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  • A61K35/14—Blood; Artificial blood
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  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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• • A—HUMAN NECESSITIES
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/82—Translation products from oncogenes
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  • 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
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  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
• • A—HUMAN NECESSITIES
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  • 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
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2740/00—Reverse transcribing RNA viruses
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  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
  • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
  • C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• T cell exhaustion is a state of T cell dysfunction that arises during chronic infections and cancer. It is characterized by poor T cell effector function, sustained expression of inhibitory receptors, and a transcriptional state that is distinct from functional effector and memory T cells. Exhaustion negatively affects the immune system’s ability to control infection and tumor growth and metastasis.
• naive antigen-specific CD8 + T cells become activated, proliferate, acquire effector functions, and differentiate into effector CD8 + T cells. Following clearance of the acute infection, most effector CD8 + T cells will undergo apoptosis; however, about 5-10% differentiate into memory CD8 + T cells.
• CD8 + T cell responses can develop, and antigen-specific CD8 + T cells often fail to differentiate into memory CD8 + T cells.
• Loss of effector function e.g., T-cell exhaustion
• Chronic antigen exposure to tumor antigens produces a similar exhaustion phenomenon in CD8 + T cells that recognize tumor antigens expressed by cancer cells.
• T cell exhaustion can also occur during acute viral infections as well.
• CD8 + T cells often exhibit diminished production of cytokines and cytotoxic molecules and exhibit similar patterns of gene expression to that observed in exhausted T cells during chronic infection.
• T cell impairment the tendency of CD8 + T cells to have significantly reduced functionality in the context of respiratory virus infection is called T cell impairment.
• compositions and methods that can decrease and/or reverse T cell exhaustion/impairment and restore effector function after or during a chronic infection and for the treatment of cancer.
• compositions and methods for treating T cell exhaustion, or T cell impairment, in a subject by administering a PTD-MYC fusion protein (e.g., an HIV TAT- MYC fusion protein) or immune cells isolated from a donor subject and treated with a PTD- MYC fusion protein.
• a PTD-MYC fusion protein e.g., an HIV TAT- MYC fusion protein
• immune cells isolated from a donor subject and treated with a PTD- MYC fusion protein.
• the T cell exhaustion is associated with a chronic microbial infection (e.g. bacterial, viral, fungal, protozoan or parasitic) or cancer.
• the T cell exhaustion, or T cell impairment is associated with an acute viral infection (e.g.
• administration of the PTD-MYC fusion protein or PTD- MYC modified immune cells reduces T cell exhaustion, or T cell impairment, in the subject.
• administration of the PTD-MYC fusion protein or PTD- MYC modified immune cells reduces the number of exhausted cells (e.g., exhausted T cells) in the subject.
• administration of the PTD-MYC fusion protein or PTD- MYC modified immune cells increases the immune response against a pathogen associated with a chronic microbial infection. In some embodiments, administration of the PTD-MYC fusion protein or PTD-MYC modified immune cells alleviate one or more symptoms of a chronic microbial infection.
• a subject in need thereof, comprising administering an effective amount of one or more modified immune cells to the subject, wherein the one or more modified immune cells comprises a MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the subject is identified as having altered expression of at least one immune cell marker of T cell exhaustion, or T cell impairment, compared to expression of the at least one immune cell marker in a healthy control.
• the immune cell marker is an immune checkpoint protein.
• the subject has a microbial infection.
• the microbial infection is a chronic microbial infection.
• the microbial infection is a bacterial infection, a viral infection, a fungal infection, a protozoan infection, or parasitic infection.
• the microbial infection is caused by a pathogen selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Helicobacter pylori, Staphylococcus aureus , Salmonella Typhi , Treponema pallidum, Escherichia coli, Hemophilus influenza, Pseudomonas aeruginosa, Plasmodium falciparum , Plasmodium vivax, Plasmodium ovale , Plasmodium malariae , Human Immunodeficiency Virus (HIV), Herpesviruses, Herpes Simplex Virus (HSV), Hepatitis B Virus (HBV), Hepatitis
• the T cell exhaustion, or T cell impairment is associated with an acute viral infection, such as an acute respiratory virus infection, such as infection by influenza virus, respiratory syncytial virus (RSV), pneumonia virus, respiratory vaccinia virus, parainfluenza virus, respiratory adenoviruses, severe acute respiratory syndrome corona virus (SARS-CoV), Middle East respiratory syndrome corona virus (MERS-CoV), or human metapneumovirus (HMPV)).
• the subject has cancer.
• the cancer is melanoma.
• the melanoma is relapsed refractory melanoma.
• the one or more modified immune cells are derived from immune cells isolated from the subject. In some embodiments, the immune cells isolated from the subject are obtained from the peripheral blood of the subject. In some embodiments, the immune cells isolated from the subject are obtained from the lymph node, spleen, or tumor of the subject. In some embodiments, the one or more modified immune cells are prepared by contacting the immune cells in vitro with the MYC fusion protein. In some embodiments, the one or more modified immune cells are prepared by contacting a population of peripheral blood mononuclear cells from the subject in vitro with the MYC fusion protein. In some embodiments, the methods further comprise expanding the modified immune cells in vitro prior to and/or following contacting the modified immune cells with the MYC fusion protein.
• the protein transduction domain sequence is a TAT protein transduction domain sequence.
• the MYC fusion protein comprises SEQ ID NO: 1.
• the MYC fusion protein translocates to the nucleus of one or more modified immune cells.
• the MYC fusion protein exhibits a biological activity of MYC.
• the altered expression comprises an increase in the cell surface expression of one or more immune cell receptors.
• the one or more cell surface receptors comprises PD-1, LAG-3, CD160, 2B4, or any combination thereof.
• the one or more modified immune cells are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.
• the subject is a human or a non-human animal.
• the one or more modified immune cells comprises one or more T cells.
• the one or more modified immune cells comprises one or more CD8 + T cells.
• the one or more modified immune cells comprises one or more exhausted immune cells.
• the one or more modified immune cells comprises one or more exhausted T cells.
• the one or more modified immune cells comprises one or more exhausted CD8 + T cells.
• a chronic microbial infection in a subject in need thereof comprising administering an effective amount of one or more modified immune cells to the subject, wherein the one or more modified immune cells comprise a MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the subject is identified as having altered expression of at least one immune cell marker of T cell exhaustion, or T cell impairment, compared to expression of the at least one immune cell marker in a healthy control.
• the immune cell marker is an immune checkpoint protein.
• the microbial infection is a bacterial infection, a viral infection, a fungal infection, a protozoan infection, or parasitic infection.
• the microbial infection is caused by a pathogen selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Helicobacter pylori,
• Staphylococcus aureus Salmonella Typhi , Treponema pallidum, Escherichia coli,
• HCV Herpesviruses
• HSV Herpes Simplex Virus
• HBV Hepatitis B Virus
• HCV
• the subject was previously vaccinated against the pathogen.
• the one or more modified immune cells are derived from immune cells isolated from the subject.
• the immune cells isolated from the subject are obtained from the peripheral blood of the subject.
• the immune cells isolated from the subject are obtained from the lymph node, spleen, or tumor of the subject.
• the one or more modified immune cells are prepared by contacting the T cells in vitro with the MYC fusion protein.
• the methods further comprise expanding the cells in vitro prior to and/or following contacting the cells with the MYC fusion protein.
• the protein transduction domain sequence is a TAT protein transduction domain sequence.
• the MYC fusion protein comprises SEQ ID NO: 1. In some embodiments, the MYC fusion protein translocates to the nucleus of one or more immune cells in the immune cell population. In some embodiments, the MYC fusion protein exhibits a biological activity of MYC. In some embodiments, the altered expression comprises an increase in the cell surface expression of one or more immune cell receptors. In some embodiments, the one or more cell surface receptors comprises PD-1, LAG-3, CD 160, 2B4, or any combination thereof. In some embodiments, the one or more modified immune cells are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratumorally. In some embodiments, the subject is a human or a non human animal.
• the one or more modified immune cells comprises one or more T cells. In some embodiments, the one or more modified immune cells comprises one or more CD8 + T cells. In some embodiments, the one or more modified immune cells comprises one or more exhausted immune cells. In some embodiments, the one or more modified immune cells comprises one or more exhausted T cells. In some embodiments, the one or more modified immune cells comprises one or more exhausted CD8 + T cells.
• the subject is identified as having altered expression of at least one immune cell marker of T cell exhaustion, or T cell impairment, compared to expression of the at least one immune cell marker in a healthy control.
• the immune cell marker is an immune checkpoint protein.
• the acute respiratory virus infection is caused by a pathogen selected from the group consisting of influenza virus, respiratory syncytial virus (RSV), pneumonia virus, respiratory vaccinia virus, parainfluenza virus, respiratory adenoviruses, severe acute respiratory syndrome corona virus (SARS-CoV), Middle East respiratory syndrome corona virus (MERS-CoV), and human metapneumovirus (HMPV)).
• RSV respiratory syncytial virus
• pneumonia virus respiratory vaccinia virus
• parainfluenza virus respiratory adenoviruses
• SARS-CoV severe acute respiratory syndrome corona virus
• MERS-CoV Middle East respiratory syndrome corona virus
• HMPV human metapneumovirus
• the subject was previously vaccinated against the pathogen.
• the one or more modified immune cells are derived from immune cells isolated from the subject.
• the immune cells isolated from the subject are obtained from the peripheral blood of the subject.
• the immune cells isolated from the subject are obtained from the lymph node, spleen, or tumor of the subject.
• the one or more modified immune cells are prepared by contacting the T cells in vitro with the MYC fusion protein.
• the methods further comprise expanding the cells in vitro prior to and/or following contacting the cells with the MYC fusion protein.
• the protein transduction domain sequence is a TAT protein transduction domain sequence.
• the MYC fusion protein comprises SEQ ID NO: 1.
• the MYC fusion protein translocates to the nucleus of one or more immune cells in the immune cell population.
• the MYC fusion protein exhibits a biological activity of MYC.
• the altered expression comprises an increase in the cell surface expression of one or more immune cell receptors.
• the one or more cell surface receptors comprises PD-1, LAG-3, CD 160, 2B4, or any combination thereof.
• the one or more modified immune cells are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.
• the subject is a human or a non human animal.
• the one or more modified immune cells comprises one or more T cells.
• the one or more modified immune cells comprises one or more CD8 + T cells.
• the one or more modified immune cells comprises one or more exhausted immune cells.
• the one or more modified immune cells comprises one or more exhausted T cells.
• the one or more modified immune cells comprises one or more exhausted CD8 + T cells.
• the one or more modified immune cells comprise a MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• one or more modified immune cells for treating a chronic microbial infection or acute respiratory virus infection associated with T cell exhaustion, or T cell impairment, in a subject in need thereof, wherein the one or more modified immune cells comprise a MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• FIGs. 1A-1C illustrate the level of CD86 expression in populations of immune cells isolated from donor subjects, following pre-treatment with the PTD-MYC fusion polypeptide or vehicle control, and activation under various conditions.
• FIGs. 2A-2E illustrate the level of CD279 (PD-1) expression (FIGs. 2A-2C) and CD152 (CTLA-4) expression (FIGs. 2A and 2D-2E) in populations of immune cells isolated from donor subjects, following pre-treatment with the PTD-MYC fusion polypeptide or vehicle control, and activation under various conditions.
• FIGs. 3A-3E illustrate the level of CD274 (PD-L1) expression (FIGs. 3A-3C) and CD25 (IL2RA) expression (FIGs. 3A and 3D-3E) in populations of immune cells isolated from donor subjects, following pre-treatment with the PTD-MYC fusion polypeptide or vehicle control, and activation under various conditions.
• PD-L1 CD274
• IL2RA CD25
• FIGs. 4A-4C illustrate the level of CD279 (PD-1) and CD 152 (CTLA-4) expression in peripheral blood mononuclear cells (PBMCs) isolated from patients enrolled in a relapsed refractory melanoma clinical study.
• FIG. 4A shows flow cytometry histograms of PBMC samples labeled with fluorescent antibodies to CD279 that were collected from two relapsed refractory melanoma patients (101 and 103) before and after a one-hour treatment with Tat-MYC protein (TBX-3400).
• FIG. 4B is a set of flow cytometry diagrams showing intracellular staining results for two different tags on the Tat-MYC protein (His or V5) in PBMCs isolated from a patient enrolled in a relapsed refractory melanoma trial.
• the patient’s PBMCs were either left untreated (left panels) or treated with Tat-MYC (TBX-4000) for one hour (right panels).
• FIG. 4C is a set of charts showing the results of a flow cytometry analysis of PBMC samples treated with Tat-MYC (TBX-4000) for one hour in a closed system.
• Tat-MYC samples were analyzed for intracellular levels of Tat-MYC by using fluorescent antibodies that bind to two different Tat-MYC tags (the His Tag and the V5 tag).
• the left- hand chart shows the results of cells stained for the protein on day zero (Od) and on the following day (Id).
• Tat-MYC treated cells TBX-3400; (+)
• untreated cells PBMC -No Treatment; (-)
• the expression levels of T cell activation marker, CD25, and the checkpoint markers, CD279 and CD 152 were analyzed by flow cytometry.
• the term“about” means that a value can vary +/- 20%, +/- 15%, +/- 10% or +/- 5% and remain within the scope of the present disclosure.
• “a concentration of about 200 IU/mL” encompasses a concentration between 160 IU/mL and 240 IU/mL.
• the term“administration” of an agent to a subject includes any route of introducing or delivering the agent to a subject to perform its intended function.
• Administration can be carried out by any suitable route, including intravenously,
• Administration includes self- administration and the administration by another.
• amino acid refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine.
• Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
• amino acids forming a polypeptide are in the D form.
• the amino acids forming a polypeptide are in the L form.
• a first plurality of amino acids forming a polypeptide are in the D form and a second plurality are in the L form.
• polypeptide “peptide,” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues.
• the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog.
• the terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
• a“control” is an alternative sample used in an experiment for comparison purpose.
• a control can be“positive” or“negative.”
• a positive control a composition known to exhibit the desired therapeutic effect
• a negative control a subject or a sample that does not receive the therapy or receives a placebo
• the term“effective amount” or“therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a desired therapeutic effect.
• the amount of a therapeutic protein administered to the subject can depend on the type and severity of the infection and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It can also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
• polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins.
• expression can include splicing of the mRNA in a eukaryotic cell.
• the expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample.
• the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample.
• the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of the compositions disclosed herein.
• the term“expression” also refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g, by transcription) within a cell; (2) processing of an RNA transcript (e.g, by splicing, editing, 5’ cap formation, and/or 3’ end formation) within a cell; (3) translation of an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presentation of a polypeptide or protein on the cell surface; and (6) secretion or presentation or release of a polypeptide or protein from a cell.
• the term“linker” refers to synthetic sequences (e.g ., amino acid sequences) that connect or link two sequences, e.g., that link two polypeptide domains.
• the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of amino acid sequences.
• Immune cells refers to any cell that plays a role in the immune response.
• Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes.
• lymphocyte refers to all immature, mature, undifferentiated and differentiated white lymphocyte populations including tissue specific and specialized varieties. It encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells.
• lymphocytes include all B cell lineages including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-l cells, and B-2 cell populations.
• T-cell includes naive T cells, CD4+ T cells, CD8+ T cells, memory T cells, activated T cells, exhausted T cells, tolerant T cells, chimeric T cells, and antigen-specific T cells.
• B cell refers to, by way of non-limiting example, a pre-B cell, progenitor B cell, early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, naive B cells, plasma B cells, activated B cells, exhausted B cells, tolerant B cells, chimeric B cells, antigen-specific B cells, memory B cell, B-l cell, and B-2 cell populations.
• the adoptive cell therapeutic composition refers to any composition comprising cells suitable for adoptive cell transfer.
• the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of a tumor infiltrating lymphocyte (TIL), TCR (i.e. heterologous T-cell receptor) modified lymphocytes and CAR (i.e. chimeric antigen receptor) modified lymphocytes.
• TIL tumor infiltrating lymphocyte
• CAR i.e. heterologous T-cell receptor
• the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells, exhausted T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, and regulatory T-cells.
• TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition.
• the adoptive cell therapeutic composition comprises T cells.
• the adoptive cell therapeutic composition may be a composition comprising one or more primary immune cells isolated from a donor subject which have been contacted with a PTD-MYC fusion protein, comprising (i) a protein transduction domain; (ii) a MYC polypeptide sequence.
• the term“exhausted immune cell,”“exhausted T cell,” and “exhausted B cell” refer to dysfunctional T cells and B cells, and does not encompass anergic immune cells, anergic T cells, or anergic B cells.
• Exhausted immune cells, exhausted T cells, and exhausted B cells are characterized by progressive loss of effector functions during chronic infections or cancer with some functions that are exhausted early (e.g., IL-2, cytotoxicity, and proliferation), whereas others (e.g., IFN-g) persist longer.
• Anergic immune cells, anergic T cells, or anergic B cells can arise when immune cells receive initial TCR signals in the absence of co-stimulation, leading to a state of hyporesponsiveness.
• MYC and“MYC gene” are synonyms. They refer to a nucleic acid sequence that encodes a MYC polypeptide.
• a MYC gene comprises a nucleotide sequence of at least 120 nucleotides that is at least 60% to 100% identical or homologous, e.g. , at least 60, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 70% to about 100% identical to sequences of NCBI
• the MYC gene is a proto-oncogene.
• a MYC gene is found on chromosome 8, at 8q24.21.
• a MYC gene begins at 128,816,862 bp from pter and ends at 128,822,856 bp from pter.
• a MYC gene is about 6 kb.
• a MYC gene encodes at least eight separate mRNA sequences— 5 alternatively spliced variants and 3 unspliced variants.
• MYC protein “MYC polypeptide,” and“MYC sequence” are synonyms and refer to the polymer of amino acid residues disclosed in NCBI Accession Number UniProtKB/Swiss-Prot:P01106.1 (MYC isoform 1) or NP_002458.2 (UniProtKB/Swiss-Pro PO 1106.2; MYC isoform 2), and functional homologs, analogs or fragments thereof.
• the sequence of or UniProtKB/Swiss-Prot:P01106.1 is:
• NP_002458.2 (UniProtKB/Swiss-ProfPOl 106.2) is:
• the MYC polypeptide is a complete MYC polypeptide sequence. In some embodiments, the MYC polypeptide is a partial MYC polypeptide sequence. In some embodiments, the MYC polypeptide comprises at least 400 consecutive amino acids of SEQ ID NO: 2 OR 11. In some embodiments, the MYC polypeptide comprises at least 400 consecutive amino acids of SEQ ID NO: 2 OR 11 and retains at least one MYC activity. In some embodiments, the MYC polypeptide comprises at least 400, at least 410, at least 420, at least 430, or at least 450 consecutive amino acids of SEQ ID NO: 2 OR 11.
• the MYC polypeptide comprises at least 400, at least 410, at least 420, at least 430, or at least 450 consecutive amino acids of SEQ ID NO: 2 OR 11 and retains at least one MYC activity.
• the MYC polypeptide is c-MYC.
• the MYC polypeptide sequence comprises the sequence shown below:
• the MYC polypeptide sequence comprises the sequence shown below:
• a MYC polypeptide comprises an amino acid sequence that is at least 40% to 100% identical, e.g ., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or any other percent from about 40% to about 100% identical to the sequence of NCBI Accession Number NP002458.2 or UniProtKB/Swiss-Prot Accession Number
• MYC polypeptide refers to a polymer of 439 amino acids, a MYC polypeptide that has not undergone any post-translational modifications. In some embodiments, MYC polypeptide refers to a polymer of 439 amino acids that has undergone post-translational modifications. In some embodiments, the MYC polypeptide is 48,804 kDa. In some embodiments, the MYC polypeptide contains a basic Helix-Loop-Helix Leucine Zipper (bHLH/LZ) domain. In some embodiments, the bHLH/LZ domain comprises the sequence of:
• the MYC polypeptide is a transcription factor (e.g, Transcription Factor 64). In some embodiments, the MYC polypeptide contains an E-box DNA binding domain. In some embodiments, the MYC polypeptide binds to a sequence comprising CACGTG. In some embodiments, the MYC polypeptide promotes one or more of cell survival and/or proliferation.
• a transcription factor e.g, Transcription Factor 64
• the MYC polypeptide contains an E-box DNA binding domain.
• the MYC polypeptide binds to a sequence comprising CACGTG. In some embodiments, the MYC polypeptide promotes one or more of cell survival and/or proliferation.
• a MYC polypeptide includes one or more of those described above, and includes one or more post-translational modifications (e.g ., acetylation).
• the MYC polypeptides comprise one or more additional amino acid residues at the N-terminus or C-terminus of the polypeptide.
• the MYC polypeptides are fusion proteins.
• the MYC polypeptides are linked to one or more additional peptides at the N-terminus or C- terminus of the polypeptide.
• Proteins suitable for use in the methods described herein also includes functional variants, including proteins having between 1 to 15 amino acid changes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, deletions, or additions, compared to the amino acid sequence of any protein described herein.
• the altered amino acid sequence is at least 75% identical, e.g, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein inhibitor described herein.
• sequence-variant proteins are suitable for the methods described herein as long as the altered amino acid sequence retains sufficient biological activity to be functional in the compositions and methods described herein. Where amino acid substitutions are made, the substitutions can be conservative amino acid substitutions.
• a“conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
• the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff et al. (1992), Proc. Natl Acad.
• the BLOSUM62 substitution frequencies are used to define conservative amino acid substitutions that, in some embodiments, are introduced into the amino acid sequences described or disclosed herein.
• the language“conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
• an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
• preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g ., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
• the E-box sequence comprises CACGTG.
• the basic helix-loop-helix domain of a transcription factor encoded by MYC binds to the E-box sequence.
• the E-box sequence is located upstream of a gene (e.g, p21, Be 1-2, or ornithine decarboxylase).
• the MYC polypeptide contains an E-box DNA binding domain.
• the E-box DNA binding domain comprises the sequence of KRRTHNVLERQRRN (SEQ ID NO: 6).
• the binding of the transcription factor encoded by MYC to the E-box sequence allows RNA polymerase to transcribe the gene downstream of the E-box sequence.
• MYC activity or“MYC biological activity” or“biologically active MYC” or“biological activity of MYC” includes one or more of enhancing or inducing cell survival, cell proliferation, and/or antibody production.
• MYC activity includes enhancement of expansion of anti-CD3 and anti-CD28 activated T-cells and/or increased proliferation of long-term self-renewing hematopoietic stem cells.
• MYC activity also includes entry into the nucleus of a cell, binding to a nucleic acid sequence (e.g, binding an E-box sequence), and/or inducing expression of MYC target genes.
• the terms“patient,”“subject,”“individual,” and the like are used interchangeably herein, and refer to an animal, typically a mammal.
• the patient, subject, or individual is a mammal.
• the patient, subject or individual is a human.
• the patient, subject or individual is an animal, such as, but not limited to, domesticated animals, such as equine, bovine, murine, ovine, canine, and feline.
• PTD protein transduction domain
• transporter peptide sequence also known as cell permeable proteins (CPP) or membrane translocating sequences (MTS)
• CPP cell permeable proteins
• MTS membrane translocating sequences
• a nuclear localization signal can be found within the protein transduction domain, which mediates further translocation of the molecules into the cell nucleus.
• “treating” or“treatment” covers the treatment of a disease in a subject, such as a human, and includes: (i) inhibiting a disease, i.e., arresting its development; (ii) relieving a disease, i.e., causing regression of the disease; (iii) slowing progression of the disease; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease.
• “treating” or“treatment” also encompasses regression of a tumor, slowing tumor growth, inhibiting metastasis of a tumor, inhibiting relapse or recurrent cancer and/or maintaining remission.
• the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean“substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
• the treatment can be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
• therapeutic means a treatment and/or prophylaxis.
• a therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
• the present disclosure relates to the treatment of T cell exhaustion, or T cell impairment, in a subject using a MYC fusion protein comprising a protein transduction domain and a MYC polypeptide sequence.
• the T cell exhaustion, or T cell impairment results from chronic conditions, such as chronic viral infection or cancer.
• the T cell exhaustion, or T cell impairment results from acute respiratory viral infection.
• T cell exhaustion, or T cell impairment occurs following vaccination.
• T cell exhaustion, or T cell impairment occurs during active infection in an individual that has been previously vaccinated.
• the present disclosure is based, at least in part, on the discovery, that contacting T cells isolated from a donor subject with a PTD-MYC fusion polypeptide containing a MYC polypeptide and a protein transduction domain (PTD), such as the HIV TAT protein transduction domain, advantageously results in a decrease in the expression of cell surface immune checkpoint proteins, including but not limited to, Programmed cell death protein 1 (PD-1), also known as CD279 (cluster of differentiation 279), or cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), also known as CD152 (cluster of differentiation 152).
• PD-1 and CTLA-4 are inhibitory receptors that promote T cell exhaustion during chronic microbial infection and in cancer.
• a MYC fusion protein to downregulate these receptors, thus decreasing the negative signaling pathways that result in T cell exhaustion, or T cell impairment.
• a MYC fusion protein provided herein is employed to reverse immune cell exhaustion/impairment.
• a MYC fusion protein provided herein is employed to prevent or ameliorate immune cell exhaustion/impairment.
• the present disclosure provides a method for treating or preventing T cell exhaustion, or T cell impairment, in a subject in need thereof, wherein the method comprises administering an effective amount of one or more modified immune cells (e.g., T cells, such as, for example, CD8 + T cells) to the subject, wherein the one or more modified immune cells comprise a PTD-MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the subject is identified as having altered expression of at least one or more immune cell markers associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control.
• the one or more modified immune cells are derived from immune cells isolated from the subject.
• immune cells are isolated from an allogenic donor.
• the immune cells can be obtained from the peripheral blood, lymph node, spleen, or a tumor.
• the immune cells comprise one or more lymphocytes.
• the one or more lymphocytes comprise a T cell, a B cell, an NK cell, or any combination thereof.
• the one or more lymphocytes comprise a T cell.
• the one or more lymphocytes comprise a CD8 + T cell.
• the one or more lymphocytes comprise one or more exhausted lymphocytes from the subject (e.g.
• the one or more modified immune cells may be prepared by contacting a population of immune cells (e.g., CD8 + T cells) in vitro with the MYC fusion protein following isolation from the subject.
• the method may further include expanding the immune cells in vitro prior to contacting the cells with the MYC fusion protein.
• the method may further include expanding the immune cells in vitro following contacting the primary immune cells with the MYC fusion protein.
• the present disclosure provides a method for treating or preventing T cell exhaustion, or T cell impairment, in a subject in need thereof, wherein the method comprises administering an effective amount of a MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the subject is identified as having altered expression of at least one or more immune cell markers associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control.
• the MYC fusion protein or one or more modified immune cells comprising the MYC fusion protein may be administered by any appropriate method, e.g., intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratumorally.
• Exemplary PTD-MYC fusion proteins for use in the methods are provided herein.
• the PTD-MYC fusion proteins provided are able to enter immune cells (e.g., T cell) and translocate to the nucleus, where the fusion protein can activate one or more MYC-responsive genes.
• the protein transduction domain sequence of the MYC fusion protein comprises a TAT protein transduction domain sequence.
• the MYC polypeptide portion of the MYC fusion protein comprises the amino sequence set forth in SEQ ID NO: 2 or 11.
• the PTD-MYC fusion protein comprises the amino sequence set forth in SEQ ID NO: 1.
• subjects for treatment with a MYC fusion protein provided herein or a modified immune cell comprising a MYC fusion protein provided herein exhibit increased expression one or more immune cell markers, wherein increased or sustained expression of the immune cell marker is associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control (e.g., a non-exhausted T cell).
• the one or more immune cell markers that are increased is an immune checkpoint protein.
• the one or more immune cell markers that are increased is selected from the group consisting of PD1, LAG3, CD160, 2B4 (CD244) and a combination of any two or more thereof.
• the one or more immune cell markers that are increased is selected from the group consisting of PD-1, CTLA-4, LAG-3, CD160, 2B4, IL-7ra, IL-15ra, KLRG-1, TIM-3, Eomes and a combination of any two or more thereof.
• the one or more immune cell markers that are increased in an exhausted T cell is selected from the group consisting of CD244, PDCD1, CTLA4, GP49B, PTGER4, CD 160, LAG3, IL7R, IL15RA, HAVCR2, PTGER2, CD7, TNFRSF9, GLYCOP, VCAM1, TNFSF6, ITM2A, MOX2, ITGAV, CD9,CCL3, CXCR4, CCL4, CCRL2, SMAD1, RGS16, GPR56, TANK, DUSP1, GPR65, PTPN13, PRKWNK, IL6ST, ITPR5, JAK3, MAP3K1, SH2D2A, SOCS3, ACTN1, ISG20, G1P2, ICSBP1, SERPINA, TCRG-V4, TCRB- VI, PBX3, EOMES, ATF1, AHR, EGR2, HFATC1, ZFP91, HIST1H2, BCL2, CASP3,
• treatment with a MYC fusion protein provided herein or a modified immune cell comprising a MYC fusion protein results in a decrease of at least one or more immune cell markers selected from the group consisting of CD244, PDCD1, CTLA4, GP49B, PTGER4, CD 160, LAG3, IL7R, IL15RA, HAVCR2, PTGER2, CD7, TNFRSF9, GLYCOP, VCAMl, TNFSF6, ITM2A, MOX2, ITGAV, CD9,CCL3, CXCR4, CCL4,
• CCRL2 SMADl, RGS16, GPR56, TANK, DUSP1, GPR65, PTPN13, PRKWNK, IL6ST, ITPR5, JAK3, MAP3K1, SH2D2A, SOCS3, ACTN1, ISG20, G1P2, ICSBP1, SERPINA, TCRG-V4, TCRB-Vl, PBX3, EOMES, ATF1, AHR, EGR2, HFATC1, ZFP91, HIST1H2, BCL2, CASP3, GAS2, CKS2, CAR2, PLSCR1, SLC12A2, ART3, GPD2, NDUFA5,
• subjects for treatment with a MYC fusion protein provided herein or a modified immune cell comprising a MYC fusion protein provided herein exhibit decreased expression one or more immune cell markers, where decreased expression of the immune cell marker is associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control (e.g., a non-exhausted T cell).
• the one or more immune cell markers that is decreased is selected from the group consisting of IGSF10, SELPL, H2-Q8, FCGR2B, CD3D, ITGB2, ITGA1, ITGA4, KLRK1, SEMA4A, IL18R1, KLRDl, LY6C, KLRC1, EDG6, ITGB7, ITGAX, KLRG1, CKLFSF7, LSP1, CCR5, CCR2, H2-K1, H2-D1, H2-Q10, H2-Q7, SIAT10, LGALSIO, RAC2, PIK3CD, ILK,
• MAP3K8 ARHGEFl, S100A6, STK38, LCK, ARHGAPl, S100A4, RASA3, JAK1,
• treatment with a MYC fusion protein provided herein or a modified immune cell comprising a MYC fusion protein results in an increase of at least one or more immune cell markers selected from the group consisting of IGSF10, SELPL, H2-Q8, FCGR2B, CD3D, ITGB2, ITGA1, ITGA4, KLRK1, SEMA4A, IL18R1, KLRDl, LY6C, KLRC1, EDG6, ITGB7, ITGAX, KLRG1, CKLFSF7, LSP1, CCR5, CCR2, H2-K1, H2-D1, H2-Q10, H2-Q7, SIAT10, LGALSIO, RAC2, PIK3CD, ILK, MAP3K8, ARHGEFl, S100A6, STK38, LCK, ARHGAPl, S100A4, RASA3, JAKl, S100A1, HCPH, DGKA, PTK9L, IL18RAP, MAP1LC3, ACTB, I
• ILK MAP3K
• TCEB2 EIF3S8, RPS16, EIF2S1, SFPQ, RPL10A, RPL36, EEF2, RPS7, PIGT, RPL27A, PPGB, TAGLN2, HINT1, LEF1, LSP1, TOB1, SDCBP, SEP 15, MTVR2, GABARAPL2, PLD3, ETS1, DSTN, LBR, GOLPH2, WDR1, PLP2, NME2, KCNAB2, DIAPl, TXNDC5, SMFN, STARDIO, CLIC1, TMSB10, IAP, GM2A, ATP6V0B, DNAJD1, SUPT4H, PTPRC, CRIPl, EMP1, PLAC8, and a combination of any two or more thereof, compared to that observed prior to administration.
• treatment with a MYC fusion protein provided herein or a modified immune cell comprising a MYC fusion protein results in an increase in cytokine production in the subject compared to that observed prior to administration.
• treatment with a MYC fusion protein provided herein or a modified immune cell comprising a MYC fusion protein results in an increase in production of IL-2, TNF, Granzyme B, and/or IFN gamma in the subject compared to that observed prior to administration.
• the present disclosure provides a method for treating a chronic microbial infection in a subject in need thereof, wherein the method comprises administering an effective amount of one or more modified immune cells (e.g., CD8 + T cells) to the subject, wherein the one or more modified immune cells comprise a PTD-MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the present disclosure provides a method for treating a chronic microbial infection in a subject in need thereof, wherein the method comprises administering an effective amount a PTD-MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the subject is identified as having altered expression of at least one or more immune cell markers associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control.
• the one or more modified immune cells are derived from immune cells isolated from the subject.
• the chronic microbial infection is a bacterial infection, a viral infection, a fungal infection, a protozoan infection, or parasitic infection. In some embodiments, the chronic microbial infection is chronic or latent form of a viral infection.
• the chronic microbial infection is caused by a pathogen selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Helicobacter pylori, Staphylococcus aureus , Salmonella Typhi , Treponema pallidum, Escherichia coli, Hemophilus influenza, Pseudomonas aeruginosa, Plasmodium falciparum , Plasmodium vivax, Plasmodium ovale , Plasmodium malariae , Human Immunodeficiency Virus (HIV), Herpesviruses, Herpes Simplex Virus (HSV), Hepatitis B Virus (HBV),
• HCV Human Immunodeficiency Virus
• HSV Herpesviruses
• HSV Herpes Simplex Virus
• HBV Hepatitis B Virus
• HCV Hepatitis C Virus
• Measles Virus Measles Virus
• Papovaviruses Varicella-Zoster Virus
• T-Cell Leukemia Viruses Adenoviruses
• Parvoviruses Epstein-Barr Virus
• Enterovirus Mouse Hepatitis Virus
• MHV Mouse Hepatitis Virus
• CMV Cytomegalovirus
• LCMV Lymphocytic Choriomeningitis Virus
• the chronic microbial infection is an antibiotic resistant or antiviral resistant infection (e.g., antibiotic resistant tuberculosis (TB), Methicillin-resistant Staphylococcus aureus (MRSA), Enterovirus 68, Nipah virus, Middle East respiratory syndrome (MERS)).
• antibiotic resistant tuberculosis TB
• MRSA Methicillin-resistant Staphylococcus aureus
• Enterovirus 68 68
• Nipah virus Middle East respiratory syndrome (MERS)
• the present disclosure provides a method for treating an acute respiratory viral infection in a subject in need thereof, wherein the method comprises administering an effective amount of one or more modified immune cells (e.g., CD8 + T cells) to the subject, wherein the one or more modified immune cells comprise a PTD-MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the present disclosure provides a method for treating an acute respiratory viral infection in a subject in need thereof, wherein the method comprises administering an effective amount a PTD-MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence.
• the subject is identified as having altered expression of at least one or more immune cell markers associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control.
• the one or more modified immune cells are derived from immune cells isolated from the subject.
• the subject has previously been administered one or more vaccines against the pathogen.
• the subject exhibits T cell exhaustion, or T cell impairment, following vaccination against the pathogen.
• the subject exhibits T cell exhaustion, or T cell impairment, during pathogenic infection, where the subject has been previously vaccinated against the pathogen.
• the subject having a chronic microbial infection, or acute respiratory viral infection exhibits increased expression one or more immune cell markers, where increased expression of the immune cell marker is associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control (e.g., a non-exhausted T cell).
• the one or more immune cell markers that are increased is an immune checkpoint protein. In some embodiments, the one or more immune cell markers that are increased is selected from the group consisting of PD1, LAG3, CD160, 2B4 (CD244) and a combination of any two or more thereof. In some embodiments, the one or more immune cell markers that are increased is selected from the group consisting of PD-1, CTLA-4, LAG- 3, CD 160, 2B4, IL-7ra, IL-15ra, KLRG-1, TIM-3, Eomes and a combination of any two or more thereof.
• the one or more immune cell markers that are increased is selected from the group consisting of CD244, PDCD1, CTLA4, GP49B, PTGER4, CD 160, LAG3, IL7R, IL15RA, HAVCR2, PTGER2, CD7, TNFRSF9, GLYCOP, VCAM1, TNFSF6, ITM2A, MOX2, ITGAV, CD9,CCL3, CXCR4, CCL4, CCRL2, SMADl, RGS16, GPR56, TANK, DUSP1, GPR65, PTPN13, PRKWNK, IL6ST, ITPR5, JAK3, MAP3K1, SH2D2A, SOCS3, ACTN1, ISG20, G1P2, ICSBP1, SERPINA, TCRG-V4, TCRB-V1, PBX3, EOMES, ATF1, AHR, EGR2, HFATC1, ZFP91, HIST1H2, BCL2, CASP3, GAS2, CKS2, CAR2, PLSCR1,
• the subject having a chronic microbial infection, or acute respiratory viral infection exhibits decreased expression one or more immune cell markers, where decreased expression of the immune cell marker is associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control (e.g., a non-exhausted T cell).
• a healthy control e.g., a non-exhausted T cell
• the one or more immune cell markers that is decreased is selected from the group consisting of IGSF10, SELPL, H2-Q8, FCGR2B, CD3D, ITGB2, ITGA1, ITGA4, KLRK1, SEMA4A, IL18R1, KLRDl, LY6C, KLRC1, EDG6, ITGB7, ITGAX, KLRG1, CKLFSF7, LSP1, CCR5, CCR2, H2-K1, H2-D1, H2-Q10, H2-Q7, SIAT10,
• subjects for treatment with a MYC fusion protein provided herein or a modified immune cell comprising a MYC fusion protein provided herein can be a human or a non-human animal.
• the present disclosure provides a method for treating a cancer in a subject in need thereof, wherein the method comprises administering an effective amount of one or more modified immune cells (e.g., CD8 + T cells) to the subject, wherein the one or more modified immune cells comprise a PTD-MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence, where the subject is identified as having altered expression of at least one or more immune cell markers associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control.
• modified immune cells e.g., CD8 + T cells
• the present disclosure provides a method for treating a cancer in a subject in need thereof, wherein the method comprises administering an effective amount a PTD-MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence, where the subject is identified as having altered expression of at least one or more immune cell markers associated with T cell exhaustion, or T cell impairment, compared to that observed in a healthy control.
• the one or more modified immune cells are derived from immune cells isolated from the subject.
• the cancer is a metastatic cancer.
• the cancer is a carcinoma, adenoma,
• the cancer is a basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS cancer, colon cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, gastric cancer, glial cell tumor, head and neck cancer, hepatoma, hepatic carcinoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, intra-epithelial neoplasm, kidney cancer, larynx cancer, liver cancer, small-cell lung cancer, non-small cell lung cancer, melanoma, myeloma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, cancer of the respiratory system, retinoblastoma, r
• the solid tumor is a metastatic tumor.
• Immune cells for use in the methods provided herein can be obtained using any suitable method known in the art.
• the immune cells are primary immune cells.
• the immune cells are lymphocytes, such as T and B cells.
• the immune cells are natural killer (NK) cells.
• the immune cells are a mixture of lymphocytes and NK cells.
• the immune cells are obtained from the peripheral blood of a donor subject.
• the immune cells are peripheral blood mononuclear cells (PBMC).
• PBMC peripheral blood mononuclear cells
• the immune cells are obtained from the spleen or lymph nodes of a donor subject.
• the immune cells are modified following isolation from a donor.
• the immune cells are CAR T cells.
• the immune cells are obtained from a subject that has a chronic infection.
• the chronic infection is a bacterial infection, a viral infection, a fungal infection, a protozoan infection, or parasitic infection.
• the immune cells are obtained from a subject that has a tumor.
• the immune cells are T cells that have infiltrated a tumor (e.g., tumor infiltrating lymphocytes).
• the T cells are removed during surgery of a tumor.
• the T cells are isolated after removal of tumor tissue by biopsy.
• the T cells are isolated from a sample containing a population of cells, such as a blood, lymph, spleen or tissue biopsy sample.
• T cells can be isolated from a population of cells by any means known in the art.
• the methods comprise obtaining a bulk population of T cells from a tumor sample by any suitable method known in the art.
• a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which specific cell populations can be selected.
• Suitable methods of obtaining a bulk population of T cells can include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting) the tumor, and/or aspiration (e.g., as with a needle).
• mechanically dissociating e.g., mincing
• enzymatically dissociating e.g., digesting
• aspiration e.g., as with a needle
• the bulk population of T cells obtained from a tumor sample can comprise any suitable type of T cell.
• the bulk population of T cells obtained from a tumor sample comprises tumor infiltrating lymphocytes (TILs).
• the population of immune cells can be obtained from any mammal.
• mammal refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits; the order Carnivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and Swines (pigs); or of the order Perssodactyla, including Equines (horses).
• the mammals can be non human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
• the mammal can be a mammal of the order Rodentia, such as mice and hamsters.
• the mammal is a non-human primate or a human.
• An exemplary mammal is a human.
• the subject to receive the immune cells is also the donor of the immune cells (i.e., autologous ACT). In some embodiments, the subject to receive the immune cells is different than the donor of the tumor sample (i.e. allogenic ACT).
• T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, and tumors.
• T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation.
• cells from the circulating blood of an individual are obtained by apheresis or leukopheresis.
• the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
• the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
• the cells are washed with phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• the wash solution lacks calcium and can lack magnesium or can lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation.
• a washing step can be accomplished by methods known to those in the art, such as by using a semi -automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer's instructions.
• the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
• apheresis sample can be removed and the cells directly resuspended in culture media.
• T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
• a specific subpopulation of T cells such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
• T cells are isolated by incubation with anti- CD3/anti-CD28 (i.e., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, or XCYTE DYNABEADSTM for a time period sufficient for positive selection of the desired T cells.
• the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time period is 24 hours.
• the incubation time period is 24 hours.
• TIL tumor infiltrating lymphocytes
• Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
• the method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
• a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CDl lb, CD16, HLA-DR, and CD8.
• monocyte populations i.e., CD14+ cells
• monocyte populations can be depleted from blood preparations by a variety of methodologies, including anti-CD 14 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal.
• the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes.
• the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name DynabeadsTM.
• other non-specific cells are removed by coating the paramagnetic particles with“irrelevant” proteins (e.g., serum proteins or antibodies).
• Irrelevant proteins and antibodies include those proteins and antibodies or fragments thereof that do not specifically target the T cells to be isolated.
• the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.
• such depletion of monocytes is performed by preincubating T cells isolated from whole blood, apheresed peripheral blood, or tumors with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20: 1 beadxell ratio) for about 30 minutes to 2 hours at 22 to 37 degrees C, followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles.
• Such separation can be performed using standard methods available in the art. For example, any magnetic separation methodology can be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after depletion.
• the concentration of cells and surface can be varied.
• it can be desirable to significantly decrease the volume in which beads and cells are mixed together i.e., increase the concentration of cells, to ensure maximum contact of cells and beads.
• a concentration of 2 billion cells/mL is used.
• a concentration of 1 billion cells/mL is used.
• greater than 100 million cells/mL is used.
• a concentration of 2 billion cells/mL is used.
• a concentration of 1 billion cells/mL is used.
• greater than 100 million cells/mL is used.
• a concentration of cells and surface e.g., particles such as beads
• concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used.
• a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used.
• concentrations of 125 or 150 million cells/mL can be used.
• Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
• use of high cell concentrations allows more efficient capture of cells that can weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue). Such populations of cells can have therapeutic value and would be desirable to obtain.
• using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
• the concentration of cells used is 5x l0 6 /mL. In other embodiments, the concentration used can be from about 1 x 10 5 /ml to 1 x 10 6 /mL, and any integer value in between. Thus, the concentration used may be from about 1 c 10 5 /mL, about l.
• T cells can also be frozen.
• the freeze and subsequent thaw step can provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
• the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media, the cells then are frozen to -80°
• cells are directly labeled with an epitope-specific reagent for isolation and/or enrichment by flow cytometry followed by characterization of cell phenotypes.
• immune cells are isolated by contacting the immune cell specific antibodies. Sorting of any cells of the present invention, can be carried out using any of a variety of commercially available cell sorters, including, but not limited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAriaTM, FACSArrayTM, FACSVantageTM, BDTM LSR II, and FACSCaliburTM (BD Biosciences, San Jose, Calif.).
• the method further comprises expanding the numbers of T cells in the enriched cell population.
• the T cells can be expanded before or after treatment of the cells with the PTD-MYC fusion polypeptide.
• the numbers of T cells can be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold), more preferably at least about 100- fold, more preferably at least about 1,000-fold, or most preferably at least about 100,000-fold.
• the numbers of T cells can be expanded using any suitable method known in the art.
• ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion.
• the T cells can be stimulated or activated by a single agent.
• T cells are stimulated or activated with two agents, one that induces a primary signal and a second that is a co- stimulatory signal.
• Ligands useful for stimulating a single signal or stimulating a primary signal and an accessory molecule that stimulates a second signal can be used in soluble form.
• Ligands can be attached to the surface of a cell, to an Engineered Multivalent Signaling Platform (EMSP), or immobilized on a surface.
• EMP Engineered Multivalent Signaling Platform
• both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell.
• the molecule providing the primary activation signal can be a CD3 ligand
• the co-stimulatory molecule can be a CD28 ligand or 4- IBB ligand.
• the cells are expanded by stimulation with one or more antigens, such as a melanoma tumor antigen or antigens derived from the patient’s tumor.
• the isolated immune cells are immediately treated with the PTD-MYC fusion polypeptide following isolation. In other embodiments, the isolated immune cells are stored in a suitable buffer and frozen prior to treatment with the PTD-MYC fusion polypeptide. In some embodiments, the isolated immune cells are immediately treated with the PTD-MYC fusion polypeptide following isolation and the treated cells are stored in a suitable buffer and frozen until needed for administration to the patient.
• the isolated immune cells e.g., a mixed population immune cells or isolated types, such as CD8 + T cells
• a composition containing a PTD-MYC fusion polypeptide for a period of time sufficient to be taken up by the cells.
• the immune cells are contacted with a composition containing a PTD-MYC fusion polypeptide for less than about 24 hours, less than about 23 hours, less than about 22 hours, less than about 21 hours, less than about 20 hours, less than about 19 hours, less than about 18 hours, less than about 17 hours, less than about 16 hours, less than about 15 hours, less than about 14 hours, less than about 13 hours, less than about 12 hours, less than about 11 hours, less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.
• a composition containing a PTD-MYC fusion polypeptide for less than about 24 hours, less than about 23 hours, less than about 22 hours, less than about 21 hours, less than about 20 hours, less than about 19 hours, less than about 18 hours, less than about 17 hours, less than about 16 hours, less than about 15 hours, less than
• the immune cells are contacted with a composition containing a PTD-MYC fusion polypeptide for less than about 55 minutes, less than about 50 minutes, less than about 45 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 29 minutes, less than about 28 minutes, less than about 27 minutes, less than about 26 minutes, less than about 25 minutes, less than about 24 minutes, less than about 23 minutes, less than about 22 minutes, less than about 21 minutes, less than about 20 minutes, less than about 19 minutes, less than about 18 minutes, less than about 17 minutes, less than about 16 minutes, less than about 15 minutes, less than about 14 minutes, less than about 13 minutes, less than about 12 minutes, less than about 11 minutes, or less than about 10 minutes.
• the immune cells are contacted with a composition containing a PTD-MYC fusion polypeptide for about 1 hour.
• the immune cells are contacted with a composition containing a PTD-MYC fusion polypeptide for 24 hours or longer. In certain embodiments, the immune cells are contacted with a composition containing a PTD-MYC fusion
• polypeptide for less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 2 days, or less than about 1 day.
• the cells are contacted with a PTD-MYC fusion polypeptide at a concentration of 0.5pg/ml to 500 pg/ml.
• 260pg/ml at least 280pg/ml, at least 300pg/ml, at least 320pg/ml, at least 340pg/ml, at least
• 360pg/ml at least 380pg/ml, at least 400pg/ml, at least 420pg/ml, at least 440pg/ml, at least
• the immune cells that are contacted with a composition containing a PTD-MYC fusion polypeptide are T cells with genetically modified antigen receptors, including chimeric antigen receptor (CAR)-T cells.
• CAR chimeric antigen receptor
• Various strategies can, for example, be employed to genetically modify T cells by altering the specificity of the T cell receptor (TCR), for example, by introducing new TCR a and b chains with selected peptide specificity (see, e.g ., U.S. Patent No.
• Chimeric antigen receptors can be used in order to generate immunoresponsive cells, such as T cells, specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (see, e.g, U.S. Patent Nos.
• CAR T cells comprising a Myc fusion polypeptide (e.g, PTD) as described herein.
• PTD-MYC fusion polypeptide can improve the expansion of the CAR T cells prior to administration to the subject.
• CARs are comprised of an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen binding domain that is specific for a predetermined target.
• the antigen-binding domain of a CAR is often an antibody or antibody fragment (e.g, a single chain variable fragment, scFv)
• the binding domain is not particularly limited so long as it results in specific recognition of a target.
• the antigen-binding domain may comprise a receptor, such that the CAR is capable of binding to the ligand of the receptor.
• the antigen-binding domain may comprise a ligand, such that the CAR is capable of binding the endogenous receptor of that ligand.
• the T cells expressing a desired CAR are selected through co-culture with g-irradiated activating and propagating cells (AaPC), which co-express the antigen and co-stimulatory molecules.
• AaPC g-irradiated activating and propagating cells
• the engineered CAR T-cells are expanded, for example by co-culture on AaPC in presence of soluble factors, such as IL-2 and IL-21. This expansion can for example be carried out so as to provide memory CAR+ T cells.
• CAR T cells can be provided that have specific cytotoxic activity against antigen bearing cells (optionally in conjunction with production of desired chemokines such as interferon-g).
• the CAR T-cells are contacted with a PTD-MYC fusion polypeptide provided herein in vitro to generation a modified CAR T cells for the treatment of a disease or condition associated with T cell exhaustion or T cell impairment (e.g., a chronic viral infection, cancer, or acute respiratory viral infection).
• a disease or condition associated with T cell exhaustion or T cell impairment e.g., a chronic viral infection, cancer, or acute respiratory viral infection.
• the modified CAR T cells can be administered according any suitable method, including the methods for administration of the PTD-MYC fusion polypeptide-modified immune cells as described above.
• the PTD-MYC fusion polypeptide comprises a protein transduction domain (PTD), a MYC polypeptide that promotes one or more of cell survival or proliferation, and optionally a protein tag domain, e.g ., one or more amino acid sequences that facilitate purification of the fusion protein.
• PTD protein transduction domain
• MYC polypeptide that promotes one or more of cell survival or proliferation
• a protein tag domain e.g ., one or more amino acid sequences that facilitate purification of the fusion protein.
• a cell contacted with MYC polypeptide exhibits increased survival time (e.g, as compared to an identical or similar cell of the same type that was not contacted with MYC), and/or increased proliferation (e.g, as compared to an identical or similar cell of the same type that was not contacted with MYC).
• the MYC fusion protein comprises (a) a protein
• the MYC fusion protein is a polypeptide of Formula (I):
• a MYC fusion protein disclosed herein comprises (a) a protein transduction domain; (b) a MYC polypeptide sequence; and (c) one or more molecules that link the protein transduction domain and the MYC polypeptide sequence.
• the MYC fusion protein is a polypeptide of Formula (II):
• -X- is a molecule that links the protein transduction domain and the MYC
• a MYC fusion protein disclosed herein comprises (a) a protein transduction domain; (b) a MYC polypeptide sequence; (c) at least two protein tags; and (d) optionally linker(s).
• the MYC fusion protein is a polypeptide of F ormul a (III- VI) :
• -X- is a linker.
• -X- is one or more amino acids.
• a MYC fusion protein disclosed herein comprises (a) a protein transduction domain; (b) a MYC polypeptide sequence; (c) a 6-histidine tag; (d) a V5 epitope tag: and (e) optionally linker(s).
• the MYC fusion protein is a polypeptide of Formula (VII-XIV):
• -X- is a linker.
• -X- is one or more amino acids.
• the MYC fusion protein comprises one or more linker sequences.
• the linker sequences can be employed to link the protein transduction domain, MYC polypeptide sequence, V5 epitope tag and/or 6-histidine tag of the fusion protein.
• the linker comprises one or more amino acids.
• the amino acid sequence of the linker comprises KGELNSKLE.
• the linker comprises the amino acid sequence of RTG.
• PTD Protein Transduction Domain
• the MYC fusion protein includes a protein transduction domain.
• Peptide transport provides an alternative for delivery of small molecules, proteins, or nucleic acids across the cell membrane to an intracellular compartment of a cell.
• PTD protein transduction domain
• One non limiting example and well-characterized protein transduction domain (PTD) is a TAT-derived peptide.
• Frankel et al. see, e.g., U.S. Pat. No. 5,804,604, U.S. Pat. No. 5,747,641, U.S. Pat. No. 5,674,980, U.S. Pat. No. 5,670,617, and U.S. Pat. No.
• TAT comprises an amino acid sequence of MRKKRRQRRR (SEQ ID NO: 7).
• Penetratin can transport hydrophilic macromolecules across the cell membrane (Derossi et al. , Trends Cell Biol., 8:84- 87 (1998) incorporated herein by reference in its entirety). Penetratin is a 16 amino acid peptide that corresponds to amino acids 43-58 of the homeodomain of Antennapedia, a Drosophila transcription factor which is internalized by cells in culture.
• VP22 a tegument protein from Herpes simplex virus type 1 (HSV-1), has the ability to transport proteins and nucleic acids across a cell membrane (Elliot et al. , Cell 88:223-233, 1997, incorporated herein by reference in its entirety). Residues 267-300 of VP22 are necessary but cannot be sufficient for transport. Because the region responsible for transport function has not been identified, the entire VP22 protein is commonly used to transport cargo proteins and nucleic acids across the cell membrane (Schwarze et al. , Trends Pharmacol Sci, 21 :45-48, 2000).
• HSV-1 Herpes simplex virus type 1
• the PTD-MYC fusion polypeptide includes a protein transduction domain.
• a protein transduction domain By way of example, but not by way of limitation, in some
• the protein transduction domain comprises the protein transduction domain of one or more of TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP16, and Antennapedia. In some embodiments, the protein transduction domain comprises the protein transduction domain of one or more of TAT, penetratin, VP22, vpr, and EPTD. In some embodiments, the protein transduction domain comprises the protein transduction domain of at least one of TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP 16, and Antennapedia. In some
• the protein transduction domain comprises a synthetic protein transduction domain (e.g ., polyarginine or PTD-5). In particular embodiments, the protein transduction domain comprises a TAT protein transduction domain. In some embodiments, the protein transduction domain is covalently linked to the MYC polypeptide. In some embodiments, the protein transduction domain is linked to the MYC polypeptide via a peptide bond. In some embodiments, the protein transduction domain is linked to the MYC polypeptide via a linker sequence. In some embodiments, the linker comprises a short amino acid sequence. By way of example, but not by way of limitation, in some embodiments, the linker sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.
• the PTD-MYC fusion protein of the present technology can be arranged in any desired order.
• the MYC fusion protein can be arranged in order of a) the protein transduction domain linked in frame to the MYC polypeptide, b) the MYC polypeptide linked in frame to the V5 domain, and c) the V5 domain linked in frame to the 6-histidine epitope tag.
• the MYC fusion protein has an order of components of a) the MYC polypeptide linked in frame to the protein transduction domain, b) the protein transduction domain linked in frame to the V5 domain, and c) the V5 domain linked in frame to the 6-histidine epitope tag.
• the protein transduction domain is a TAT protein transduction domain. In some embodiments, the protein transduction domain is TAT [ 48-57 ] . In some embodiments, the protein transduction domain is TAT [ 57-48 ].
• the MYC fusion protein comprises a protein tag domain that comprises one or more amino acid sequences that facilitate purification of the fusion protein.
• the protein tag domain comprises one or more of a
• exemplary tags include one or more of a V5, a histidine-tag (e.g, a 6-histidine tag), HA (hemagglutinin) tags, FLAG tag, CBP (calmodulin binding peptide), CYD (covalent yet dissociable NorpD peptide), Strepll, or HPC (heavy chain of protein C).
• a histidine-tag e.g, a 6-histidine tag
• HA hemagglutinin
• FLAG tag e.g, a 6-histidine tag
• CBP calmodulin binding peptide
• CYD covalent yet dissociable NorpD peptide
• Strepll heavy chain of protein C
• the protein tag domain comprises about 10 to about 20 amino acids in length.
• the protein tag domain comprises 2 amino acids to 40 amino acids in length, for example 6-20 amino acids in length. In some embodiments, the protein tag domain comprises 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, or 40 amino acids. In some embodiments, two of the above listed tags (for example, V5 and the 6-histidine-tag) are used together to form the protein tag domain.
• two of the above listed tags are used together to form the protein tag domain.
• the histidine tag is a 6-histidine tag.
• the histidine tag comprises the sequence HHHHHH (SEQ ID NO: 8).
• the MYC fusion protein disclosed herein comprises a V5 epitope tag.
• the V5 tag comprises the amino acid sequence of: GKPIPNPLLGLDST (SEQ ID NO:9).
• the V5 tag comprises the amino acid sequence of
• the protein tags can be added to the fusion protein disclosed herein by any suitable method.
• a TAT-MYC polypeptide sequence is cloned into an expression vector encoding one or more protein tags, e.g ., a polyHis-tag and/or a V5 tag.
• a polyhistidine tag and/or a V5 tag is added by PCR (i.e., the PCR primers comprise a polyhistidine sequence and/ or V5 sequence).
• PTD-MYC fusion polypeptides e.g, TAT-MYC fusion polypeptide
• TAT-MYC fusion polypeptide can be constructed by methods well known in the art.
• a nucleotide sequence encoding a TAT-MYC fusion polypeptide can be generated by PCR.
• a forward primer for a human MYC sequence comprises an in frame N-terminal 9-amino-acid sequence of the TAT protein transduction domain (e.g, RKKRRQRRR).
• a reverse primer for a human MYC sequence is designed to remove the stop codon.
• the PCR product is cloned into any suitable expression vector.
• the expression vector comprises a polyhistidine tag and a V5 tag.
• a MYC fusion protein disclosed herein comprises (a) TAT, and (b) c-MYC. In some embodiments, a MYC fusion protein disclosed herein comprises (a) TAT [48-57], and (b) c-MYC. In some embodiments, a MYC fusion protein disclosed herein comprises (a) TAT[57-48], and (b) c-MYC.
• a MYC fusion protein disclosed herein comprises (a) TAT, (b) c-MYC, (c) linker(s), (d) V5 tag, and (e) 6-histidine tag.
• a MYC fusion protein disclosed herein comprises (a) TAT [48-57], (b) c-MYC, (c) linker(s), (d) V5 tag, and (e) 6-histidine tag.
• a MYC fusion protein disclosed herein comprises (a) TAT[57-48], (b) c-MYC, (c) linker(s), (d) V5 tag, and (e) 6-histidine tag.
• the PTD-MYC fusion polypeptide comprises SEQ ID NO: 1; in some embodiments, the PTD-MYC fusion polypeptide is SEQ ID NO: 1.
• the fusion protein can be modified during or after synthesis to include one or more functional groups.
• the protein can be modified to include one or more of an acetyl, phosphate, acetate, amide, alkyl, and/or methyl group. This list is not intended to be exhaustive, and is exemplary only. In some
• the protein includes at least one acetyl group.
• a PTD-MYC fusion polypeptide can be generated by any suitable method known the art, e.g. by recombinant protein expression in a cell, such as a bacterial cell, an insect cell, or mammalian cell.
• a PTD-MYC fusion polypeptide is recombinantly produced by microbial fermentation.
• microbial fermentation is performed in a fermentation volume of from about 1 to about 10,000 liters, for example, a fermentation volume of about 10 to about 1,000 liters.
• the fermentation can utilize any suitable microbial host cell and culture medium.
• E. coli is utilized as the microbial host cell.
• microorganisms can be used, e.g. , S. cerevisiae, P. pastoris, Lactobacilli, Bacilli and Aspergilli.
• the microbial host cell is BL-21 StarTM A. coli strain (Invitrogen).
• Invitrogen In an exemplary
• the microbial host cell is BLR DE3 E. coli. strain.
• the host cells are modified to provide tRNAs for rare codons, which are employed to overcome host microbial cell codon bias to improve translation of the expressed proteins.
• the host cells e.g, E. coli
• a plasmid such as pRARE (CamR), which express tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons.
• pRARE CamR
• Additional, suitable plasmids or constructs for providing tRNAs for particular codons are known in the art and can be employed in the methods provided.
• Integrative or self-replicative vectors can be used for the purpose of introducing the PTD-MYC fusion polypeptide expression cassette into a host cell of choice.
• the coding sequence for the PTD-MYC fusion polypeptide is operably linked to promoter, such as an inducible promoter.
• Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g ., the presence or absence of a nutrient or a change in temperature.
• the nucleic acid encoding the PTD-MYC fusion polypeptide is codon optimized for bacterial expression.
• promoters that are recognized by a variety of potential host cells are well known. These promoters can be operably linked to PTD-MYC fusion polypeptide encoding DNA by removing the promoter from the source DNA, if present, by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Promoters suitable for use with microbial hosts include, but are not limited to, the b-lactamase and lactose promoter systems (Chang et al. (1978) Nature, 275:617-624; Goeddel et al.
• promoters for use in bacterial systems can contain a Shine-Dalgarno (S.D.) sequence operably linked to the coding sequence.
• the inducible promoter is the lacZ promoter, which is induced with Isopropyl b-D-l-thiogalactopyranoside (IPTG), as is well-known in the art. Promoters and expression cassettes can also be synthesized de novo using well known techniques for synthesizing DNA sequences of interest.
• the expression vector for expression of the PTD-MYC fusion polypeptides herein is pETlOl/D- Topo (Invitrogen).
• the microbial host containing the expression vector encoding the PTD-MYC fusion polypeptide is typically grown to high density in a fermentation reactor.
• the reactor has controlled feeds for glucose.
• a fermenter inoculum is first cultured in medium supplemented with antibiotics (e.g, overnight culture). The fermenter inoculum is then used to inoculate the fermenter culture for expression of the protein. At an OD600 of at least about 15, usually at least about 20, at least 25, at least about 30 or higher, of the fermenter culture, expression of the recombinant protein is induced.
• IPTG is added to the fermentation medium to induce expression of the PTD-MYC fusion polypeptide.
• the IPTG is added to the fermenter culture at an OD600 which represents logarithmic growth phase.
• induced protein expression is maintained for around about 2 to around about 5 hours post induction, and can be from around about 2 to around about 3 hours post-induction. Longer periods of induction may be undesirable due to degradation of the recombinant protein.
• the temperature of the reaction mixture during induction is preferably from about 28°C to about 37°C, usually from about 30°C to about 37°C. In particular embodiments, induction is at about 37°C.
• the PTD-MYC fusion polypeptide is typically expressed as cytosolic inclusion bodies in microbial cells. To harvest inclusion bodies, a cell pellet is collected by
• the cells are lysed by conventional methods, e.g., sonication, homogenization, etc.
• the lysate is then resuspended in solubilization buffer, usually in the presence of urea at a concentration effective to solubilize proteins, e.g, from around about 5M, 6M, 7M, 8M, 9M or greater. Resuspension may require mechanically breaking apart the pellet and stirring to achieve homogeneity.
• the cell pellet is directly resuspended in urea buffer and mixed until homogenous.
• the resuspension/solubilization buffer is 8M Urea, 50 mM Phosphate pH 7.5 and the suspension is passed through a homogenizer.
• the homogenized suspension is sulfonylated.
• the homogenized suspension is adjusted to include 200 mM Sodium Sulfite and 10 mM Sodium Tetrathionate.
• the solution is then mixed at room temperature until homogeneous.
• the mixed lysate is then mixed for an additional period of time to complete the sulfonylation (e.g, at 2-8°C for > 12 hours).
• the sulfonylated lysate was then centrifuged for an hour.
• the supernatant containing the sulfonylated PTD-MYC fusion polypeptides is then collected by centrifugation and the cell pellet discarded.
• the supernatant is then passed through a filter, e.g., 0.22 pm membrane filter to clarify the lysate.
• the solubilized protein is then purified.
• Purification methods may include affinity chromatography, reversed-phase chromatography, gel exclusion chromatography, and the like.
• affinity chromatography is used.
• the protein is provided with an epitope tag or 6-histidine-tag for convenient purification.
• exemplary PTD-MYC fusion polypeptides comprise a 6-histidine-tag for purification using Ni affinity chromatography using Ni-resin.
• the Ni-resin column is equilibrated in a buffer containing urea.
• the equilibration buffer is 6M Urea, 50 mM
• Phosphate 500 mM NaCl, and 10% Glycerol solution.
• the sulfonylated and clarified supernatant comprising the PTD-MYC fusion polypeptide is then loaded onto the Ni-resin column.
• the column is then washed with a wash buffer, e.g, 6M Urea, 50mM Phosphate,
• exemplary subsequent washed can include 6M Urea, 50mM Phosphate, 10% Glycerol, and 2M NaCl, pH 7.5, followed another wash of 6M Urea, 50mM Phosphate, 10% Glycerol, 50mM NaCl, and 30mM Imidazole, pH 7.5.
• the PTD-MYC fusion polypeptide is eluted from the column by addition of elution buffer, e.g, 6M Urea, 50mM Phosphate, 10% Glycerol, and 50mM NaCl, pH 7.5 with a gradient from 100 to 300 mM Imidazole, and collecting fractions.
• elution buffer e.g, 6M Urea, 50mM Phosphate, 10% Glycerol, and 50mM NaCl, pH 7.5 with a gradient from 100 to 300 mM Imidazole.
• the protein containing fractions to be pooled are then filtered through a 0.22 pm membrane.
• Assessment of protein yield can be measured using any suitable method, e.g, spectrophotometry at UV wavelength 280.
• one or more additional purification methods can be employed to further purify the isolated PTD-MYC fusion polypeptides.
• the pooled fractions from the Ni-Sepharose chromatography step are further purified by anion exchange chromatography using a Q-Sepharose resin.
• the pool is prepared for loading onto the Q-Sepharose column by diluting the samples to the conductivity of the Q Sepharose buffer (17.52 +/- 1 mS/cm) with the second wash buffer (e.g, 6M Urea, 50mM Phosphate, 10% Glycerol, 2M NaCl, pH 7.5) from the Ni Sepharose chromatography step.
• the second wash buffer e.g, 6M Urea, 50mM Phosphate, 10% Glycerol, 2M NaCl, pH 7.5
• the diluted pool is then loaded onto the Q-Sepharose column, followed by two chase steps using a chase buffer (e.g ., 6M Urea, 50mM Phosphate, 300mM NaCl, and 10% Glycerol), with further sequential applications of the chase buffer until the UV trace reaches baseline, indicating that the protein has eluted from the column.
• a chase buffer e.g ., 6M Urea, 50mM Phosphate, 300mM NaCl, and 10% Glycerol
• the present disclosure provides a method for treating an T cell exhaustion, or T cell impairment, in a subject in need thereof, wherein the method comprises administering an effective amount of a MYC fusion protein comprising (i) a protein transduction domain; and (ii) a MYC polypeptide sequence or one or more immune cells comprising the MYC fusion protein.
• the methods comprise
• any method known to those in the art for contacting a cell, organ or tissue with one or more PTD-MYC fusion polypeptides disclosed herein may be employed. Suitable methods include in vitro , ex vivo , or in vivo methods. In vivo methods typically include the administration of one or more PTD-MYC fusion polypeptides to a mammal, suitably a human. In vitro or ex vivo methods typically include the administration of one or more modified immune cells that have been treated with a PTD-MYC fusion polypeptides to a mammal, suitably a human.
• the one or more PTD-MYC fusion polypeptides or PTD-MYC modified immune cells described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect).
• effective amounts i.e., amounts that have desired therapeutic effect.
• the dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular PTD-MYC fusion polypeptide used, e.g., its therapeutic index, and the subject’s history.
• the effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.
• An effective amount of one or more PTD-MYC fusion polypeptides or PTD-MYC modified immune cells useful in the methods disclosed herein may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds and cells.
• the PTD-MYC fusion polypeptides or PTD-MYC modified immune cells may be administered systemically or locally.
• any suitable method for administration of cells to a subject may be employed.
• suitable methods for adoptive cell therapy typically involve systemic infusion (e.g., intravenous or intraperitoneal infusion) of the modified immune cells together with a pharmaceutically acceptable carrier, diluent or excipient.
• the PTD- MYC modified immune cells are administered locally, for example, intratumorally or at the site of infection.
• the one or more PTD-MYC fusion polypeptides described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a microbial infection.
• the one or more PTD-MYC fusion polypeptides or modified immune cells described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of immune cell exhaustion.
• Such compositions typically include the active agent and a pharmaceutically acceptable carrier.
• “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
• compositions are typically formulated to be compatible with its intended route of administration.
• routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
• antibacterial agents such as benzyl alcohol or methyl parabens
• antioxidants
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• the dosing formulation can be provided in a kit containing all necessary equipment (e.g ., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g, 7 days of treatment).
• compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the
• suitable carriers include physiological saline, bacteriostatic water,
• CREMOPHOR ELTM BASF, Parsippany, N. J.
• PBS phosphate buffered saline
• a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• compositions having one or more PTD-MYC fusion polypeptides disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• a carrier which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomera
• Glutathione and other antioxidants can be included to prevent oxidation.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Oral compositions generally include an inert diluent or an edible carrier.
• the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g ., gelatin capsules.
• Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
• compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
• a lubricant such as magnesium stearate or Sterotes
• a glidant such as colloidal silicon dioxide
• a sweetening agent such as sucrose or saccharin
• the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g. , a gas such as carbon dioxide, or a nebulizer.
• Systemic administration of PTD-MYC fusion polypeptides described herein can also be by transmucosal or transdermal means.
• transmucosal or transdermal For transmucosal or transdermal
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of nasal sprays.
• the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
• transdermal administration may be performed by iontophoresis.
• a therapeutic agent can be formulated in a carrier system.
• the carrier can be a colloidal system.
• the colloidal system can be a liposome, a phospholipid bilayer vehicle.
• the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity.
• One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al, Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al. , Liposome Technology , CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother ., 34(7- 8):915-923 (2000)).
• An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes.
• Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
• the carrier can also be a polymer, e.g. , a biodegradable, biocompatible polymer matrix.
• the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity.
• the polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids.
• Examples include carriers made of, e.g. , collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
• carriers made of, e.g. , collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
• the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA).
• the polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother ., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).
• hGH human growth hormone
• the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
• Such formulations can be prepared using known techniques.
• the materials can also be obtained commercially, e.g ., from Alza Corporation and Nova Pharmaceuticals, Inc.
• Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell- specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
• the therapeutic compounds can also be formulated to enhance intracellular delivery.
• liposomal delivery systems are known in the art, see, e.g. , Chonn and Cullis,“Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in
• Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
• Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e ., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
• IC50 i.e ., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
• levels in plasma may be measured, for example, by high performance liquid chromatography.
• an effective amount of the one or more PTD-MYC fusion polypeptides disclosed herein sufficient for achieving a therapeutic or prophylactic effect range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
• the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
• dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks.
• a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight.
• one or more PTD-MYC fusion polypeptides concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.
• An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
• a therapeutically effective amount of one or more PTD- MYC fusion polypeptides may be defined as a concentration of inhibitor at the target tissue of 10 32 to 10 6 molar, e.g, approximately 10 7 molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g, parenteral infusion or transdermal application).
• treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
• the mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits.
• the mammal is a human.
• the PTD-MYC fusion polypeptides or the PTD-MYC modified immune cells are administered with an additional therapeutic agent.
• additional therapeutic agent is administered prior to, simultaneously with, intermittently with, or following treatment with the PTD-MYC fusion polypeptides or the PTD-MYC modified immune cells.
• the additional therapeutic agent is an immunomodulator, such as an interleukin (e.g. IL-2, IL-7, IL-12), a cytokine, a chemokine, or and immunomodulatory drug.
• the additional therapeutic agent is an anticancer agent (e.g., chemotherapy, radiation therapy, oncolytic agent, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti cancer viruses or microorganisms, and any combinations thereof), an antibacterial agent or an antiviral agent.
• an anticancer agent e.g., chemotherapy, radiation therapy, oncolytic agent, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti cancer viruses or microorganisms, and any combinations thereof
• an antibacterial agent e.g., chemotherapy, radiation therapy, oncolytic agent, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti cancer viruses or microorganisms, and any combinations thereof
• an antibacterial agent e.g., chemotherapy, radiation therapy, oncolytic agent, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti cancer viruses or microorganisms
• Kits according to this embodiment can comprise a carrier means, such as a box, carton, tube, having in close confinement therein one or more containers, such as vials, tubes, ampoules, bottles, syringes, or bags.
• the kits can also comprise associated instructions for using the PTD-MYC fusion polypeptides and/or PTD-MYC fusion polypeptide-modified immune cells of the present technology.
• the kit comprises an effective amount of an adoptive cell therapy, such as MYC-fusion polypeptide-modified immune cells.
• the kit comprises one for more reagents for the detection of the administered MYC-fusion polypeptides and/or MYC-fusion polypeptide-modified immune cells.
• the present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.
• the examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compositions and systems of the present technology.
• the examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims.
• the examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above.
• the variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.
• a whole blood sample was isolated from a donor subject and mixed with the blood anticoagulant, ethylenediaminetetraacetic acid (EDTA). After allowing the cells to incubate at least 24 hours at 37°C, 5% CO2, the whole blood sample is then separated into peripheral blood mononuclear cells (PBMCs; immune cells), which include lymphocytes (e.g., T cells, B cells, NK cells) and monocytes, and waste (i.e., red blood cells, platelets, plasma, etc.) using a density-gradient solution (DGS) on a SEPAX-100 cell processing system
• PBMCs peripheral blood mononuclear cells
• lymphocytes e.g., T cells, B cells, NK cells
• monocytes i.e., red blood cells, platelets, plasma, etc.
• DGS density-gradient solution
• the PBMCs were washed twice during the cell separation process with a 2.5% (w/v) HSA (Human Serum Albumin) solution in saline.
• HSA Human Serum Albumin
• the immune cells were resuspended in the 2.5% (w/v) HSA solution at a concentration of 1 c 10 6 cells/ml to provide a cell suspension.
• samples of the immune cells were treated with DPBS (negative control) or TAT-MYC fusion protein (25 pg/mL for 10 6 cells) and incubated at room temperature for 1 hour.
• the treated immune cells (called TBX-3400) were then re-washed on the SEPAX-100, and excess TAT-MYC was washed off of the cells with the 2.5% (w/v) HSA solution.
• the TBX-3400 were
• a 24-well plate was coated with a solution of an anti-CD3e antibody (500 pL, 5 pg/mL; BD Biosciences) in sterile DPBS.
• an anti-CD3e antibody 500 pL, 5 pg/mL; BD Biosciences
• sterile DPBS 500 pL, 5 pg/mL
• the plates were allowed to incubated overnight at 4°C prior to removing the solutions.
• Each well was then washed twice with 2 mL of sterile DPBS.
• Cells were then resuspended in complete RPMI medium (cRPMI) at a concentration of 2 x 10 6 cells/mL, and subsequently washed with 1 mL of DPBS.
• cRPMI complete RPMI medium
• a solution of an anti-CD28 antibody (100 pL, 200 pg/mL; BD Biosciences) was prepared in cRPMI and serially diluted 10-fold to make two stock solutions of the CD28 antibody in cRPMI (20 pg/mL, and 2 pg/mL).
• 10 pL of the appropriate CD28 antibody solution or DPBS (controls or singly activated cells) was added to the designated wells.
• FIGs. 2A-2E illustrate that the TBX-3400 demonstrates decreased expression levels of PD-1 and CTLA-4, compared to control immune cells, as determined by flow cytometry after cell activation.
• PBMCs Peripheral blood mononuclear cells
• samples of the immune cells are treated with DPBS (negative control) or TAT-MYC fusion protein (25 pg/mL for 10 6 cells) and incubated at room temperature for 1 hour.
• the treated immune cells (called TBX-3400) are then re-washed on the SEPAX-100, and excess TAT-MYC is washed off of the cells with the 2.5% (w/v) HSA solution.
• the TBX-3400 are resuspended in in the 2.5% (w/v) HSA solution at a concentration of 1 c 10 6 cells/ml.
• a 24-well plate is coated with a solution of an anti-CD3e antibody (500 pL, 5 pg/mL; BD Biosciences) in sterile DPBS.
• an anti-CD3e antibody 500 pL, 5 pg/mL; BD Biosciences
• DPBS sterile DPBS
• the plates were allowed to incubate overnight at 4°C prior to removing the solutions.
• Each well is then washed twice with 2 mL of sterile DPBS.
• Cells are then resuspended in complete RPMI medium (cRPMI) at a concentration of 2 x 10 6 cells/mL, and subsequently washed with 1 mL of DPBS.
• cRPMI complete RPMI medium
• a solution of an anti-CD28 antibody (100 pL, 200 pg/mL; BD Biosciences) is prepared in cRPMI and serially diluted 10-fold to make two stock solutions of the CD28 antibody in cRPMI (20 pg/mL, and 2 pg/mL).
• 10 pL of the appropriate CD28 antibody solution or DPBS (controls or singly activated cells) is added to the designated wells.
• Assay plates are then incubated at 37°C, 5% CO2 for 48 or 72 hours, followed by staining with the antibodies to LAG3, CD 160, 2B4, IL-7ra, IL-15ra, KLRG-1, TIM-3, and Eomes for visualization of the magnitude of cell surface expression of these immune cell markers of immune cell dysfunction proteins by FACS analysis. It is expected that TAT-MYC treated cells (TBX-3400) exhibit decreased expression levels of LAG3, CD160, 2B4, IL-7ra, IL-15ra, KLRG-1, TIM-3, and Eomes, compared to control immune cells, as determined by flow cytometry after cell activation.
• mice will be treated with the PTD-MYC fusion protein of the present technology to determine whether the T cells in mice can be rescued from T cell exhaustion characterized by the increased expression of immune cell markers of T cell dysfunction after a chronic viral infection.
• 2xl0 5 plaque-forming units (PFU) of mouse hepatitis virus (MHV), cytomegalovirus (CMV), hepatitis C virus (HEPC) or lymphocytic choriomeningitis vims (LCMV) are injected i.v. into the mice to initiate chronic infection.
• MHV mouse hepatitis virus
• CMV cytomegalovirus
• HEPC hepatitis C virus
• LCMV lymphocytic choriomeningitis vims
• mice 1-10 weeks post infection
• the mice are separated into two groups and are treated with 50 ug/kg PTD-MYC fusion protein (e.g., daily, weekly or biweekly) or not treated (NT; control group) via s.c. tail vein injection.
• NT chronic microbial infection
• the PTD-MYC fusion proteins disclosed herein are useful in methods for treating chronic viral infection, characterized by an increase in immune checkpoint proteins, a decrease cell surface expression of cell markers of immune cell dysfunction, and ameliorating and/or reversing immune cell exhaustion in a subject in need thereof. Further, it is expected that treatment with a MYC fusion protein results in an increase in cytokine production (e.g., IL-2, TNF),
• cytokine production e.g., IL-2, TNF
• Granzyme B, and/or IFN gamma expression in the subject compared to no treatment are considered Granzyme B, and/or IFN gamma expression in the subject compared to no treatment.
• Example 4 Mice Treated with TAT-MYC-Treated Immune Cells for Treatment of
• mice having a chronic viral infection are treated with immune cells isolated from the lymph node or spleen and treated with the PTD-MYC fusion protein (e.g., TBX-3400).
• the PTD-MYC fusion protein e.g., TBX-3400.
• 2xl0 5 plaque-forming units (PFU) of mouse hepatitis virus (MHV), cytomegalovirus (CMV), hepatitis C virus (HEPC) or lymphocytic choriomeningitis virus (LCMV) are injected i.v. into the mice to initiate chronic infection.
• MHV mouse hepatitis virus
• CMV cytomegalovirus
• HEPC hepatitis C virus
• LCMV lymphocytic choriomeningitis virus
• Immune cells isolated from the lymph node or spleen that have been treated with the PTD- MYC fusion protein are administered to the mice intravenously via tail vein injection either during the initial wave of infection (to prevent chronic disease) or after the viral spike has passed and T cell function begins to decline (to treat chronic disease).
• the mice are assayed for expression of one or more immune checkpoint proteins and/or markers of immune cell exhaustion.
• mice treated with TBX-3400 will exhibit decreased viral titers and/or a decrease in one or more symptoms of chronic viral infection compared to control mice that are not treated with TBX-3400. Accordingly, it is expected that the treated mice will exhibit an increase in one or more immune checkpoint proteins, a decrease cell surface expression of cell markers of immune cell dysfunction, and amelioration and/or reversal of immune cell exhaustion compared to control mice that are not treated with TBX-3400.
• cytokine production e.g., IL-2, TNF
• Granzyme B e.g., IFN gamma expression
• mice will be treated with the PTD-MYC fusion protein of the present technology to determine whether the T cells in mice can be rescued from the increased expression of immune cell markers of dysfunction after microbial vaccination.
• BALB ⁇ c mice are vaccinated withM bovis bacille Calmette-Guerin (BCG) either via intranaseal (i.n.) vaccination or subcutaneous (s.c.) vaccination.
• BCG M bovis bacille Calmette-Guerin
• the i.n. vaccination is carried out by applying a total of 30 mL of the BCG vaccine suspension containing lxlO 6 CFU drop- wise to the external nares using a micropipette and allowing the mouse to inhale the suspension into the lungs naturally.
• BCG M bovis bacille Calmette-Guerin
• mice 50 mL of a BCG suspension is injected into the both, the right and left flanks, using a needle to deliver a total of lxlO 6 CFU.
• the vaccine diluent-administered or naive mice serve as controls.
• BCG-vaccinated and control mice are separated into two groups and are treated with 50 ug/kg PTD-MYC fusion protein or not injected (NT-BCG-vaccinated; control group) via s.c. tail vein injection.
• the mice are challenged by the airway i.n. route to deliver 5xl0 4 CFU oiMtb Erdman.
• Lung or spleen cells harvested from BCG-vaccinated and control mice are then stained with the appropriate fluorochrome-labeled antibodies for visualization of cell surface expression levels of immune checkpoint proteins by FACS analysis. It is anticipated that NT- BCG-vaccinated mice will have elevated levels of immune cell markers of dysfunction including but not limited to, PD-1, CTLA-4, and KLRG-1. Further, it is expected that mice treated with the PTD-MYC fusion protein of the present technology will demonstrated decreased levels of immune cell markers of dysfunction.
• a MYC fusion protein results in an increase in cytokine production (e.g., IL-2, TNF), Granzyme B, and/or IFN gamma expression in the subject compared to no treatment.
• Example 6 Mice Treated with TAT-MYC-Treated Immune Cells for Treatment of
• mice having a chronic microbial infection are treated with immune cells isolated from the lymph node or spleen and treated with the PTD-MYC fusion protein (e.g., TBX-3400).
• the PTD-MYC fusion protein e.g., TBX-3400.
• mice are infected with a microbial pathogen (e.g., M. bovis bacille Calmette-Guerin (BCG)) to initiate chronic infection.
• BCG M. bovis bacille Calmette-Guerin
• the mice are separated into groups.
• Immune cells isolated from the lymph node or spleen that have been treated with the PTD-MYC fusion protein e.g.
• TBX-3400 are administered to the mice intravenously via tail vein injection either during the initial wave of infection (to prevent chronic disease) or after the initial spike of infection has passed and T cell function begins to decline (to treat chronic disease).
• the mice are assayed for expression of one or more immune checkpoint proteins and/or markers of immune cell exhaustion.
• mice treated with TBX-3400 will exhibit decreased CFU and/or a decrease in one or more symptoms of chronic microbial infection compared to control mice that are not treated with TBX-3400. Accordingly, it is expected that the treated mice will exhibit an increase in one or more immune checkpoint proteins, a decrease cell surface expression of cell markers of immune cell dysfunction, and amelioration and/or reversal of immune cell exhaustion compared to control mice that are not treated with TBX-3400.
• mice will be treated with the PTD-MYC fusion protein of the present technology to determine whether the T cells in mice can be rescued from T cell impairment characterized by the increased expression of immune cell markers of T cell dysfunction after a chronic viral infection.
• a respiratory virus e.g., influenza virus, respiratory syncytial virus (RSV), pneumonia virus, respiratory vaccinia virus, parainfluenza virus, respiratory adenoviruses, severe acute respiratory syndrome corona virus (SARS-CoV), Middle East respiratory syndrome corona virus (MERS-CoV), or human metapneumovirus (HMPV)
• a respiratory virus e.g., influenza virus, respiratory syncytial virus (RSV), pneumonia virus, respiratory vaccinia virus, parainfluenza virus, respiratory adenoviruses, severe acute respiratory syndrome corona virus (SARS-CoV), Middle East respiratory syndrome corona virus (MERS-CoV), or human metapneumovirus (HMPV)
• SARS-CoV severe
• mice Immune cells isolated from virus-infected mice and control mice are then stained with the appropriate fluorochrome-labeled antibodies for visualization of the magnitude of cell surface expression of immune checkpoint proteins by FACS analysis. It is anticipated that mice which exhibit T-cell impairment during an acute respiratory viral infection (NT; control group) will demonstrate elevated levels of immune cell markers of dysfunction including, but not limited to, PD-1, CTLA-4, and KLRG-1. Further, it is expected that mice treated with the PTD-MYC fusion protein of the present technology will demonstrated decreased levels of immune cell markers of dysfunction.
• PTD- MYC fusion proteins disclosed herein are useful in methods for treating chronic viral infection, characterized by an increase in immune checkpoint proteins, a decrease cell surface expression of cell markers of immune cell dysfunction, and amelioration and/or reversal of immune cell exhaustion/impairment in a subject in need thereof.
• treatment with TBX-3400 results in an increase in cytokine production (e.g., IL-2, TNF), Granzyme B, and/or IFN gamma expression in the subject compared to no treatment.
• mice will be treated with the PTD-MYC fusion protein of the present technology to determine whether the T cells in mice can be rescued from T cell impairment characterized by the increased expression of immune cell markers of T cell dysfunction after a chronic viral infection.
• a respiratory virus e.g., influenza virus, respiratory syncytial virus (RSV), pneumonia virus, respiratory vaccinia virus, parainfluenza virus, respiratory adenoviruses, severe acute respiratory syndrome corona virus (SARS-CoV), Middle East respiratory syndrome corona virus (MERS-CoV), or human metapneumovirus (HMPV)
• a respiratory virus e.g., influenza virus, respiratory syncytial virus (RSV), pneumonia virus, respiratory vaccinia virus, parainfluenza virus, respiratory adenoviruses, severe acute respiratory syndrome corona virus (SARS-CoV), Middle East respiratory syndrome corona virus (MERS-CoV), or human metapneumovirus (HMPV)
• SARS-CoV severe
• mice treated with TBX-3400 will exhibit decreased viral titers and/or a decrease in one or more symptoms of viral infection compared to control mice that are not treated with TBX-3400. Accordingly, it is expected that the treated mice will exhibit an increase in one or more immune checkpoint proteins, a decrease cell surface expression of cell markers of immune cell dysfunction, and amelioration and/or reversal of immune cell exhaustion compared to control mice that are not treated with TBX-3400. Further, it is expected that treatment with TBX-3400 results in an increase in cytokine production (e.g., IL- 2, TNF), Granzyme B, and/or IFN gamma expression in the subject compared to no treatment.
• cytokine production e.g., IL- 2, TNF
• Granzyme B e.g., IL- 2, TNF
• IFN gamma expression e.g., IFN gamma expression
• Example 9 Isolated Immune Cells from Patients with Relapsed Refractory Melanoma
• the cells are activated using plate bound anti-human CD3 antibody and soluble anti-CD28 antibodies for 4-6 days. Once the cells have been activated, they are collected, washed, and stained for flow cytometric analysis.
• the FACS analysis relies on the use of monoclonal antibodies directly conjugated to fluorochromes.
• FIG. 4C shows the results of a study on PBMCs treated with Tat-MYC (TBX- 4000) for one hour in a closed system. Samples were analyzed by flow cytometry for intracellular levels of the protein by using fluorescent antibodies that bind to two different tags encoded in Tat-MYC (His Tag and V5 Tag). As shown in FIG. 4C (left panel), over 25% of the cells stained positive for the protein on day zero (Od) and similar levels were detected the following day (Id).
• a range includes each individual member.
• a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
• a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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