WO2025043177A1 - Protéines de fusion akt1 et procédés d'utilisation - Google Patents

Protéines de fusion akt1 et procédés d'utilisation Download PDF

Info

Publication number
WO2025043177A1
WO2025043177A1 PCT/US2024/043631 US2024043631W WO2025043177A1 WO 2025043177 A1 WO2025043177 A1 WO 2025043177A1 US 2024043631 W US2024043631 W US 2024043631W WO 2025043177 A1 WO2025043177 A1 WO 2025043177A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
fusion protein
cells
akt1
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/043631
Other languages
English (en)
Inventor
Brian Curtis Turner
Yosef Refaeli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ascensus Therapeutics Inc
Original Assignee
Ascensus Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ascensus Therapeutics Inc filed Critical Ascensus Therapeutics Inc
Priority to IL326720A priority Critical patent/IL326720A/en
Publication of WO2025043177A1 publication Critical patent/WO2025043177A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/033—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/915—Fusion polypeptide containing a motif for post-translational modification containing a motif for acylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/505—CD4; CD8
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase

Definitions

  • 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.
  • 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.
  • anergy in T cells, 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.
  • Cytokine pathway signaling is also involved in the function of immunosuppressive regulatory T cells (Tregs).
  • Tregs immunosuppressive regulatory T cells
  • cytokine signaling e.g., IL-2 pathway signaling
  • IL-2 pathway signaling e.g., IL-2 pathway signaling
  • IL-2 pathway signaling e.g., IL-2 pathway signaling
  • IL-2 pathway signaling e.g., IL-2 pathway signaling
  • Increasing cytokine signaling in this context can restore the function of immunosuppressive Tregs, thereby supporting the treatment of autoimmune disease.
  • 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.
  • ATP adenosine triphosphate
  • 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).
  • a signaling activator comprising a constitutively active AKT1 polypeptide or a functional fragment or variant thereof
  • PTD protein transduction domain
  • the constitutively active AKT1 polypeptide or functional fragment or variant thereof is phosphatase resistant.
  • 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.
  • 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.
  • 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.
  • 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 constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a deletion of the pleckstrin homology (PH) domain of AKT1.
  • 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.
  • the constitutively active AKT1 polypeptide or functional fragment or variant thereof comprises a substitution that prevents AKT-induced neoplasia.
  • 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).
  • the threonine residue at a position corresponding to position 308 of wild-type human AKT1 is substituted by aspartic acid (T308D).
  • 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).
  • the serine residue at a position corresponding to position 473 of wild-type human AKT1 is substituted by aspartic acid (S473D).
  • 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.
  • 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.
  • the PTD comprises a cationic PTD, a hydrophobic PTD, or a cell-type specific PTD.
  • 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.
  • the PTD comprises an HIV-1 Tat peptide or a functional variant thereof.
  • the PTD comprises the amino acid sequence of SEQ ID NO: 11 or 12.
  • 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.
  • a hydrophobic PTD e.g., atransportan peptide, a MAP peptide, a TP 10 peptide, or a functional variant of any of the foregoing.
  • the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 24-40 and 64.
  • 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.
  • 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.
  • the cell is a bacterial cell.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of any one of the foregoing embodiments and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides a composition comprising the fusion protein of any of the foregoing embodiments and an immune cell.
  • the composition further comprises a pharmaceutically acceptable carrier or excipient.
  • 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.
  • the method further comprises cryopreserving the cell therapeutic composition and, optionally, thawing the cell therapeutic composition.
  • the step of contacting the immune cell with the fusion protein occurs prior to cryopreservation.
  • the step of contacting the immune cell with the fusion protein occurs after thawing the cell therapeutic composition.
  • 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.
  • the contacting step comprises contacting the immune cell with a medium comprising 0.05-500 pg/mL of the fusion protein.
  • the disclosure provides a cell therapeutic composition generated by any one of the foregoing methods.
  • 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.
  • the immune cell is in a resting state prior to step (a).
  • the step of contacting the immune cell with the fusion protein induces the immune cell to enter the G1 phase of the cell cycle.
  • 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.
  • the vector is RNA.
  • step (b) of the method comprises contacting the cell with a liposome encapsulating the vector.
  • 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.
  • the mitogenic stimulus is an anti- CD3 antibody and/or an anti-CD28 antibody.
  • the growth medium further comprises one or more cytokines, e.g., IL-2, IL-4, IL-7, and/or IL-15.
  • 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.
  • the growth medium comprises the fusion protein and the mitogenic stimulus.
  • step (a) is carried out prior to step (b).
  • 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.
  • 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 is the same growth medium used in step (a).
  • 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.
  • 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.
  • the cytokine is IL-2.
  • the activation of signaling through the IL-2 signaling pathway occurs independently of IL-2 -mediated activation of the signaling pathway.
  • the step of contacting the immune cell occurs in vivo or ex vivo.
  • 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.
  • 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.
  • the immune cell is a genetically modified immune cell.
  • 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.
  • 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.
  • 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.
  • a composition e.g., a pharmaceutical composition
  • the cancer is selected from breast cancer (e.g., triple negative breast cancer), colorectal cancer, and lung cancer (e.g, NSCLC).
  • 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.
  • 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.
  • a composition e.g, a pharmaceutical composition
  • 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.
  • 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.
  • a composition e.g., a pharmaceutical composition
  • 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).
  • Staphylococcus aureus Streptococcus pnuemoniae
  • Heamophila influenzae Neisseria meningitidis
  • Klebsiella pneumoniae Mycobacterium tuberculosis
  • Escherichia coli and group B Streptococci
  • 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).
  • a chronic viral infection e.g., an infection of a virus selected from Hepatitis A Virus Hepatitis B Virus, Hepatitis C Virus, LCMV
  • 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.
  • the infection is a protozoan infection.
  • 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.
  • 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.
  • 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.
  • 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.
  • the immune cell is a genetically modified immune cell.
  • 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.
  • CAR chimeric antigen receptor
  • the target cell can be, for example, a cancer cell or an infected cell.
  • 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.
  • 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.
  • a composition e.g., a pharmaceutical composition
  • 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.
  • 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.
  • the T cell is a CD25+ CD4+ Treg.
  • the immune cell is a genetically modified immune cell.
  • 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.
  • CAR chimeric antigen receptor
  • 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.
  • the autoimmune disease is Type 1 diabetes.
  • 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.
  • 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.
  • the disclosure provides a use of a fusion protein of any of the foregoing embodiments in genetically modifying an immune cell.
  • the disclosure provides a use of a fusion protein of any of the foregoing embodiments in expanding an immune cell in a culture.
  • 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.
  • 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).
  • 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
  • Bcl-2 production FIG. 3B were measured 30-minutes post-treatment via western blotting with anti-phospho-AKTl and anti -Bel -2 antibodies.
  • 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.
  • DN AKT dominant-negative form of AKT1
  • 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.
  • 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).
  • 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”).
  • T cells 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”).
  • pMIG empty vector
  • pMIG-Akt* a vector encoding a constitutively active Myr-Akt
  • pMIG-Bcl2 a vector encoding Bcl2
  • Non-transduced T cells were used as a control (“None”).
  • 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”).
  • 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.
  • 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”).
  • FIG. 9 depicts the results of a non-reducing SDS-PAGE for the detection of purified 6His-tagged PTD4-MyrAkt.
  • 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.
  • 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.
  • Media medium without the fusion protein
  • Denatured Protein denatured fusion protein
  • 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.
  • 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.
  • 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).
  • Tregs primary human regulatory T cells
  • 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”).
  • 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.
  • cytokine receptors 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.
  • constitutively active 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).
  • 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.
  • a phosphatase-resistant form of Aktl is considered to be a constitutively active form of Aktl .
  • a wild-type polypeptide that is phosphorylated e.g., phosphorylated Aktl
  • Aktl phosphorylated Aktl
  • Activator generally refers to the ability of a polypeptide to induce, enhance or promote the function of a given target or signaling pathway.
  • 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.
  • 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.
  • 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.
  • a cell or population of cells is “autologous” to the subject from which the cell or population of cells was derived.
  • 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.
  • cancer 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • An “engineered,” “modified,” and “genetically modified” cell or cells 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.
  • 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.
  • 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.
  • coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • non-coding strand used as the template for transcription, of a gene or cDNA
  • 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.
  • 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).
  • 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.
  • 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.”
  • expression may be characterized as intracellular, extracellular or membrane.
  • intracellular means inside a cell.
  • extracellular means outside a cell.
  • membrane means at least a portion of a polypeptide is contacting or embedded in a cell membrane.
  • cytoplasmic means residing within the cell membrane, outside the nucleus.
  • 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.
  • 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.
  • 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
  • Kerat activity 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.
  • 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).
  • 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.
  • the pharmaceutical composition facilitates administration of the fusion polypeptide or cell or population of cells to an individual.
  • 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.
  • the subject is a human.
  • phrases “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • treating refers 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.
  • 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
  • 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.
  • 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.
  • both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.
  • 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).
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • immunoblotting for expression of gene products
  • suitable analytical methods to test the expression of an introduced nucleic acid or its corresponding gene product (e.g. , enzyme or protein).
  • fusion proteins having a signaling activator comprising an AKT1 polypeptide or a functional fragment or variant thereof, and a protein transduction domain (PTD).
  • PTD protein transduction domain
  • 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.
  • PH N- terminal pleckstrin homology
  • PI3Ks class I phosphoinositide 3-kinases
  • PIP2 phosphatidylinositol-4, 5- bisphosphate
  • 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.
  • Akt Akt-dependent kinase 1
  • mT0RC2 mammalian target of rapamycin complex 2
  • 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).
  • PPI Protein Phosphatase 1
  • the AKT polypeptide is a human AKT1 polypeptide.
  • the AKT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, or a functional fragment or variant thereof.
  • the AKT1 polypeptide is a variant of a naturally occurring AKT1 polypeptide (e.g., a variant of human AKT1).
  • 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.
  • 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.
  • 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.
  • 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.
  • the AKT1 polypeptide is a fragment of a naturally occurring polypeptide, or a variant thereof.
  • 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.
  • the AKT1 polypeptide comprises the residues 131 through 477 ofhuman AKT1 (z.e., SEQ ID NO: 2).
  • the AKT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 2.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AKT1 polypeptide is constitutively active.
  • 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.
  • the constitutively active AKT1 polypeptide is phosphatase resistant.
  • 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.
  • the AKT1 polypeptide comprises a substitution and/or an amino acid sequence that facilitates sequestration of the fusion protein at the plasma membrane.
  • the AKT1 polypeptide comprises an amino acid sequence having a myristoylation sequence, e.g., a Src myristoylation sequence or a Gag myristoylation sequence.
  • the Src myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 5 (MGSSKSKPKDPSQRSE), or a functional fragment or variant thereof.
  • the Src myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 6 (MGSSKSKPKSR), or a functional fragment or variant thereof.
  • the Gag myristoylation sequence comprises the amino acid sequence of SEQ ID NO: 7 (
  • 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.
  • the myristoylation sequence is fused to full-length AKT1, e.g., an AKT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • 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.
  • the myristoylation sequence is fused to an AKT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 4.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AKT1 polypeptide comprises an amino acid substitution that renders the protein constitutively active.
  • the AKT1 polypeptide comprises a substitution associated with an oncogenic or cancer- associated form of the protein.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • the AKT1 polypeptide or functional fragment or variant thereof comprises any combination of the foregoing substitutions.
  • 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.
  • 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.
  • the variant AKT1 polypeptide, or the functional fragment thereof further comprises one or more substitutions that prevent AKT-induced neoplasia in a subject.
  • 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).
  • 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).
  • 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.
  • 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, dihexy
  • 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.
  • 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.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • AKT1 polypeptide e.g., as in Table 1
  • PTD e.g., as in Table 2
  • the fusion protein used in the methods of the disclosure comprises a cationic PTD, a hydrophobic PTD, or a cell-type specific PTD.
  • 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.
  • the hydrophobic PTD is derived from a transportan peptide, a MAP peptide, a TP 10 peptide, or a variant thereof.
  • the PTD has a sequence set forth in any one of SEQ ID NOs: 11-20.
  • the fusion protein further comprises a linker and/or a tag.
  • the fusion protein comprises a sequence of any one of SEQ ID NOs: 24-40 and 64 (shown in Table 3).
  • the fusion protein used in a method is capable of penetrating a plasma membrane of a cell.
  • the fusion polypeptide is capable of inducing activation of cytokine pathway signaling independently of the presence of a ligand.
  • the ligand is selected from IL-2, IL-4, IL-7, and IL- 15. a. Activation of Cytokine Pathway Signaling
  • 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.
  • the contacting occurs in vivo. In some embodiments, the contacting occurs ex vivo.
  • activation of cytokine pathway signaling after the contacting occurs independently of ligand-mediated activation of the cytokine pathway.
  • the ligand is selected from the group consisting of IL-2, IL-4, IL-7, and IL-15.
  • the cell exhibits of one or more properties associated with cytokine pathway signaling after the contacting.
  • 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).
  • 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.
  • 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).
  • 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
  • the immune cell is a cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor.
  • TCR T-cell receptor
  • the immune cell expresses a TCR.
  • the immune cell expresses CD3.
  • 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).
  • the contacting is performed in vivo. In some embodiments, the contacting is performed ex vivo.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the target cell is a cell intended to be targeted to be killed in accordance with a therapeutic method of the disclosure.
  • the target cell is a cancer cell or an infected cell.
  • 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.
  • 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.
  • 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.
  • the population of cells e.g., CAR-T cells
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the step of contacting the cell or population of cells occurs after cryopreservation.
  • the contacting step occurs after thawing the cell therapeutic composition.
  • 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.
  • 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/m
  • 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.
  • biomarker e.g. , a cell surface protein
  • 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.
  • a binding agent e.g., an antibody
  • flow cytometry e.g., fluorescence activated cell sorting
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 30 days, within 29 days, within 28 days, within 27 days, within 26 days, within 25 days, within 24 days, within 23 days,
  • 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.
  • the cell therapeutic composition e.g., a composition of CAR-T cells
  • 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.
  • Also provided herein is a cell, a population of cells, or a cell therapeutic composition prepared by any of the methods described herein.
  • fusion proteins e.g., PTD- MyrAkt fusion protein
  • 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).
  • methods for using the disclosed fusion protein, e.g. , PTD-MyrAkt fusion protein to treat freshly thawed CAR-T cells e.g. , PTD-MyrAkt fusion protein to treat freshly thawed CAR-T cells.
  • 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.
  • fusion proteins e.g., the PTD-MyrAkt fusion protein
  • CAR-T cells are currently administered to patients following lymphodepletion.
  • these individuals are given up to 6 injections of systemic high dose IL-2 (maximum tolerated doses).
  • 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.
  • 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.
  • step (a) and/or step (b) is performed ex vivo.
  • the vector can, for example, be any vector suitable for stably or transiently introducing an ectopic nucleic acid into the cell.
  • the vector is a viral vector, such as an adenoviral vector or a retroviral vector (e.g., a type-C retroviral vector).
  • the vector is RNA.
  • step (b) comprises contacting the cell with a liposome encapsulating the vector.
  • the immune cell is in a resting state prior to the step of being contacted with the fusion protein.
  • 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.
  • 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.
  • NK natural killer
  • 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.
  • the cell is autologous to a subject to be treated with the immune cell.
  • the cell is allogeneic to a subject to be treated with the immune cell.
  • the cell is an immune cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor.
  • TCR T-cell receptor
  • the immune cell expresses a TCR.
  • the immune cell expresses CD3.
  • the vector comprises a nucleic acid encoding a chimeric antigen receptor (CAR).
  • the CAR comprises an antigen-binding site, wherein the antigen-binding site specifically binds an antigen on the surface of a target cell.
  • the target cell is a cell intended to be targeted to be killed in accordance with a therapeutic method of the disclosure.
  • the target cell is a cancer cell or an infected cell.
  • 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.
  • step (a) and/or step (b) is performed ex vivo.
  • 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.
  • 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 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 heterologous nucleic acid e.g., a nucleic acid encoding a CAR
  • 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.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • TALEN Transcription activator-like effector nuclease
  • ZFN zinc -finger nuclease
  • HE homing endonuclease
  • 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.
  • 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.
  • 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.
  • the cell is autologous to a subject to be treated with the cell.
  • the cell is allogeneic to a subject to be treated with the cell.
  • the cell is an immune cell (e.g., a T cell) expressing a T-cell receptor (TCR) and/or a T-cell co-receptor.
  • TCR T-cell receptor
  • the immune cell expresses a TCR.
  • the immune cell expresses CD3.
  • the cell is modified by the genomic editing system to express a CAR.
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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//
  • step (a) is carried out before step (b).
  • 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 p
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • steps (a) and (b) results in an enhanced expansion efficiency, as compared to the expansion of immune cells that are not contacted with the fusion protein.
  • 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.
  • contacting the immune cell with a fusion protein of the disclosure 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.
  • a fusion protein of the disclosure 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.
  • 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.
  • NK natural killer
  • 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.
  • 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).
  • the cell is autologous to a subject to be treated with the immune cell.
  • the cell is allogeneic to a subject to be treated with the immune cell.
  • the levels or viral gene transduction may be improved by the addition of the disclosed fusion proteins to the viral transduction culture.
  • 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.
  • 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.
  • fusion proteins e.g., PTD-MyrAkt fusion protein
  • PTD-MyrAkt fusion protein PTD-MyrAkt fusion protein
  • 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.
  • contacting the immune cell with a fusion protein of the disclosure 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.
  • a fusion protein of the disclosure 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.
  • the disclosed fusion proteins e.g., PTD-MyrAkt fusion protein
  • the target Treg cell population may be naturally occurring CD25+CD4+ Treg cells.
  • 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.
  • fusion proteins e.g., PTD-MyrAkt fusion protein
  • 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.
  • fusion proteins e.g., PTD-MyrAkt fusion protein
  • 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.
  • 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.
  • contacting the T cells e.g., Tregs
  • a fusion protein of the disclosure e.g., a PTD-MyrAkt fusion protein
  • 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.
  • the cell or population of cells includes one or more immune cells.
  • 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.
  • a T cell e.g., a T cell
  • 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).
  • the one or more immune cells are autologous to a subject to be treated with the one or more immune cells.
  • the one or more immune cells are allogeneic to a subject to be treated with the one or more immune cells.
  • 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.
  • 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.
  • the cell or population of cells are human.
  • the cell or population of cells from a non-human mammal may be, for example, a dog, cat, horse, cattle, dairy cow, swine, sheep, lamb, goat, primate, mouse, or rat.
  • the disease or disorder is an ischemia reperfusion injury, a cancer, an infection, or an autoimmune disease or disorder.
  • 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).
  • the AKT1 polypeptide is constitutively active.
  • the AKT1 polypeptide is phosphatase resistant.
  • 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.
  • 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.
  • 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.
  • the hydrophobic PTD is derived from a transportan peptide, a MAP peptide, a TP 10 peptide, or a variant thereof.
  • the PTD has a sequence set forth in SEQ ID NOs: 11-20.
  • the fusion protein further comprises a linker and/or a tag.
  • 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.
  • the fusion polypeptide is capable of inducing activation of cytokine pathway signaling independently of the presence of a ligand.
  • the ligand is selected from a ligand selected from the group consisting of IL-2, IL- 4, IL-7, and IL- 15.
  • 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).
  • the population of cells exhibits activation of cytokine pathway signaling after the contacting.
  • 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.
  • 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.
  • 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.
  • the population of cells include one or more immune cells.
  • 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.
  • a T cell e.g., a T cell selected from a CD4+ T cell, a CD8+
  • 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.
  • 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.
  • the population of cells is genetically modified. In other embodiments, the population of cells is not genetically modified.
  • the population of cells is autologous to the subject. In some embodiments, the population of cells is allogenic to the subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • a. Ischemia Reperfusion Injury 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.
  • 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.
  • Ischemia reperfusion 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.
  • the ischemia reperfusion injury is a myocardial ischemia, a cerebral ischemia, a hepatic ischemia, a pulmonary ischemia, or a nephritic ischemia.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • biomarkers of ischemic reperfusion injury include, but are not limited to, NGAL, KIM-1, IL- 18, RBP, FABP4, cystatin C and creatinine.
  • the biomarker is a biomarker associated with a myocardial ischemia, a cerebral ischemia, a hepatic ischemia, a pulmonary ischemia, or a nephritic ischemia.
  • a method of treating a subject having an infection 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.
  • 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.
  • 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.
  • 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.
  • infectious agents including, but not limited to, bacteria, fungi, protozoa, and viruses.
  • the infection is a bacterial infection, a viral infection, a fungal infection, a protozoan infection, or a parasite infection.
  • the infection treated by the methods described herein is a bacterial infection.
  • 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.
  • 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.
  • the infection treated by the methods described herein is a viral infection.
  • 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
  • herpes simplex type II HSV- II
  • rinderpest 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
  • HV-III human immunodeficiency virus type II
  • agents of viral diseases such as viral meningitis, encephalitis, dengue or small pox.
  • the viral infection is a chronic viral infection.
  • 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).
  • the viral infection is an acute viral infection.
  • 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.
  • 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.
  • 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.
  • the fungal infection is an infection from a fungal pathogen selected from Candida albicans, Aspergillus, Candida auris, Pneumocystis jirovecii, Cryptococcus neoformans, or Sporothrix.
  • a fungal pathogen selected from Candida albicans, Aspergillus, Candida auris, Pneumocystis jirovecii, Cryptococcus neoformans, or Sporothrix.
  • the infection treated by the methods described herein is a protozoan infection.
  • the protozoan infection is an infection from a protozoa selected from Giardia intestinalis, Entamoeba hystolitica, Cyclospora cayatanenensis, or cryptosporidium.
  • the infection treated by the methods described herein is a parasitic infection.
  • 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.
  • a parasite selected from Taenia, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichomoniasis, Babesiosis, Blastocytosis, Cryptospridium, Trypanosomes, Trichonomas, Sarcocystis, Rhinosporodium, Malaria, Leishmania, Giardia, or an amoeban parasites.
  • a method of treating a cancer 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), or a cell or population of cells that has been contacted ex vivo with the fusion protein.
  • the cell or population of cells are autologous to the cancer subject.
  • the cell or population of cells are allogeneic to the cancer subject.
  • the cancer to be treated is a solid cancer.
  • 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.
  • 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,
  • the cancer is non-Hodgkin’s lymphoma, such as a B-cell lymphoma or a T- cell lymphoma.
  • 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.
  • B-cell lymphoma such as a diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphom
  • 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.
  • 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
  • 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.
  • 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.
  • the cancer is non-metastatic. In some embodiments, the cancer is metastatic.
  • the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC)).
  • the cancer is breast cancer (e.g., triple negative breast cancer or HER2 -negative breast cancer).
  • the cancer is colorectal cancer.
  • the method comprises administering an immune cell that has been contacted ex vivo with a fusion protein of the disclosure.
  • 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.
  • 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.
  • 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.
  • a method of treating an autoimmune disease or disorder in a subject 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.
  • the cell or population of cells are autologous to the subject having the autoimmune disease.
  • the cell or population of cells are allogeneic to the subject having the autoimmune disease.
  • 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.
  • the autoimmune disease is Type 1 diabetes.
  • the autoimmune disease is rheumatoid arthritis (e.g., stage 2 rheumatoid arthritis or stage 3 rheumatoid arthritis).
  • the method comprises administering an immune cell that has been contacted ex vivo with a fusion protein of the disclosure.
  • 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.
  • the T cell is a CD25+ CD4+ Treg.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • another therapeutic agent which also includes a therapeutic regimen
  • the overall benefit experienced by the subject is additive of the combination or in other embodiments, the subject experiences a synergistic benefit.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fusion protein described herein may be administered sequentially with an agent that reduces cytokine pathway signaling.
  • 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.
  • cytokine pathway signaling may be fine-tuned so as to optimize the therapeutic benefit to the subject.
  • dosages of the co-administered therapies vary depending on the type of therapy, on the disorder or condition being treated, and so forth.
  • 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.
  • the combination methods are not to be limited to the use of only two agents; the use of multiple therapeutic combinations are also envisioned.
  • 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.
  • 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.
  • the subject to be treated is an animal, such as a mammal.
  • the mammal is a dog, cat, horse, cattle, dairy cow, swine, sheep, lamb, goat, primate, mouse, rat, or human.
  • the subject is a human.
  • 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.
  • T cells 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 2xl0 7 cells/mL.
  • 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.
  • GFP green fluorescent protein
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • GFP Wgh populations were isolated by high-speed cell sorting.
  • AKT-ER T cells were cultured with or without 100 nM of TMX.
  • IL-2 family cytokines promote T-cell survival and activate AKT
  • AKT acts downstream of cytokine receptors (e.g., IL-2R) to stimulate cell proliferation and survival.
  • cytokine receptors e.g., IL-2R
  • 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.
  • a dominant negative AKT blocks cytokine -mediated T-cell survival signaling
  • DN AKT dominant negative form of AKT
  • MIG empty vector control
  • AKT activation stimulates T-cell proliferation and survival in the absence of cytokine stimulation
  • 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”).
  • TMX synthetic steroid 4-Hydroxytamoxifen
  • 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.
  • FIG. 5A expression of the constitutively active Myr-Akt fusion protein promoted T-cell survival and proliferation, even in the absence of cytokine stimulation.
  • 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).
  • This example describes the ability of T cells expressing constitutively active Aktl to inhibit Non-Hodgkin’s Lymphoma (NHL) tumor formation.
  • constitutively active Aktl e.g., MyrAkt
  • Aktl can restore antigen responsiveness in an anergic lymphoid cell population.
  • mice carrying the E i -MY C transgene were obtained from the Jackson
  • mice express MYC in a B cell-specific manner, beginning at the Pre/Pro-B cell stage.
  • BCR HEL mice MD4
  • sHEL mice ML5
  • 3A9 mice were also obtained.
  • MD4 mice express a pre-rearranged murine BCR from the endogenous immunoglobulin promoter
  • 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 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 5xl0 6 cells/mouse intravenously.
  • 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.
  • 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.
  • CFSE CarboxyFluorescein Succinimidyl Ester
  • 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.
  • Assays in which doxycycline was added to the cultures contained 100 ng/ml of doxycycline (Sigma, St. Louis MO) added to the media.
  • 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*.
  • T cells expressing constitutively active Aktl can inhibit NHL tumor development
  • 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.
  • 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).
  • 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).
  • the anergic T cells 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”).
  • pMIG-Akt* a constitutively active Akt
  • pMIG-Bcl2 a vector encoding Bcl2
  • pMIG-Bcl2 a vector encoding Bcl2
  • pMIG empty vector as a control
  • 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.
  • 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.
  • 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).
  • lysis buffer 8 M urea, 100 mM NaH2PO4, 10 mM Tris pH to 8.0
  • 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.
  • T-cells 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 2xl0 7 cells/mL. T-cells were activated in vitro with 1 mg/mL of anti-CD3 (Pharmingen) for 1 day.
  • 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.
  • a PTD-cytokine fusion 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.
  • 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.
  • Myr-Akt constitutively active form of Akt
  • Tat PTD a Tat PTD
  • 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.
  • 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.
  • Myr-Akt constitutively active form of Akt
  • Tat4 PTD a Tat4 PTD
  • 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.
  • 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.
  • 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.
  • Spleens and lymph nodes 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 A 6 cells/ml in 24 well plates (1 ml/well).
  • 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.
  • 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 A 6 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.
  • PTD4-MyrAkt fusion protein 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.
  • 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.
  • 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.
  • 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).
  • RPMI-based complete lymphocyte media
  • the amino acid sequence of the Tat-MYC fusion protein was: MRKKRRQRRRMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQSELQPPAPSED IWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGG DMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARG HSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTES SPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKP PHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSD TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAE
  • mice 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.
  • 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.
  • affinity-purified CD4+CD25+ human regulatory T cells 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).
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hematology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des protéines de fusion présentant (i) un activateur de signalisation comportant un polypeptide AKT1 et (ii) un domaine de transduction protéique. Les protéines de fusion peuvent activer la signalisation de la voie des cytokines dans une cellule, indépendamment de la liaison de la cytokine à un récepteur de cytokine. La présente invention concerne également les acides nucléiques et le vecteur codant pour les protéines de fusion, ainsi que les cellules et les compositions contenant la protéine de fusion. En outre, La présente invention concerne des procédés d'utilisation des protéines de fusion pour activer la signalisation des cytokines dans une cellule, préparer des cellules thérapeutiques et traiter une maladie ou un trouble chez un sujet.
PCT/US2024/043631 2023-08-23 2024-08-23 Protéines de fusion akt1 et procédés d'utilisation Pending WO2025043177A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
IL326720A IL326720A (en) 2023-08-23 2024-08-23 AKT1 fusion proteins and methods of use

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202363578244P 2023-08-23 2023-08-23
US63/578,244 2023-08-23
US202363609934P 2023-12-14 2023-12-14
US63/609,934 2023-12-14
US202463665555P 2024-06-28 2024-06-28
US63/665,555 2024-06-28

Publications (1)

Publication Number Publication Date
WO2025043177A1 true WO2025043177A1 (fr) 2025-02-27

Family

ID=94732690

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/043631 Pending WO2025043177A1 (fr) 2023-08-23 2024-08-23 Protéines de fusion akt1 et procédés d'utilisation

Country Status (2)

Country Link
IL (1) IL326720A (fr)
WO (1) WO2025043177A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055116A1 (en) * 2006-04-13 2010-03-04 Liou Hsiou-Chi Methods and Compositions for Targeting c-Rel
US20130267030A1 (en) * 2010-12-03 2013-10-10 Kyoto University Efficient method for establishing induced pluripotent stem cells
WO2019068066A1 (fr) * 2017-09-29 2019-04-04 National Health Research Institutes Méthodes et compositions permettant d'améliorer la survie et la fonctionnalité de lymphocytes t anti-tumoraux et anti-viraux
WO2020102802A1 (fr) * 2018-11-16 2020-05-22 The Brigham And Women's Hospital, Inc. Substances et méthodes pour des pathologies musculaires suite à une lésion, une maladie ou au vieillissement

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055116A1 (en) * 2006-04-13 2010-03-04 Liou Hsiou-Chi Methods and Compositions for Targeting c-Rel
US20130267030A1 (en) * 2010-12-03 2013-10-10 Kyoto University Efficient method for establishing induced pluripotent stem cells
WO2019068066A1 (fr) * 2017-09-29 2019-04-04 National Health Research Institutes Méthodes et compositions permettant d'améliorer la survie et la fonctionnalité de lymphocytes t anti-tumoraux et anti-viraux
WO2020102802A1 (fr) * 2018-11-16 2020-05-22 The Brigham And Women's Hospital, Inc. Substances et méthodes pour des pathologies musculaires suite à une lésion, une maladie ou au vieillissement

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HART JONATHAN R., VOGT PETER K.: "Phosphorylation of AKT: a Mutational Analysis", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 2, no. 6, 30 June 2011 (2011-06-30), United States , pages 467 - 476, XP093284056, ISSN: 1949-2553, DOI: 10.18632/oncotarget.293 *
SIDDIKA TARANA, BALASURIYA NILEEKA, FREDERICK MALLORY I., ROZIK PETER, HEINEMANN ILKA U., O’DONOGHUE PATRICK: "Delivery of Active AKT1 to Human Cells", CELLS, MDPI AG, vol. 11, no. 23, pages 3834, XP093284055, ISSN: 2073-4409, DOI: 10.3390/cells11233834 *
YIN, K-J ET AL.: "AP 25-35 alters Akt activity, resulting in Bad translocation and mitochondrial dysfunction in cerebrovascular endothelial cells", JOURNAL OF CEREBRAL BLOOD FLOW & METABOLISM, vol. 25, no. 11, 2005, pages 1445 - 1455, XP055052905, DOI: 10.1038/sj.jcbfm.9600139 *

Also Published As

Publication number Publication date
IL326720A (en) 2026-04-01

Similar Documents

Publication Publication Date Title
TWI846697B (zh) 工程化細胞外囊泡及其用途
US20230348537A1 (en) Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
AU2019301147B2 (en) ROR-1 specific chimeric antigen receptors and uses thereof
JP7320947B2 (ja) 新規な免疫原性ＣＤ１ｄ結合ペプチド
JP7269806B2 (ja) 結核組成物及びそれを使用する方法
CN107148427B (zh) 新型免疫原性肽
KR20190111022A (ko) T-세포 조절 다량체 폴리펩타이드 및 이의 사용 방법
US20180100158A1 (en) Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
JP2019519516A (ja) がんの治療のためのｍＲＮＡ併用療法
CN101511872A (zh) 用于抑制凋亡的药物组合物及其递送方法
US11629170B2 (en) Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
JP5964233B2 (ja) Ｈｐｖにより惹起される疾患の治療用組成物
KR20190126798A (ko) 당뇨병 치료를 위한 펩티드 및 방법
US20070105775A1 (en) Methods for fusion polypeptide delivery into a cell
CN112442129B (zh) 肿瘤酶响应型重组焦亡蛋白递药系统及其抗肿瘤用途
JP2020506901A (ja) 自家ｔ細胞を用いた多発性硬化症の処置方法
JP4740595B2 (ja) 疾患の治療または予防のための可溶型ｃｄ８３タンパク質およびそれをコードする核酸の使用
Bleifuss et al. The translocation motif of hepatitis B virus improves protein vaccination
JP2017516752A (ja) 単離されたドナーｍｈｃ由来ペプチド及びその使用
KR20090028624A (ko) 자가면역질환, 알러지성 질환 및 염증성 질환 치료용 약제 조성물 및 이의 전달 방법
WO2025043177A1 (fr) Protéines de fusion akt1 et procédés d'utilisation
CN121969742A (zh) Akt1融合蛋白及使用方法
EP2706113B1 (fr) Peptide synthétique capable d'induire l'expression du récepteur de tnf de type-2 et son utilisation
WO2022111475A1 (fr) Tcr capable de reconnaître un antigène hpv
CN105555796A (zh) 抑制肽对治疗炎性疾病的用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24857363

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 326720

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 2024857363

Country of ref document: EP