308] This example describes the ability of a PTD4-MyrAkt fusion protein to promote cell survival during T cell activation ex vivo, and to promote expansion of activated T cells ex vivo in the absence of added cytokines.

Materials and Methods

[00309] Spleens and lymph nodes (pairs of axillary, inguinal, and sacral) were obtained from two female C57BL6/J mice (6 weeks old). Lymphoid organs were homogenized using a sieve and a single cell suspension was generated. Cells were washed in PBS, and red blood cells were lysed using a hypotonic buffer. The resulting white blood cell population was plated at 2xlO^A6 cells/ml in 24 well plates (1 ml/well).

[00310] White blood cells were activated via the addition of PMA (10 ng/ml) and ionomycin (250 ng/ml) in the presence or absence of recombinant purified PTD4-MyrAkt produced in Example 5 (0.5 pg/ml or 2.5 pg/ml), or in the presence of 2.5 pg/ml of denatured protein (incubated at 92 °C for 12 minutes). The cells were incubated at 37 °C and 5% CO2 for 72 hours. Cells were then collected, washed in PBS, and incubated in 7AAD to measure number of apoptotic cells through 7AAD uptake. Cells were analyzed by FACS.

[00311] Additionally, the primary T cells that were activated in the absence of added PTD4-MyrAkt protein were collected, washed twice in PBS, live-cell -enriched by Ficoll cushion centrifugation, and washed twice in media. The cells were subsequently plated at 10^A6 cells/ml in either media alone, or in media supplemented with PTD4-MyrAkt (0.5, 1.0, 2.0, or 4.0 pg/ml). Cells were washed in media. Cells were then incubated at 37 °C and 5% CO2 for 48 hours.

Cells were analyzed by FACS-evaluating their forward- and side-scatter characteristics to quantify viable cells, as described in Example 1.

Results

[00312] In order to determine whether PTD4-MyrAkt fusion protein can promote cell survival during T cell activation, primary lymphocytes obtained from C57BL6/J mice were activated for 72 hours with PMA and ionomycin in the presence or absence of PTD4-MyrAkt, or in the presence of denatured protein as a negative control. Cells were stained with 7AAD and analyzed by FACS to measure apoptotic cells. As shown in FIG. 10, incubation with 2.5 pg/ml PTD4-MyrAkt reduced the number of apoptotic cells after 3 days of T-cell activation. [00313] In order to determine whether treatment with PTD4-MyrAkt fusion protein stimulates T-cell expansion in the absence of added cytokines, primary T cells that were activated in the absence of PTD4-MyrAkt were collected, live-cell -enriched, and incubated in medium with or without PTD4-MyrAkt for 48 hours, or in medium supplemented with denatured protein as a negative control. Viable cells were quantified by FACS-evaluation of forward and side scatter characteristics. As shown in FIG. 11, treatment of activated primary murine T cells with PTD4-MyrAkt promoted cell expansion in the absence of activated cytokines.

Example 7

[00314] This example describes the ability of a PTD-MyrAkt fusion protein (corresponding to SEQ ID NO: 29) to promote survival and proliferation of T cells, as compared to exogenous cytokines. Briefly, primary CD4+ T cells were incubated with either purified PTD4-MyrAkt produced as described in Example 5 (0.5, 1.0, 2.0, or 4.0 pg/ml) or with human IL-2 (50 U/mL), using the protocol described in Example 6. Cells incubated with medium alone, with denatured PTD4-MyrAkt, or with heat-inactivated IL-2 were used as negative controls. Viable cells were quantified by FACS-evaluation of forward and side scatter characteristics.

[00315] As shown in FIG. 12 rand as summarized in Table 7, treatment of primary CD4+ T cells with PTD4-MyrAkt promoted T-cell survival and expansion ex vivo in a dose-dependent manner. Moreover, the positive effect on survival and expansion was greater than that resulting from treatment with exogenous IL-2, when PTD4-MyrAkt was used at concentrations greater than 0.5 pg/mL. These results demonstrate that treatment with the constitutively active, recombinant protein can promote T-cell survival and proliferation better than treatment with exogenous IL-2.

Table 7: Experimental results from Example 7 ex vivo study

Example 8

[00316] This example describes the ability of a PTD-MyrAkt fusion protein (corresponding to SEQ ID NO: 29) to support lymphocyte-based tumor-killing in vivo. Briefly, mice were injected intravenously with 200,000 MC38 colorectal cancer cells. 7 days later, spleens and lymph nodes were collected from certain of the tumor-bearing mice (4 pairs of lymph nodes from every mouse: axillary and brachial, inguinal, and cervical). The spleens and lymph nodes were pushed through a fine metal screen and resuspended in saline to generate a single cell suspension. Red blood cells were lysed by hypotonic methods, and cells were then washed twice in complete lymphocyte media (RPMI-based). Cells were incubated for 1 hour at 37 °C with either PTD-MyrAkt (2.5 pg/mL; produced as described in Example 5), or with a fusion protein comprising the cytokine signaling pathway protein MY C fused to an HIV Tat PTD (“Tat-MYC;” 25 pg/mL). The amino acid sequence of the Tat-MYC fusion protein was: MRKKRRQRRRMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSED IWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGG DMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARG HSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTES SPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKP PHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSD TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAE EQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH (SEQ ID NO: 65).

[00317] Cells were washed twice in saline and administered intravenously to MC38- treated mice at a dose of 5xl0^A5 cells/kg (10 mice per treatment group). Two additional control groups were also included in the study including (1) a wild type (“WT”) group of mice that did not undergo intravenous injection of colorectal cancer cells and (2) a no treatment (“No-Tx”) group of mice that received intravenous injection of colorectal cancer cells, but did not receive a subsequent treatment involving a fusion protein (e.g., no PTD-MyrAkt or Tat-MYC).

[00318] All animals were maintained for observation and followed at least once a day. Specifically, mice were monitored for survival and for externally evident clinical signs of disease (scruffy fur, hunched posture, labored breathing, difficulty walking, externally evident lymphadenopathy or splenomegaly). Mice were to be euthanized if found with at least 4 of the externally evident clinical signs, although no mice ultimately needed to be euthanized during the course of the study. The number of surviving mice overtime is summarized in FIG. 13.

[00319] As shown in FIG. 13, administration of cells treated with either fusion protein (PTD-MyrAkt or Tat-MYC) significantly improved survival in comparison to the No-Tx group. Moreover, administration of PTD-MyrAkt-treated cells resulted in a significantly improved rate of survival as compared to administration with Tat-MY C-treated cells, even though PTD- MyrAkt was used at a 10-fold lower concentration than Tat-MYC.

Example 9

[00320] This example describes the ability of a PTD-MyrAkt fusion protein (corresponding to SEQ ID NO: 29) to support expansion of human regulatory T cells. Briefly, affinity-purified CD4+CD25+ human regulatory T cells (Tregs) were isolated from the peripheral blood of two normal healthy human volunteers (“Nl”) and from four patients who had each previously been diagnosed with stage 2 or stage 3 rheumatoid arthritis (“utf-##”). The purified Tregs were cultured in U-bottom 96-well plates, which had been coated with antihuman CD3 and anti-human CD28 antibodies by overnight incubation at 4 °C. The Tregs (100,000 Tregs/well) were cultured in RPMI medium supplemented with 10% heat inactivated fetal calf serum, 2 mM L-glutamine (Invitrogen), 100 units/ml penicillin G and streptomycin sulfate (Invitrogen), 10 mM HEPES, 0.1 mM MEM non-essential amino acids (Invitrogen), and 0.55 mM P-mercaptoethanol (Invitrogen). The culture medium was supplemented with either IL-2 (20 U/mL or 100 U/ml), the PTD-MyrAkt fusion protein (1 pg/ml or 5 pg/mL), or the Tat- MYC fusion protein described in Example 8 (10 pg/mL or 50 pg/mL). Tregs that were not supplemented with IL-2 or fusion protein were used as a control. The Tregs were cultured for 5 days and then labelled with a CCK8 reagent for 4 hours to determine the number of live cells. Plates were then analyzed by determining their optical density at UV 450 nm. [00321] As shown in FIG. 14, PTD-MyrAkt treatment promoted expansion of human Tregs from both healthy volunteers and rheumatoid arthritis patients. Additionally, treatment with PTD-MyrAkt was more effective at promoting Treg expansion than treatment with exogenous IL-2, regardless of the source of the Tregs. Moreover, the PTD-MyrAkt protein’s positive effect on Treg expansion was comparable to the effect resulting from treatment with the Tat-MYC construct, even though PTD-MyrAkt was used at a 10-fold lower concentration than Tat-MYC.

Claims

CLAIMS What is claimed is:

1. A fusion protein comprising:

(a) a signaling activator comprising a constitutively active AKT1 polypeptide or a functional fragment or variant thereof, and

(b) a protein transduction domain (PTD).

2. The fusion protein of claim 1, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 29, 26, 24, 25, 27, 28, and 64.

3. The fusion protein of claim 1 or 2, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof is phosphatase resistant.

4. The fusion protein of any one of claims 1-3, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution and/or an amino acid sequence that facilitates sequestration of the fusion protein at the plasma membrane.

5. The fusion protein of any one of claims 1-4, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises an Src myristoylation sequence.

6. The fusion protein of claim 5, wherein the Src myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 5 or 6.

7. The fusion protein of claim 1, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a Gag myristoylation sequence.

8. The fusion protein of claim 7, wherein the Gag myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 7.

9. The fusion protein of claim 1, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of:

(i) a glutamate residue at a position corresponding to position 17 of wild-type human AKT1 (E17);

(ii) a leucine residue at a position corresponding to position 52 of wild-type human AKT1 (L52);

(iii) a cysteine residue at a position corresponding to position 77 of wild-type human AKT1 (C77); (iv) a glutamine residue at a position corresponding to position 79 of wild-type human AKT1 (Q79); and/or

(v) a glycine residue at a position corresponding to position 171 of wild-type human AKT1 (G171).

10. The fusion protein of claim 9, wherein:

(i) the glutamate residue at a position corresponding to position 17 of wild-type human AKT1 is substituted by lysine (E17K);

(ii) the leucine residue at a position corresponding to position 52 of wild-type human AKT1 is substituted by arginine (L52R);

(iii) the cysteine residue at a position corresponding to position 77 of wild-type human AKT1 is substituted by phenylalanine (C77F);

(iv) the glutamine residue at a position corresponding to position 79 of wild-type human AKT1 is substituted by lysine (Q79K); and/or

(v) the glycine residue at a position corresponding to position 171 of wild-type human AKT1 is substituted by arginine (G171R).

11. The fusion protein of any one of claims 1-10, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a deletion of the pleckstrin homology (PH) domain of AKT1.

12. The fusion protein of claim 11, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a deletion of the residues corresponding to residues 4 through 129 of wild-type AKT1.

13. The fusion protein of any one of claims 1-12, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution that prevents AKT-induced neoplasia.

14. The fusion protein of any one of claims 1-13, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a threonine residue at a position corresponding to position 308 of wild-type human AKT1 (T308).

15. The fusion protein of claim 14, wherein the threonine residue at a position corresponding to position 308 of wild-type human AKT1 is substituted by aspartic acid (T308D).

16. The fusion protein of any one of claims 1-15, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution of a serine residue at a position corresponding to position 473 of wild-type human AKT1 (S473).

17. The fusion protein of claim 16, wherein the serine residue at a position corresponding to position 473 of wild-type human AKT1 is substituted by aspartic acid (S473D).

18. The fusion protein of any one of claims 1-6, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises the amino acid sequence of any one of SEQ ID NOs: 2-4 or 8.

19. The fusion protein of any one of claims 1-6 or 18, wherein the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises the amino acid sequence of any one of SEQ ID NOs: 9-10 or 61.

20. The fusion protein of any one of claims 1-19, wherein the PTD comprises a cationic PTD, a hydrophobic PTD, or a cell-type specific PTD.

21. The fusion protein of any one of claims 1-20, wherein the PTD comprises a cationic PTD.

22. The fusion protein of any one of claims 1-21, wherein the PTD comprises a VP- 16 peptide, an antennapedia peptide, a PTD-5 peptide, a polylysine peptide, a polyarginine peptide, an HIV VPR peptide, an HIV Tat peptide, or a functional variant of any of the foregoing.

23. The fusion protein of any one of claims 1-22, wherein the PTD comprises an HIV-1 Tat peptide or a functional variant thereof.

24. The fusion protein of any one of claims 1-23, wherein the PTD comprises the amino acid sequence of SEQ ID NO: 11 or 12.

25. The fusion protein of any one of claims 1-20, wherein the PTD comprises a hydrophobic PTD.

26. The fusion protein of any one of claims 1-20 or 25, wherein the PTD comprises a transportan peptide, a MAP peptide, a TP 10 peptide, or a functional variant of any of the foregoing.

27. The fusion protein of any one of claims 1-6 or 18-19, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 24-40 and 64.

28. The fusion protein of claim 2, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 26.

29. The fusion protein of claim 2 or 28, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 29.

30. A nucleic acid encoding the fusion protein of any one of claims 1-29.

31. A vector comprising the nucleic acid of claim 30.

32. A cell comprising the vector of claim 31.

33. The cell of claim 32, wherein the cell is a bacterial cell.

34. A pharmaceutical composition comprising the fusion protein of any one of claims 1-29 and a pharmaceutically acceptable carrier or excipient.

35. A composition comprising the fusion protein of any one of claims 1-29 and an immune cell.

36. The composition of claim 35, further comprising a pharmaceutically acceptable carrier or excipient.

37. A method of preparing a cell therapeutic composition, the method comprising a step of contacting an immune cell with the fusion protein of any one of claims 1-29.

38. The method of claim 37, further comprising cry opreserving the cell therapeutic composition.

39. The method of claim 38, further comprising thawing the cell therapeutic composition.

40. The method of claim 38 or 39, wherein the step of contacting the immune cell with the fusion protein occurs prior to cryopreservation.

41. The method of claim 39, wherein the step of contacting the immune cell with the fusion protein occurs after thawing the cell therapeutic composition

42. The method of any one of claims 39-41, wherein the thawed immune cell exhibits increased surface expression of CD25, CD44, and/or CD69, as compared to a frozen and thawed immune cell that was not contacted with the fusion protein.

43. The method of any one of claims 37-42, wherein the contacting step comprises contacting the immune cell with a medium comprising 0.05-500 pg/mL of the fusion protein.

44. A cell therapeutic composition generated by the method of any one of claims 37-43.

45. A method of genetically modifying an immune cell, the method comprising:

(a) contacting an immune cell with the fusion protein of any one of claims 1-29, thereby generating an activated immune cell; and

(b) contacting the activated immune cell with a vector encoding a gene of interest.

46. The method of claim 45 wherein, prior to step (a), the immune cell is in a resting state.

47. The method of claim 45 or 46, wherein the step of contacting the immune cell with the fusion protein induces the immune cell to enter the G1 phase of the cell cycle.

48. The method of any one of claims 45-47, wherein the vector is a viral vector.

49. The method of claim 48, wherein the viral vector is an adenoviral vector or a retroviral vector (e.g., atype-C retroviral vector).

50. The method of any one of claims 45-47, wherein the vector is RNA.

51. The method of any one of claims 45-47 or 50, wherein step (b) comprises contacting the cell with a liposome encapsulating the vector.

52. A method of expanding an immune cell in a culture, the method comprising:

(a) contacting the immune cell with a growth medium comprising a mitogenic stimulus, and

(b) contacting the immune cell with the fusion protein of any one of claims 1-29.

53. The method of claim 52, wherein the mitogenic stimulus is an anti-CD3 antibody and/or an anti-CD28 antibody.

54. The method of claim 52 or 53, wherein the growth medium further comprises one or more cytokines.

55. The method of claim 54, wherein the one or more cytokines comprise IL-2, IL-4, IL-7, and/or IL- 15.

56. The method of any one of claims 52-55, wherein the immune cell is incubated in the growth medium for at least 3 days.

57. The method of claim 56, wherein the immune cell is incubated in the growth medium for 3 to 5 days.

58. The method of any one of claims 54-57, wherein additional copies of the fusion protein and/or the one or more cytokines are added to the culture every 72-120 hours.

59. The method of any one of claims 52-58, wherein steps (a) and (b) are carried out simultaneously.

60. The method of claim 59, wherein the growth medium comprises the fusion protein and the mitogenic stimulus.

61. The method of any one of claims claim 52-58, wherein step (a) is carried out prior to step (b).

62. The method of claim 61, wherein step (b) comprises incubating the immune cell in a medium comprising the fusion protein for at least 5 minutes, e.g., at least 5, 15, 30, 45, or 60 minutes.

63. The method of claim 62 wherein, following step (b), the immune cell is removed from the medium comprising the fusion protein, washed, and incubated in a second growth medium comprising the mitogenic stimulus.

64. The method of claim 63, wherein the second growth medium is the same growth medium used in step (a).

65. The method of any one of claims 52-64, wherein following steps (a) and (b), the immune cell expresses a higher level of CD25, CD44, and/or CD69 relative to an immune cell which was contacted with the growth medium comprising the mitogenic stimulus without being contacted with the fusion protein.

66. The method of any one of claims 52-65, wherein following steps (a) and (b), the immune cell exhibits increased survival and/or proliferation relative to an immune cell which was contacted with the growth medium comprising the mitogenic stimulus without being contacted with the fusion protein.

67. A method of activating a cytokine signaling pathway in an immune cell, the method comprising contacting the immune cell with the fusion protein of any one of claims 1-29.

68. The method of claim 67, wherein the cytokine is IL-2.

69. The method of claim 68, wherein the activation of signaling through the IL-2 signaling pathway occurs independently of IL-2 -mediated activation of the signaling pathway.

70. The method of any one of claims 37-43 or 45-69, wherein the step of contacting the immune cell occurs in vivo or ex vivo.

71. The method of any one of claims 37-43 or 45-70, wherein the immune cell is selected from a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a mast cell, an NKT cell, a myeloid cell, a hematopoietic stem cell, and a red blood cell.

72. The method of any one of claims 37-43 or 45-71, wherein the immune cell is a T cell.

73. The method of claim 72, wherein the T cell is selected from a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, and an activated T cell.

74. The method of any one of claims 37-43 or 45-73, wherein the immune cell is a genetically modified immune cell.

75. The method of any one of claims 37-43 or 45-74, wherein the immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

76. The method of claim 75, wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell.

77. The method of claim 76, wherein the target cell is a cancer cell or an infected cell.

78. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the fusion protein of any one of claims 1-29 or the composition of claim 34.

79. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the composition of claim 35 or 36, or an immune cell that was contacted ex vivo with the fusion protein of any one of claims 1-29 or the composition of claim 34.

80. The method of claim 78 or 79, wherein the cancer is selected from breast cancer (e.g. , triple negative breast cancer), colorectal cancer, and lung cancer (e.g., NSCLC).

81. A method of treating or preventing ischemia reperfusion injury in a subject in need thereof, the method comprising administering to the subject the fusion protein of any one of claims 1-29 or the composition of claim 34.

82. A method of treating or preventing ischemia reperfusion injury in a subject in need thereof, the method comprising administering to the subject the composition of claim 35 or 36, or an immune cell that was contacted ex vivo with the fusion protein of any one of claims 1-29 or the composition of claim 34.

83. A method of treating an infection in a subject in need thereof, the method comprising administering to the subject the fusion protein of any one of claims 1-29 or the composition of claim 34.

84. A method of treating an infection in a subject in need thereof, the method comprising administering to the subject the composition of claim 35 or 36, or an immune cell that was contacted ex vivo with the fusion protein of any one of claims 1-29 or the composition of claim 34.

85. The method of claim 83 or 84, wherein the infection is selected from a bacterial infection, a viral infection, a fungal infection, a protozoan infection, and a parasitic infection.

86. The method of claim 85, wherein the infection is a bacterial infection selected from an infection of Staphylococcus aureus, Streptococcus pnuemoniae, Heamophila influenzae, Neisseria meningitidis, Klebsiella pneumoniae , Mycobacterium tuberculosis, Escherichia coli, and group B Streptococci.

87. The method of claim 83 or 84, wherein the infection is a chronic viral infection or an acute viral infection.

88. The method of any one of claims 83-84 or 87, wherein the chronic viral infection is an infection of a virus selected from Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, LCMV, herpes virus (e.g. HSV, Epstein Barr Virus, or Kaposi’s sarcoma-associated herpesvirus (KSHV)), Human Immunodeficiency Virus (HIV), or Human Papilloma Virus (HPV).

89. The method of any one of claims 83-84 or 87, wherein the acute viral infection is an infection from a virus selected from an influenza virus, West Nile Virus, Respiratory syncytial virus (RSV), a coronavirus, measles, Dengue virus, Ebola virus, Japanese encephalitis virus (JEV), or a rhinovirus.

90. The method of claim 83 or 84, wherein the fungal infection is an infection from a fungal pathogen selected from Candida albicans, Aspergillus, Candida auris, Pneumocystis jirovecii, Cryptococcus neoformans, or Sporothrix.

91. The method of claim 83 or 84, wherein the parasitic infection is an infection from a parasite selected from Taenia, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichomoniasis, Babesiosis, Blastocytosis, Cryptospridium, Trypanosomes, Trichonomas, Sarcocystis, Rhinosporodium, Malaria, Leishmania, Giardia, or an amoeban parasites.

92. The method of any one of claims 79, 82, or 84, wherein the immune cell is selected from a T cell, a B cell, a natural killer (NK) cell, an NKT cell, a dendritic cell, and a mast cell..

93. The method of any one of claims 79, 82, 84, or 92, wherein the immune cell is a T cell.

94. The method of claim 93, wherein the T cell is selected from a CD4+ T cell, a CD8+ T cell, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, and an activated T cell..

95. The method of any one of claims 79, 82, 84, or 92-94, wherein the immune cell is a genetically modified immune cell.

96. The method of any one of claims 79, 82, 84, or 92-95, wherein the immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

97. The method of claim 96, wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell.

98. The method of claim 97, wherein the target cell is a cancer cell or an infected cell.

99. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject the fusion protein of any one of claims 1-28 or the composition of claim 34.

100. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject the composition of claim 35 or 36, or an immune cell that was contacted ex vivo with the fusion protein of any one of claims 1-28 or the composition of claim 34.

101. The method of claim 100, wherein the immune cell is selected from a T cell, a B cell, a natural killer (NK) cell, an NKT cell, a dendritic cell, and a mast cell.

102. The method of claim 100 or 101, wherein the immune cell is a T cell.

103. The method of claim 102, wherein the T cell is selected from a regulatory T cell (Treg), an induced Treg, a primary T cell, an expanded primary T cell, a T cell derived from PBMC cells, a T cell derived from cord blood cells, and an activated T cell.

104. The method of claim 103, wherein the T cell is a CD25+ CD4+ Treg.

105. The method of any one of claims 100-104, wherein the immune cell is a genetically modified immune cell.

106. The method of any one of claims 100-105, wherein the immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

107. The method of claim 106, wherein the CAR comprises an extracellular domain comprising an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell.

108. The method of any one of claims 99-107, wherein the autoimmune disease is A T-cell dependent autoimmune disease selected from Type 1 diabetes, rheumatoid arthritis, LADA, multiple sclerosis, lupus, scleroderma pigmentosa, Myasthenia Gravis, Guillain Barre Syndrome, amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, or a chronic inflammatory disorder of the central nervous system.

109. The method of any one of claims 99-108, wherein the autoimmune disease is Type 1 diabetes.

110. A use of the fusion protein of any one of claims 1-29 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

111. A use of the fusion protein of any one of claims 1-29 in the manufacture of a medicament for the treatment of an autoimmune disease in a subject in need thereof.

112. A use of the fusion protein of any one of claims 1-29 for genetically modifying an immune cell.

113. A use of the fusion protein of any one of claims 1-29 for expanding an immune cell in a culture.