EP4713352A1 - Compositions de récepteurs antigéniques chimériques, cellules car-mast ainsi formées et procédés d'utilisation associés - Google Patents

Compositions de récepteurs antigéniques chimériques, cellules car-mast ainsi formées et procédés d'utilisation associés

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Publication number
EP4713352A1
EP4713352A1 EP24731778.7A EP24731778A EP4713352A1 EP 4713352 A1 EP4713352 A1 EP 4713352A1 EP 24731778 A EP24731778 A EP 24731778A EP 4713352 A1 EP4713352 A1 EP 4713352A1
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EP
European Patent Office
Prior art keywords
cell
cells
car
mast
antigen
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EP24731778.7A
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German (de)
English (en)
Inventor
Xiaolei SU
Jianjian GUO
Yiwei XIONG
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Yale University
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Yale University
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Publication date
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Publication of EP4713352A1 publication Critical patent/EP4713352A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4258—Gangliosides, e.g. GM2, GD2 or GD3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3084—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the disclosed invention is generally in the field of immunology and specifically in the area of engineered therapeutic mast cells.
  • CAR Chimeric antigen receptor
  • TME immune-suppressive tumor microenvironment
  • CAR CAR-killer cells
  • CAR-NK cells CAR-killer cells
  • CAR-macrophages CAR-macrophages
  • NK cells are short lived which might compromise the long-term anti-tumor effect whereas macrophages can sometimes promote tumor growth and suppress T cell responses 12 .
  • the clinical outcomes of these new types of CAR cells remained to be fully evaluated. Therefore, a need remained for developing alternative CAR carriers that could provide new strategies to address the above issues.
  • compositions and methods for improved therapeutic CAR cells are provided.
  • CAR chimeric antigen receptors
  • mast cells can specifically kill cancer cells in vitro and release chemokines that recruit T cells and NK cells, e.g., to the tumor tissues and inhibit solid tumor growth and in mouse xenograft models.
  • fusion polypeptides also referred to as fusion proteins
  • fusion proteins including (a) a mast cell intracellular signaling domain; and (b) an amino acid sequence that is heterologous to the mast cell intracellular signaling domain are provided.
  • (a) includes the intracellular domain of IgE receptor (FcsRI), a cytokine receptor (c- KIT), a G-protein-coupled receptor, or a toll-like receptor expressed in mast cells, or functional fragment or variant thereof.
  • the amino acid sequence of (a) includes an IT AM sequence such as DGVYTGLSTRNQETYETLKHE (SEQ ID NO: 11) or a functional variant thereof, or variant thereof having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:11.
  • the amino acid sequence of (a) includes the amino acid sequence of any one of SEQ ID NOS: 11-21, or a functional fragment thereof, or a variant thereof having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOS: 11-21.
  • the (b) includes one or more of an extracellular domain, transmembrane domain, and/or a further intracellular domain.
  • the fusion polypeptide is a chimeric antigen receptor (CAR) (also referred to herein as a
  • the CAR includes an intracellular cellular domain comprising (a), and (b) includes a transmembrane domain, and an extracellular domain.
  • the CAR is specific for an antigen selected from the group consisting of a cancer antigen, an inflammatory disease antigen, a neuronal disorder antigen, HIV/AIDS, a diabetes antigen, a cardiovascular disease antigen, an infectious disease antigen (including a viral antigen, a protozoan antigen, a bacterial antigen, and an allergen), an autoimmune disease antigen and an autoimmune disease antigen, or combinations thereof.
  • an antigen selected from the group consisting of a cancer antigen, an inflammatory disease antigen, a neuronal disorder antigen, HIV/AIDS, a diabetes antigen, a cardiovascular disease antigen, an infectious disease antigen (including a viral antigen, a protozoan antigen, a bacterial antigen, and an allergen), an autoimmune disease antigen and an autoimmune disease antigen, or combinations thereof.
  • target antigens include, for example, CD19, GD2, B7H3, AFP, AKAP 4, ALK, Androgen receptor, B7H3, BCMA, Bcr Abl, BORIS, Carbonic, CD123, CD138, CD174, CD20, CD22, CD30, CD33, CD38, CD80, CD86, CEA, CEACAM5, CEACAM6, Cyclin, CYP1B1, EBV, EGFR, EGFR806, EGFRvIII, EpCAM, EphA2, ERG, ETV6 AML, FAP, Fos related antigenl, Fucosyl, fusion, GD3, GloboH, GM3, gplOO, GPC3, HER 2/neu, HER2, HMWMAA, HPV E6/E7, hTERT, Idiotype, IL12, IL13RA2, IM19, IX, LCK, Legumain, IgK, LMP2, MAD CT 1, MAD CT 2, MAGE, MelanA/M
  • the target antigen is a cancer antigen, such as CD 19, GD2, B7H3, 41BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C MET, CCR4, CD152, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HER, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI CAM, IL 13, IL 6, insulin
  • the CAR includes an extracellular domain having an anti-CD19, anti-GD2, anti-B7H3, or anti-HER2 antigen binding domain, optionally of SEQ ID NOS:2, 8, 9, and SEQ ID NO:34, respectfully, or a fragment or variant thereof comprising the complementary determining regions (CDRs) thereof.
  • Nucleic acids encoding the fusion polypeptides such as RNA, including, but not limited to mRNA, and DNA nucleic acids, are also provided.
  • the nucleic acid includes an expression control sequence(s) such as promoter.
  • the nucleic acid is a vector or a transposon.
  • Exemplary vectors including expression vectors such plasmids, cosmids, and replicons, and viral vectors such as a lentiviral vector, an Adeno-associated virus (AAV) vector, an adenovirus vector, a Herpes Simplex virus (HSV) vector, a vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV), or a chimeric vector including a combination of any two or more of an Adeno-associated virus (AAV) vector, Herpes Simplex virus (HSV) vector, vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV).
  • AAV Adeno-associated virus
  • HSV Herpes Simplex virus
  • VSV vesicular stomatitis
  • human Bocavirus vector hBoV
  • Isolated cells including or expression the polypeptides and/or nucleic acids are also provided.
  • the cells are mast cells, mast progenitor cells, or hematopoietic stem cells (HSC).
  • HSC hematopoietic stem cells
  • the cells can be isolated from a subject in need of adoptive cell therapy.
  • Pharmaceutical compositions comprising a population of isolated cells and a pharmaceutically acceptable buffer, carrier, diluent, or excipient are also provided, as are methods of use thereof.
  • An exemplary method of treating a subject having a disease, disorder, or condition can include administering to the subject an effective amount of the pharmaceutical composition including isolated cells.
  • the subject has a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen, and the method includes administering to the subject an effective amount of cells including a CAR-mast targeting the antigen.
  • the cells are isolated or derived from the subject having the disease, disorder, or condition prior to the introduction to the cell or from a healthy donor.
  • the cells are mast cells isolated as mast progenitor cells or hematopoietic stem cells and differentiated ex vivo into mast cells.
  • the subject is a human.
  • the subject has cancer, optionally a cancer one or more solid tumors associated therewith.
  • Figures 1A-1C illustrate the generation of exemplary CAR-mast cells.
  • Figure 1 is a schematic of the generation of mouse CAR-mast cells.
  • Figure IB is a micrograph image of mouse mast cells stained with toluidine blue 49 days after in vitro culture. Granules were indicated by purple condensates around the nucleus.
  • Figure 1C is a density plot showing surface staining of c-kit and FcsR 1 a of Bulk bone marrow cells and spleen cells cultured at day 0 (left) and day 59 (right).
  • Figures 2A-2E illustrate exemplary CD19 CAR-mast cells.
  • Figure 2A is a schematic of an exemplary CD 19 CAR construct.
  • Figure 2B is a graph showing CD 19 CAR expression on mouse mast cells.
  • Figure 2C is a bar graph showing TNFa release determined by ELISA.
  • Figure 2D is a bar graph showing cytotoxicity of CAR-mast cells determined by luciferase assay.
  • Figure 2E is a heat map showing chemokine and cytokine profiling of CAR-mast cells.
  • Figure 3A-3F show exemplary GD2 CAR-mast cells.
  • Figure 3A is a schematic of an exemplary GD2 CAR construct.
  • Figure 3B is a graph showing the GD2 CAR expression on mouse mast cells.
  • Figure 3C are bar graphs showing surface expression of CD 107a release (left) and TNFa release (right).
  • Figure 3D is a line graph showing the cytotoxicity of CAR- mast cells determined by luciferase assay.
  • Mouse CAR mast cells (CAR+) or wildtype mast cells (CAR-) were cocultured with GD2+MC38 or plain MC38 cells for 2 Days at 37 °C.
  • Figure 3E is a heat map of chemokine and cytokine profiling of CAR-mast cells.
  • Figure 3F shows killing by MC38-GD2-mCherry-Luciferase cells (GD2+) after 24 hr coculture at 37 °C at the E:T of 0.008:1, 0.04:1, 0.2:1, 1:1, 5: 1 respectively.
  • Figures 4A-4F show exemplary B7H3 CAR-mast cells.
  • Figure 4A is a schematic of an exemplary B7H3 CAR construct.
  • Figures 4B is a graph showing B7H3 CAR expression on mouse mast cells.
  • Figure 4C are bar graphs showing surface expression of CD107a release (left) and TNFa release (right).
  • Figure 4D is a line graph showing cytotoxicity of CAR-mast cells determined by luciferase assay.
  • Mouse CAR mast cells (CAR+) or wildtype mast cells (CAR-) were cocultured with GD2+MC38 or plain MC38 cells for 2 days at 37 °C.
  • Figure 4E is a heat map showing chemokine and cytokine profiling of CAR-mast cells.
  • Figure 4F is a graph showing CAR mast cells killing of a mixture of MC38-B7H3 cells (Antigen+) and MC38-mCherry-Luciferase cells (Antigen-) after 21 hr coculture at 37 °C at the E:T of 1:1, 3:1, 6:1 respectively.
  • Figures 5A-5C show the anti-tumor effect of exemplary B7H3 CAR-mast cells.
  • Figure 5A is a schematic of an experimental timeline of CAR-mast cell treatment in a mouse xenograft model.
  • Figure 5B is a line graph of tumor growth in individual mouse receiving 2 doses of CAR-mast cells, wildtype mast cells or PBS.
  • Figure 5C is a survival curve of mice monitored in Figure 5B. Endpoint was determined by death of the animal or exceeding of tumor size over 2000 mm 3 .
  • Figure 6A is a heat map showing the results of cytokine profiling of B7-H3 CAR mast cells (B7H3 CAR) or GD2 CAR mast cells (GD2 CAR) activated by either coculturing with MC38-B7H3-mCherry-Luciferase cells (B7H3-MC38) or MC38-GD2- mCherry-Luciferase cells (GD2-MC38) by coculture at E:T of 1: 1 respectively, or by IgE, where the CAR mast cells are sensitized with lug/mL anti-DNP IgE for 1 hr then cocultured with 20ng/mL DNP for 20 hr at 37 °C.
  • FIG. 6B is a graph showing antigen- specific killing of MC38-B7H3-mCherry-Luciferase cells (B7H3+) treated with either B7H3 CAR mast cells (CAR), wildtype mast cells (WT) or IgE-activated wildtype mast cells (WT+IgE) at the E:T of 1:1, 3:1, 6:1 respectively with cancer cells total number at 0.02M.
  • Figure 6C is a diagram of mouse CAR-mast cell anaphylactic responses evaluation in mouse MC38-B7H3 tumor model. Timing of mast cell injections are indicated by the arrows.
  • Figure 6E is a bar graph showing plasma histamine concentration of the treated mice.
  • Figure 7A is a series of scatter plots showing HER2 expression on CAR-mast cells compared to Control CD 19 CAR-mast cells.
  • Figure 7B is a scatter plot showing surface expressions of lineage markers (e.g., cKit, FceRa) on HER2 CAR-mast cells.
  • Figure 7C is a line graph showing the results of a Degranulation/ Activation assay, as measured by CD 107 staining for CAR-mast cells.
  • Figure 7D is a bar graph showing TNFa production in the supernatant of mouse CAR mast cells cocultured with HER2- EMT6 cells at the indicated effector-to-target (E:T) ratios after 48 hr.
  • Figure 7E is a heat map showing the results of cytokine and chemokine profiles of HER2 CAR-mast cells incubated with and without HER2-EMT6 cells after 48 hr.
  • Figures 8A and 8B is a line graph and a bar graph respectively, showing cytotoxicity of HER2 CAR-mast cells after 48 hr of coculture with HER2-EMT6 cells at the indicated ratios ( Figure 8A), and against HER2-EMT6 cells in the presence of varying concentrations of Soybean Trypsin Inhibitor (SBTI) ( Figure 8B).
  • SBTI Soybean Trypsin Inhibitor
  • Figure 9B is a graph showing average tumor burden of mice through a 25 -day period after FFluc+-HER2-EMT6 cells inoculation.
  • Figure 9C is a series of plots showing tumor burden of individual mice through a 61-day period post inoculation. Each line represents one mouse.
  • Figure 9D is a survival curve of mice through a 90-day period post tumor cells inoculation.
  • Figure 11A is a schematic representation of wild-type (HER2 WT) and cytoplasmic domain-truncated HER2 (HER2 dCyto) constructs used for ectopic expression in EMT6 cells.
  • Figures 11B and 11C are line graphs showing cytotoxicity (% lysis) and TNFa secretion, respectively, of HER2 CAR-mast cells after 48 hr of coculture with HER2 dCyto-EMT6 cells at the indicated E:T ratios in comparison to CD19 CAR control.
  • CAR-mast cells were generated from spleen-derived (SPL) mast cells or bone marrow-derived (BM) mast cells.
  • Figure 12B shows a schematic transwell setting (left) and results of the assay (right) in which HER2-EMT6 cells were seeded on the insert membrane (3 um pore) and lower bottom of the same transwell.
  • Chimeric Antigen Receptor (CAR)-T cell therapies showed remarkable effects for hematological cancers that are resistant to traditional treatments.
  • CARs targeting solid tumors showed limited efficacies.
  • Major challenges include T cells’ poor infiltration, low persistence, and exhaustion in a hostile tumor microenvironment.
  • CAR-mast cells are provided. Mast cells are long-lived and tissueresident immune cells. When activated by allergens, they release a variety of effectors to induce broad immune reactions.
  • Chimeric antigen receptors (CARs) got mast cells (CAR-mast) that activate mast cells in an antigen-dependent manner are provided.
  • the CAR-mast typically include a mast intracellular signaling domain.
  • CAR-mast cells can specifically kill cancer cells in vitro and release chemokines that recruit T cells and NK cells, e.g., to the tumor tissues and in mouse xenograft models, and inhibit solid tumor growth. Together, these results illustrated CAR-mast cells’ efficacy against solid tumors both in vitro and in vivo. As described in more detail below, these results lead to a positive impact on cancer immunology by providing a new strategy for applying CAR therapies to the treatment of cancers including those with solid tumors.
  • “Introduce” in the context of genome modification refers to bringing in to contact.
  • to introduce a gene editing composition to a cell is to provide contact between the cell and the composition.
  • the term encompasses penetration of the contacted composition to the interior of the cell by any suitable means, e.g., via transfection, electroporation, transduction, gene gun, nanoparticle delivery, etc.
  • homologous means derived from a common ancestor.
  • a homologous trait is any characteristic of organisms that is inherited by two or more species from a common ancestor species.
  • Homologous sequences can be orthologous or paralogous.
  • Homologous sequences are orthologous if they were separated by a speciation event: when a species diverges into two separate species, the divergent copies of a single gene in the resulting species are said to be orthologous.
  • Orthologs, or orthologous genes are genes in different species that are similar to each other because they originated from a common ancestor.
  • Homologous sequences are paralogous if they were separated by a gene duplication event: if a gene in an organism is duplicated to occupy two different positions in the same genome, then the two copies are paralogous.
  • Heterologous means having a different relation, relative position, or structure. Thus, unless otherwise specified, heterologous includes joining or linking of two or more amino acid or nucleic acid sequences from that organism (e.g., species) that are not normally found joined or linked (e.g., together) as well as joining or linking of two or more amino acid or nucleic acid sequences from different species. “Endogenous” refers to any material from or produced inside an organism, cell, tissue or system.
  • Exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.
  • extracellular domain and ectodomain refer to any protein structure that is thermodynamically stable in outside of the cell membrane (i.e., in the extracellular space).
  • intracellular domain refers to any protein structure that is thermodynamically stable in inside of the cell membrane (i.e., in the intracellular cytosol).
  • CAR Chimeric Antigen Receptor
  • a CAR refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a cancer cell, or other specific cell, and with intracellular signal generation.
  • a CAR includes at least an antigen binding domain such as an extracellular binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as "an intracellular signaling domain”) including a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule.
  • the term “antigen binding domain” is used in the context of a CAR to refer to the portion of a CAR that specifically recognizes and binds to an antigen of interest.
  • the “antigen binding domain” of a CAR may be derived from a binding protein such as an antibody or fragment thereof.
  • the “binding domain” of a CAR is a single-chain variable fragment (scFv).
  • the “binding domain” of a CAR includes the complementarity determining regions of a binding protein disclosed herein.
  • the cytoplasmic signaling domain of the CAR further includes one or more functional signaling domains derived from at least one costimulatory molecule.
  • the CAR includes a chimeric fusion protein including an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain including a functional signaling domain derived from a stimulatory molecule.
  • CARs are fusion proteins of single-chain variable fragments (scFv) fused to a CD3-zeta transmembrane domain.
  • scFv single-chain variable fragments
  • the term “antigen” as used herein is defined as a molecule capable of being bound by an antibody or T-cell receptor.
  • An antigen can additionally be capable of provoking an immune response. This immune response can involve either antibody production, or the activation of specific immunologically competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which includes a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene.
  • compositions and methods includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response.
  • an antigen need not be encoded by a “gene” at all.
  • an antigen can be generated synthesized or can be derived from a biological sample.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • “antigen” refers to an antigenic substance that is produced in a tumor cell, which can therefore trigger an immune response in the host.
  • cancer antigens can be useful as markers for identifying a tumor cell, which could be a potential candidate/target during treatment or therapy.
  • TSA tumor specific antigens
  • TAA tumor associated antigens
  • the chimeric antigen receptors are specific for tumor specific antigens.
  • the chimeric antigen receptors are specific for tumor associated antigens.
  • the chimeric antigen receptors are specific both for one or more tumor specific antigens and one or more tumor associated antigens.
  • immune effector cell refers to a cell that is involved in an immune response (e.g. promotion of an immune effector response).
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic- derived phagocytes.
  • the immune effector cell (s) is allogenic.
  • the immune effector cell(s) is autologous.
  • Immune effector cells such as T cells may be activated and expanded generally using methods previously described, such as for example, as described in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318;
  • Bi-specific chimeric antigen receptor refers to a CAR that includes two domains, wherein the first domain is specific for a first ligand/antigen/target, and wherein the second domain is specific for a second ligand/antigen/target.
  • the ligand is a B-cell specific protein, a tumor- specific ligand/antigen/target, a tumor associated ligand/antigen/target, or combinations thereof.
  • a bispecific CAR is specific to two different antigens.
  • a multi- specific or multivalent CAR is specific to more than one different antigen, e.g., 2, 3, 4, 5, or more.
  • a multi-specific or multivalent CAR targets and/or binds three or more different antigens.
  • Encoding refers to the property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • locus is the specific physical location of a DNA sequence e.g., of a gene) on a chromosome. It is understood that a locus of interest can not only qualify a nucleic acid sequence that exists in the main body of genetic material i.e., in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting examples.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences.
  • a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha- anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
  • cDNA complementary DNA
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • isolated also refers to a cell altered or removed from its natural state. That is, the cell is in an environment different from that in which the cell naturally occurs, e.g., separated from its natural milieu such as by concentrating to a concentration at which it is not found in nature. “Isolated cell” is meant to include cells that are within samples that are substantially enriched for the cell of interest and/or in which the cell of interest is partially or substantially purified.
  • transformed As used herein, “transformed,” “transduced,” and “transfected” encompass the introduction of a nucleic acid or other material into a cell by one of a number of techniques known in the art.
  • a “vector” is a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” encompasses an autonomously replicating plasmid or a virus.
  • the term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus (AAV) vectors, retroviral vectors, and the like.
  • Tumor burden refers to the number of cancer cells, the size or mass of a tumor, or the total amount of tumor/cancer in a particular region of a subject. Methods of determining tumor burden for different contexts are known in the art, and the appropriate method can be selected by the skilled person. For example, in some forms, tumor burden can be assessed using guidelines provided in the Response Evaluation Criteria in Solid Tumors (RECIST).
  • RECIST Response Evaluation Criteria in Solid Tumors
  • subject includes, but is not limited to, animals, plants, parasites and any other organism or entity.
  • the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
  • the subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans).
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • a patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • the subject can be any organism in which the disclosed method can be used to genetically modify the organism or cells of the organism.
  • inhibitor or other forms of the word such as “inhibiting” or “inhibition” means to decrease, hinder or restrain a particular characteristic such as an activity, response, condition, disease, or other biological parameter. It is understood that this is typically in relation to some standard or expected value, i.e., it is relative, but that it is not always necessary for the standard or relative value to be referred to. “Inhibits” can also mean to hinder or restrain the synthesis, expression or function of a protein relative to a standard or control. Inhibition can include, but is not limited to, the complete ablation of the activity, response, condition, or disease.
  • “Inhibits” can also include, for 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 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, 100%, or any amount of reduction in between as compared to native or control levels.
  • “inhibits expression” means hindering, interfering with or restraining the expression and/or activity of the gene/gene product pathway relative to a standard or a control.
  • Treatment means to administer a composition to a subject or a system with an undesired condition (e.g., cancer).
  • the condition can include one or more symptoms of a disease, pathological state, or disorder.
  • Treatment includes medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • active treatment that is, treatment directed specifically toward the improvement of a disease, pathological state, or disorder
  • causal treatment that is, treatment directed toward removal of the cause of the associated disease, pathological state, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological state, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological state, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological state, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological state, or disorder.
  • Such measurements and assessments can be made in qualitative and/or quantitative terms.
  • characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
  • “Prevention” or “preventing” means to administer a composition to a subject or a system at risk for an undesired condition (e.g., cancer).
  • the condition can include one or more symptoms of a disease, pathological state, or disorder.
  • the condition can also be a predisposition to the disease, pathological state, or disorder.
  • the effect of the administration of the composition to the subject can be the cessation of a particular symptom of a condition, a reduction or prevention of the symptoms of a condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or reduction of the chances that a particular event or characteristic will occur.
  • the terms “effective amount” or “therapeutically effective amount” means a quantity sufficient to alleviate or ameliorate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and/or physiological effect. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise quantity will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, weight, etc.), the disease or disorder being treated, as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.
  • subject-dependent variables e.g., age, immune system health, weight, etc.
  • the disease or disorder being treated as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • polypeptides includes proteins and functional fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T),
  • the term “functional fragment” or “functional variant” means a fragment or variant of a polypeptide, such as a full-length or native polypeptide, that retains one or more functional properties of the full-length or native polypeptide.
  • variants refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains one or more functional properties (e.g., functional or biological activity).
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological or functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties (e.g., functional or biological activity).
  • Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution).
  • certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within + 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments.
  • the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide.
  • substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: lie, Leu).
  • Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above.
  • embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.
  • “conservative” amino acid substitutions are substitutions wherein the substituted amino acid has similar structural or chemical properties.
  • non-conservative amino acid substitutions are those in which the charge, hydrophobicity, or bulk of the substituted amino acid is significantly altered.
  • identity is a relationship between two or more polypeptide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences.
  • Identity can also mean the degree of sequence relatedness of a polypeptide compared to the full-length of a reference polypeptide.
  • Identity and similarity can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (j.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure.
  • a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%.
  • Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide.
  • fusion protein For example, if a fusion protein is disclosed and discussed and a number of modifications that can be made to a number of molecules including the fusion protein are discussed, each and every combination and permutation of the fusion protein and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary.
  • A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated.
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.
  • These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
  • use of the word “can” indicates an option or capability of the object or condition referred to. Generally, use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to.
  • use of the word “may” indicates an option or capability of the object or condition referred to. Generally, use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of “may” herein does not refer to an unknown or doubtful feature of an object or condition.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.
  • Mast cells are a type of tissue-resident granulocytes that have a lifespan of up to years. They are recognized for their large quantity of intracellul r granules. A wide range of mediators are stored in these granules including proteases, cytotoxic factors, chemokines, cytokines, and bioactive chemicals 13 . These granules can be rapidly released into the extracellular space, a process called degranulation, upon the stimuli from allergens and pathogens 14 15 . After granule release, interestingly, mast cells can refill the granules and undergo repetitive release for multiple rounds 16 . Functionally, mast cells are best-known for their roles in triggering allergic inflammation and in defending parasite infection 17,18 .
  • mast cells have either positive, negative, or no roles in predicting patient outcomes and these observations are highly tumor-type specific 19-24 .
  • a cause-and-effect relationship has been established between mast cells and solid tumors: adoptively transferred mast cells, when primed by LPS or IgE, inhibit tumor growth in xenograft models of melanoma and breast cancer 25,26 , indicating an antitumor function of mast cells.
  • mast cells are advantageous as effector cells because (1) they release cytotoxic factors including granzyme B and TNFa that induce target cell deaths 27-29 , (2) they release chemokines and cytokines including CCL3, CCL4, and CXCL10 that recruit T cells and NK cells into the tumor and remodel TME 25,30,31 , (3) they are long-lived (up to years) in tissues, release mediators repeatedly 32,33 , and could confer a sustainable antitumor effect, and (4) do not express conventional inhibitory receptors including PD- 1 , CTLA-4, TIM-3, or LAG-3 at either resting or activated states 34 and are thus not susceptible to these immune suppressive signals in the TME.
  • Fusion proteins including, but not limited to CAR polypeptides incorporating a mast cell intracellular signaling domain together with one or more heterologous sequences are provided.
  • Recombinant constructs including nucleic acids expressing or encoding the polypeptides.
  • Viral genomes including the recombinant constructs, recombinant viruses including the constructs, and vaccine formulations formed thereof are also provided, as are cells, particularly mast cells, including the nucleic acids and/or the fusion polypeptides are also provided.
  • compositions and methods are especially applicable to development of chimeric antigen receptor engineered mast cell therapy (CAR-mast).
  • CAR-mast chimeric antigen receptor engineered mast cell therapy
  • Compositions typically include or encode a polypeptide including mast cell intracellular signaling domain gene product or variant thereof, referred to herein as “mast cell intracellular signaling domain polypeptides”, are provided.
  • the mast cell intracellular signaling domain polypeptide is between about 10 amino acids and about 500 amino acids, or any specific integer number of amino acids therebetween, including, but not limited to 20, 25, 30, 35, 40, 45, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, or 450 amino acids; preferably, at least a length that is sufficient to promote intracellular signal transduction in mast cells, preferably leading to enhanced mast cell activation when forming a fusion protein with CAR.
  • the mast cell intracellular signaling domain polypeptides, nucleic acids encoding the same, and delivery vehicles thereof, and cells including them can optionally include one or more additional heterologous proteins, polypeptides, or other amino acid sequences.
  • the presence of one or more mast cell intracellular signaling domain polypeptides within the cytoplasmic domain of a recombinant fusion peptide will impart an increased cell activation and/or signaling to/from the fusion peptide.
  • mast cell intracellular signaling domain polypeptides are described as part of a fusion protein.
  • the native receptors that activate mast cells include the high affinity IgE receptor (FcsRI), cytokine receptors (e.g., c- KIT), G-protein-coupled receptors, and toll-like receptors (Gilfillan, and Tkaczyk, Nat Rev Immunol 6:218-230 (2006)).
  • the mast intracellular signaling domain can be or include the intracellular domain of any of IgE receptor (FCERI), cytokine receptors (e.g., c- KIT), G-protein-coupled receptors, and tolllike receptors (e.g., TLR2, TLR4, TLR6, etc.), preferably ones that are expressed by mast cells.
  • FceRI is of particular interest because 1) the pathway by which FceRI triggers degranulation is well characterized and 2) FceRI contains the immunoreceptor tyrosine-based activation motif (IT AM). ITAM is important for the signal transduction in CAR T cells, and thus, the mast cell intracellular signaling domain can include a ITAM sequence.
  • An exemplary ITAM sequence from human FceRI is DGVYTGLSTRNQETYETLKHE (SEQ ID NO: 11).
  • the mast cell intracellular domain is the intracellular domain from mouse FceRlgamma: REKIQVRKAAIASRERADAVYTGLNTRSQETYETLKHEKPPQ (SEQ ID NO: 12), with the ITAM sequence in bold and italics.
  • the domains of a CAR for use in human cells include mast cell intracellular signaling domain from a human protein or fragment or variant thereof.
  • the corresponding segment of a human FceRI gamma intracellular domain protein is RLRLQVRKAAnSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 13), with the ITAM sequence in bold and italics.
  • the mast intracellular signaling domain is or includes a sequence from C-kit, TLR2, TLR4, or TLR6, e.g., mouse or human C-kit, TLR2, TLR4, or TLR6, preferably including or being the intracellular domain thereof or a functional fragment or variant thereof.
  • compositions typically are, or include, a mast intracellular signaling domain or a functional fragment or variant thereof, or a nucleic acid encoding the same.
  • Functional fragments and variants can be, for example, any number of amino acids sufficient to drive enhanced mast cell activation.
  • the data below supports the conclusions that CAR-mast enhances proinflammatory cytokine secretion and tumor cell killing by CAR-mast cells having a CAR-mast fusion protein.
  • the mast intracellular signaling domain is between about 20 amino acids and about 500 amino acids, or any specific integer number of amino acids therebetween.
  • Variants can have, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of the provided SEQ ID NOs, or a functional fragment thereof; or the corresponding sequence of a homologue such as an orthologue or paralogue of any of the foregoing sequences; or any combination thereof.
  • variants maintain the ability to promote intracellular signaling in a mast cell.
  • variants are identified as functional in a CAR fusion construct if they maintain or enhance cytotoxicity of a host mast cell in vitro or in vivo towards target cells, expression one or more inflammatory cytokines and/or chemokines such as those discussed in the experiments below, recruitment or attraction of T and/or NK cells e.g., into tumor tissues, or a combination thereof.
  • any of the polypeptide sequences including the amino acid sequences of any of the SEQ ID NOs provided anywhere herein can include one or more amino acid substitutions.
  • the amino acid substitutions do not reduce the ability of the polypeptide in fusion construct to promote cell activation and/or signaling, for example in the case of CAR-mast fusion proteins maintain or enhance cytotoxicity of a host mast cell in vitro or in vivo towards target cells, expression one or more inflammatory cytokines and/or chemokines such as those discussed in the experiments below, recruitment or attraction of T and/or NK cells e.g., into tumor tissues, or a combination thereof.
  • Amino acid substitutions within peptides are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions of amino acids within any of the provided SEQ ID NOs can include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, Leu).
  • Embodiments of this disclosure thus contemplate functional or biological equivalents of the provided polypeptides, e.g.
  • Fusion proteins including one or more heterologous polypeptide sequences fused to one or more mast cell intracellular signaling polypeptides are provided.
  • the term “mast cell intracellular signaling domain” is used in the context of fusion peptides that include one or more signaling domains, to refer to the component of the fusion peptide that includes a mast cell intracellular signaling domain.
  • the fusion proteins in addition to a mast cell intracellular signaling domain, include one or more extracellular polypeptide domains, and/or one or more transmembrane domains, and/or one or more additional (i.e., heterologous) intracellular domain.
  • Heterologous elements that can be associated with, linked, conjugated, or otherwise attached directly or indirectly to the mast cell intracellular signaling domain sequence(s), or nucleic acids expressing the mast cell intracellular signaling domain are disclosed.
  • Such molecules include, but are not limited to, protein domains, such as transduction domains, fusogenic peptides, targeting molecules, and sequences that enhance protein expression and/or isolation.
  • Suitable protein domains include ectodomains, transmembrane domains, cytoplasmic domains of proteins and macromolecular structures including combinations of ectodomains, transmembrane domains, and cytoplasmic domains.
  • the other protein domains are not proteins from which the mast cell intracellular signaling domain is derived.
  • the other protein domains have or have potential for one or more molecular functions or activities.
  • Such “functional” domains can be engineered to provide one or more functions or activities, as desired.
  • Exemplary functions include receptor or ligand binding, enzymic activity, and molecular transport, such as active transport of one or more molecules into or out of one or more cellular compartments.
  • the other protein domains within a mast cell intracellular signaling domain fusion protein bind to a specific substrate or molecule.
  • An exemplary molecule is an antigen or a cell-surface receptor.
  • mast cell intracellular signaling domain fusion peptides include one or more heterologous peptide domains, such as receptors at the surface of a cell, optionally including a transmembrane domain that anchors or connects the ectodomain to the cell surface and connects with the intracellular signaling domain.
  • Exemplary cell surface receptors coordinate the activity of cells upon interaction with other cells, such as immune cells, such as mast cells, T cells, etc.
  • the heterologous domain is a recombinant or engineered chimeric antigen receptor (CAR).
  • the heterologous domain includes a specific transmembrane domain (e.g., transmembrane domain of CD8 or CD28) for further enhancing signaling and receptor sensitivity in the case of CAR.
  • the heterologous domain is a co-signaling domain. Exemplary cosignaling domains have been previously described (Majzner, R. G.
  • the fusion peptides include multiple heterologous domains, such as a CAR domain (e.g., an antigen binding domain such as a single chain variable fragment targeting a cancer antigen, e.g., CD19, GD2, B7H3, or others mentioned or exemplified herein), a stalk and/or transmembrane domain (e.g,, of CD8a), or a combination thereof.
  • a CAR domain e.g., an antigen binding domain such as a single chain variable fragment targeting a cancer antigen, e.g., CD19, GD2, B7H3, or others mentioned or exemplified herein
  • a stalk and/or transmembrane domain e.g, of CD8a
  • Various domains of the fusion protein can optionally be linked with a linker.
  • exemplary linkers include, but are not limited to, a. Chimeric Antigen Receptors (CAR)
  • the fusion protein includes a Chimeric Antigen Receptor (CAR) fused with one or more mast cell intracellular signaling domains (“CAR-mast”).
  • CARs include an extracellular domain, a transmembrane domain and one or more intracellular/cytoplasmic domains.
  • the mast cell intracellular signaling domain forms part or all of the intracellular signaling domain of the CAR-mast.
  • a CAR-mast fusion protein includes the extracellular and transmembrane domains of an existing CAR fused to one or more mast intracellular signaling domains alone or in further combination with one or more additional intracellular domains.
  • CARs are engineered receptors that possess both antigen-binding and cellactivating functions. Immunotherapy using cells genetically engineered to express a CAR is rapidly emerging as a promising new treatment for hematological and non- hematological malignancies. Based on the location of the CAR in the membrane of the cell, the CAR can be divided into three main distinct domains, including an extracellular antigen-binding domain, followed by a space region, a transmembrane domain, and the intracellular signaling domain.
  • the antigen-binding domain typically contains VH and VL chains that are joined up by a linker to form the so-called “scFv.”
  • the segment interposing between the antigen-binding domain (e.g., scFv) and the transmembrane domain is a “spacer domain.”
  • the spacer domain can include the constant IgGl hinge-CH2-CH3 Fc domain. In some cases, the spacer domain and the transmembrane domain are derived from CD8.
  • the intracellular signaling domains mediating T cell activation can include a CD3 ⁇ co-receptor signaling domain derived from C-region of the TCR a and chains and one or more costimulatory domains.
  • a mast intracellular signaling domain typically supplements or replaces one or more, or all, of the intracellular domains of a traditional CAR.
  • the antigen-binding domain is derived from an antibody.
  • antibody herein refers to natural or synthetic polypeptides that bind a target antigen.
  • the term includes polyclonal and monoclonal antibodies, including intact antibodies and functional (e.g., antigen-binding) antibody fragments, including Fab fragments, F(ab')2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antigenbinding domain of a CAR can contain complementary determining regions (CDR) of an antibody, variable regions of an antibody, and/or antigen binding fragments thereof.
  • CDR complementary determining regions
  • the antigen-binding domain for a CD 19 CAR can be derived from a human monoclonal antibody to CD19, such as those described in U.S. Patent 7,109,304, which is specifically incorporated by reference herein in its entirety for use in accordance with the disclosed compositions and methods.
  • the antigen-binding domain can include an F(ab')2, Fab', Fab, Fv or scFv.
  • the CAR includes one or more spacer domain(s) (also referred to as hinge domain) that is located between the extracellular antigen-binding domain and the transmembrane domain.
  • a spacer domain is an amino acid segment that is generally found between two domains of a protein and may allow for positioning and flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such positioning, flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain can be used.
  • the spacer domain can be a spacer or hinge domain of a naturally occurring protein.
  • the hinge domain is derived from CD8a or CD28, such as, a portion of the hinge domain of CD8a or CD28, e.g., a fragment containing at least 5 (e.g., 5, 10, 15, 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a.
  • Hinge domains of antibodies such as an IgG, IgA, IgM, IgE, or IgD antibodies can also be used.
  • the hinge domain is the hinge domain that joins the constant CHI and CH2 domains of an antibody.
  • Non-naturally occurring peptides may also be used as spacer domains.
  • the spacer domain can be a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer of 3 or more, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.
  • the CAR includes a transmembrane domain that can be directly or indirectly fused to the antigen-binding domain.
  • the transmembrane domain may be derived either from a natural or a synthetic source.
  • the transmembrane domain of the CAR includes a transmembrane domain of an alpha, beta, or zeta chain of a T-cell receptor, CD8, CD4, CD28, CD137, CD80, CD86, CD152 (CTLA-4) or PD1, or a portion thereof.
  • Transmembrane domains can also contain at least a portion of a synthetic, non-naturally occurring protein segment.
  • the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet.
  • the protein segment is at least about 15 amino acids, e.g., at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids.
  • synthetic transmembrane domains are known in the art, for example in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000/032776.
  • the domains of a CAR for use in human cells includes a human protein or fragment or variant thereof.
  • the corresponding segment of a human CD28 stalk and transmembrane domain protein is 100% identical to SEQ ID NO:10, see, e.g., NCBI Reference Sequence: NP_006130.1.
  • the intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CAR.
  • effector function refers to a specialized function of a cell.
  • Effector function of a mast cell may be cytotoxicity of the mast cell in vitro or in vivo towards target cells, expression one or more inflammatory cytokines and/or chemokines such as those discussed in the experiments below, recruitment or attraction T and/or NK cells e.g., into tumor tissues, or a combination thereof.
  • the mast cell intracellular signaling domains are discussed in more detail above, and thus not repeated here.
  • an intracellular signaling domain includes the zeta chain of the T cell receptor or any of its homologs e.g., eta, delta, gamma, or epsilon), MB1 chain, B29, Fc RIII, Fc RI and combinations of signaling molecules such as CD3 ⁇ and CD28, 4- IBB, 0X40 and combination thereof, as well as other similar molecules and fragments.
  • Intracellular signaling portions of other members of the families of activating proteins can be used, such as FcyRIII and FceRI.
  • the CAR includes at least one co- stimulatory signaling domain.
  • co-stimulatory signaling domain refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function.
  • the co-stimulatory signaling domain can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.
  • the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from TLR2, TLR4, TLR6, IL3, FceRlb, c-kit, CD27, CD28, CD137, 0X40, CD30, CD40, CARs can be used in order to generate immuno-responsive cells, such as mast cells, specific for selected targets, such as malignant cells.
  • a co-stimulatory molecule selected from TLR2, TLR4, TLR6, IL3, FceRlb, c-kit, CD27, CD28, CD137, 0X40, CD30, CD40, CARs
  • First-generation CARs typically include a single-chain variable fragment of an antibody specific for an antigen, for example including a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3 ⁇ or FcRy (scFv-CD3 ⁇ or scFv- FcRy; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936, each of which is specifically incorporated by reference herein in its entirety).
  • Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD134), or 4-1BB (CD137) within the endodomain (for example scFv-CD28/OX40/4-lBB-CD3 ⁇ ; see U.S. Patent Nos.8, 911,993; 8,916,381 ; 8,975,071; 9,101,584; 9,102,760; 9,102,761, each of which is specifically incorporated by reference herein in its entirety).
  • costimulatory molecules such as CD28, 0X40 (CD134), or 4-1BB (CD137) within the endodomain (for example scFv-CD28/OX40/4-lBB-CD3 ⁇ ; see U.S. Patent Nos.8, 911,993; 8,916,381 ; 8,975,071; 9,101,584; 9,102,760; 9,102,761, each of which is specifically incorporated by reference herein in its entirety).
  • Third-generation CARs include a combination of costimulatory endodomains, such a CD3 -chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, 0X40, 4-1BB, or CD28 signaling domains (for example scFv-CD28-4-lBB-CD3 or scFv-CD28-OX40-CD3 ⁇ ; see U.S. Patent No.8,906,682; U.S. Patent No.8,399,645; U.S. Pat. No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000, each of which is specifically incorporated by reference herein in its entirety).
  • costimulatory endodomains such as CD3 -chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, 0X40, 4-1BB, or CD28 signaling domains (for example s
  • any of the first, second, or third generation CARs described above can be modified in accordance with the disclosed compositions and methods, e.g., to supplement or replace the intracellular signaling domain of such CARs with a mast intracellular signaling domain as provided herein, to form a CAR-mast.
  • the target specificity of the cell expressing a CAR is determined by the antigen recognized by the antibody/ectodomain.
  • the disclosed compositions and methods can be used to create constructs, and cells expressing the constructs, that target any antigen.
  • numerous antigens, and suitable ectodomains for targeting them are well known.
  • the majority of scFv-based CARs recognize target antigens expressed on the cell surface rather than internal antigens that are processed and presented by the cells’ MHC, however, CARs have the advantage over the classical TCR that they can recognize structures other than protein epitopes, including carbohydrates and glycolipids Doth, et al., Immunol Rev.
  • targets include antigens that are only expressed on cancer cells or their surrounding stroma (Chee ver, et al., Clin Cancer Res., 15:5323-5331 (2009)), such as the splice variant of EGFR (EGFRvIII), which is specific to glioma cells (Sampson, et al., Semin Immunol., 20(5):267-75 (2008)).
  • EGFRvIII the splice variant of EGFR
  • human antigens meet this requirement, and the majority of target antigens are expressed either at low levels on normal cells (e.g. GD2, CAIX, HER2) and/or in a lineage restricted fashion (e.g. CD19, CD20).
  • CD33 e.g., Myeloid
  • CD70 e.g., B-cell/T-cell
  • CD123 e.g., Myeloid
  • Kappa e.g., B-cell
  • Lewis Y e.g., Myeloid al., Clin Cancer Res., 12:6106-6115 (2006), Hwu, et al., Cancer Res., 55:3369-3373 (1995)
  • FAP e.g., cancer associated fibroblasts
  • FAR e.g., rhabdomyosarcoma
  • GD2 e.g., neuroblastoma, sarcoma, melanoma
  • GD3 e.g., melanoma, lung cancer
  • HMW-MAA e.g., melanoma
  • ILllRa e.g., osteosarcoma
  • the CAR targeting one or more antigens specific for cancer, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, an autoimmune disease, or combinations thereof One of skill in the art, based on general knowledge in the field and/or routine experimentation would be able to determine the appropriate antigen to be targeted by a CAR for a specific disease, disorder, or condition.
  • antigens specific for cancer that could be targeted by the CAR include, but are not limited to, 4- IBB, 5T4, adenocarcinoma antigen, alpha- fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), SISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSDFTLSINSVEPEDVG VYYCQNGHSFPLTFGAGTKLELKQTS (SEQ ID N0:9); or
  • HER2 single chain Fv KYLLPTAAAGLLLLAAQPAMAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSY WIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSS LKPSDSAVYFCARHDVGYCTDRTCAKWPEYFQHWGQGTLVTVSSGGGGSGGG GSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLL IYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFG GGTKLTVLGGSGS (SEQ ID NO:34), any of which can be utilized as the antigen binding domain in any of the disclosed CAR-mast constructs.
  • Other Protein domains any of which can be utilized as the antigen binding domain in any of the disclosed CAR-mast constructs.
  • any of the disclosed recombinant proteins can include one or more additional domains.
  • any of the disclosed proteins can include one or more linkers or spacers.
  • the term “linker” as used herein includes, without limitation, peptide linkers.
  • the peptide linker can be any size provided it does not interfere with the binding of the epitope by the variable regions.
  • the linker includes one or more glycine and/or serine amino acid residues.
  • the linker includes a glycine-glutamic acid di-amino acid sequence.
  • a linker can include 4-8 amino acids.
  • a linker includes the amino acid sequence GQSSRSS (SEQ ID NO:25).
  • the linker includes one, two or more copies the amino acid sequence GGGGS (SEQ ID NO:26). In another embodiment, a linker includes 15-20 amino acids, for example 18 amino acids.
  • Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:27), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO:28), (Gly 4 -Ser) 2 (SEQ ID NO:29) and (Gly 4 -Ser) 4 (SEQ ID NO:30), (Gly-Gly-Gly-Ser) 2 (SEQ ID NO: 31) and (Gly-Gly-Gly- Gly-Ser) 3 (SEQ ID NO:32).
  • the linkers can be used to link or connect two domains, regions, or sequences of a fusion protein.
  • Molecular biology techniques have developed so that therapeutic proteins can be genetically engineered to be expressed by microorganisms.
  • compositions disclosed herein include one or more of a signal peptide, a marker, and/or expression or solubility enhancing amino acid sequence.
  • An exemplary signal peptide is provided in the examples below, but can be substituted of another signal peptide as is known in the art.
  • Exemplary markers are include GFP, mCherry and HA, each of which is exemplified in the experiments below.
  • Exemplary expression or solubility enhancing amino acid sequences include maltose- binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and a small ubiquitin-related modifier (SUMO).
  • MBP maltose- binding protein
  • GST glutathione S-transferase
  • TRX thioredoxin
  • NUS A ubiquitin
  • Ub ubiquitin
  • SUMO small ubiquitin-related modifier
  • Nucleic acids and vectors encoding or expressing the disclosed polypeptides and fusion proteins are also described.
  • isolated nucleic acid sequences encoding mast intracellular signaling domain polypeptides and fusion peptides are disclosed.
  • the isolated nucleic acid sequences encode a CAR-mast.
  • nucleic acid encoding the disclosed SEQ ID NOs. alone or in any combination are expressly provided.
  • isolated nucleic acid refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome.
  • An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.
  • a virus e.g., a retrovirus, lentivirus, adenovirus, or herpes virus
  • an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid.
  • Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence a polypeptide or fusion peptide.
  • nucleic acids encoding the provided polypeptides e.g., the disclosed amino acid sequence SEQ ID NOs
  • the nucleic acids can be DNA, RNA, or nucleic acid analogs.
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid.
  • Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2’-deoxycytidine or 5-bromo- 2’ -deoxycytidine for deoxycytidine.
  • Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 2’-O-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained.
  • deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
  • nucleic acids encoding the provided polypeptides and fusion proteins are present within vectors.
  • the vectors encode or express a CAR-mast.
  • Vector encoding or expressing any one or more of the disclosed polypeptide sequence are also expressly provided.
  • Vectors including an isolated polynucleotide encoding a polypeptide or fusion protein for the expression of the polypeptide or fusion peptide within a host cell are described.
  • vector is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs).
  • a vector can be a DNA vector or an RNA vector. In some embodiments, a vector is a DNA plasmid.
  • One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.
  • the vector including nucleic acids encoding a polypeptide or fusion protein is an expression vector.
  • expression vector refers to any type of genetic construct including a nucleic acid coding for an RNA capable of being transcribed.
  • Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • vectors contain one or more regulatory sequences.
  • regulatory sequence refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. mRNA) into the host cell or organism.
  • this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability).
  • the vector is a non- viral vector.
  • non- viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc.
  • non-viral vectors include, but are not limited to, RNA replicon, mRNA replicon, modified mRNA replicon or selfamplifying mRNA, closed linear deoxyribonucleic acid, e.g., a linear covalently closed DNA, e.g., a linear covalently closed double stranded DNA molecule.
  • a non- viral vector is a DNA plasmid.
  • DNA plasmid which is used interchangeably with “DNA plasmid vector,” “plasmid DNA” or “plasmid DNA vector,” refers to a doublestranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell.
  • DNA plasmids used for expression of an encoded polynucleotide typically include an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene.
  • DNA plasmids examples include, but are not limited to, commercially available expression vectors for use in well-known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif.), which can be used for production and/or expression of protein in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and/or expression in Saccharomyces cerevisiae strains of yeast; MAXBAC®. complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and/or expression in insect cells; pcDNATM.
  • pSE420 Invitrogen, San Diego, Calif.
  • pYES2 Invitrogen, Thermo Fisher Scientific
  • MAXBAC® complete baculovirus expression system
  • Thermo Fisher Scientific complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and/or expression in insect cells
  • pcDNA3TM Life Technologies, Thermo Fisher Scientific
  • pVAX or pVAX-1 Life Technologies, Thermo Fisher Scientific
  • the backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and/or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and/or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).
  • a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells.
  • Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADV AX, NTC8454, etc.
  • an expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells.
  • pVAX-1 is a commonly used plasmid in DNA vaccines, and contains a strong human immediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH)-derived polyadenylation sequence (pA).
  • pVAX-1 further contains a pUC origin of replication and a kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.
  • the vector is a viral vector.
  • viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection.
  • viral vectors examples include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi-segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which include an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated and replaced by a nucleic acid encoding a heterologous polypeptide or fusion protein as described herein), arenavirus such as lymphocytic choriomeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain, etc.
  • the viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector.
  • a recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd).
  • an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc.
  • an adenovirus vector is a rhAd vector, e.g., rhAd51, rhAd52 or rhAd53.
  • a recombinant viral vector is prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide.
  • a polynucleotide encoding a provided polypeptide or fusion polypeptide is codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure.
  • the vectors e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), include any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the polypeptide or fusion protein encoded by the polynucleotide sequence 3. Regulatory Elements
  • the disclosed nucleic acids including RNAs and DNAs such as DNA vectors expressing or encoding the provided polypeptides and fusion proteins include one or more regulatory elements.
  • Regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc.
  • An isolated nucleic acid can be, and a vector can include one or more expression cassettes.
  • An “expression cassette” is part of a nucleic acid such as a vector that directs the cellular machinery to make RNA and protein.
  • An expression cassette typically includes three components: a promoter sequence, an open reading frame, and a 3 '-untranslated region (UTR) optionally including a polyadenylation signal.
  • An open reading frame is a reading frame that contains a coding sequence of a protein of from a start codon to a stop codon. Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding polypeptide or fusion protein.
  • operably linked is to be taken in its broadest reasonable context, and refers to a linkage of polynucleotide (or polypeptide, etc.) elements in a functional relationship.
  • a polynucleotide is “operably linked” when it is placed into a functional relationship with another polynucleotide.
  • a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application. a. Promotors
  • the disclosed nucleic acids can include a promoter sequence, preferably within an expression cassette, to control expression of a polypeptide or fusion polypeptide.
  • promoter is used in its conventional sense and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence.
  • a promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be a constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic.
  • a promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter can be a homologous promoter (/. ⁇ ?., derived from the same genetic source as the vector) or a heterologous promoter (/. ⁇ ?., derived from a different vector or genetic source).
  • the promoter can be endogenous to the plasmid (homologous) or derived from other sources (heterologous).
  • the promoter is located upstream of the polynucleotide encoding an a protein of interest within an expression cassette.
  • promoters examples include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.
  • a promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein.
  • a promoter can also be a tissue specific promoter, such as a kidney specific promoter, preferably a kidney epithelial cell promoter, which can be natural or synthetic.
  • tissue specific promoter such as a kidney specific promoter, preferably a kidney epithelial cell promoter, which can be natural or synthetic.
  • examples include, but are not limited to, the CDH 16 promoter, which is mostly kidney specific (it is also expressed in the thyroid) (Igarashi, et al., Am J Physiol.
  • the Pax-8 promoter which is also expressed primarily in the kidney as well as in the thyroid (Dehbi, et al., EMBO J., 15(16):4297-306 (1996)); the aquaporin 2 promoter, which drives expression specifically in principal cells of the renal collecting duct (which are the target of Tolvaptan) (Stricklett, et al., Exp Nephrol., 7(l):67-74 (1999)), and kidney tubule-specific promoters in association with gene delivery viral vectors (Watanabe, et al., PloS one, vol. 12,3 e0168638 (2017)).
  • the promoter is a strong eukaryotic promoter, such as cytomegalovirus immediate early (CMV-IE) promoter.
  • CMV-IE cytomegalovirus immediate early
  • the nucleic acids include additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcriptional-translational coupling.
  • additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcriptional-translational coupling.
  • sequences include polyadenylation signals and enhancer sequences.
  • a polyadenylation signal is typically located downstream of the coding sequence for a disclosed polypeptide or fusion protein within an expression cassette of the vector.
  • Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene.
  • An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding polypeptide or fusion protein, but downstream of a promoter sequence within an expression cassette of the vector.
  • the polyadenylation signal can be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human beta-globin polyadenylation signal.
  • a polyadenylation signal is a bovine growth hormone (bGH) poly adenylation signal or a SV40 poly adenylation signal.
  • an enhancer sequence can be a human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV.
  • a viral enhancer such as one from CMV, HA, RSV, or EBV.
  • WPRE Woodchuck HBV Post-transcriptional regulatory element
  • ApoAI intron/exon sequence derived from human apolipoprotein Al precursor
  • HTLV-1) long terminal repeat (LTR) untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit beta-globin intron, or any combination thereof.
  • an enhancer sequence is a composite sequence of three consecutive elements of the untranslated R-U5 domain of HTLV-1 LTR, rabbit betaglobin intron, and a splicing enhancer, which is referred to herein as “a triple enhancer sequence.”
  • a vector can include a polynucleotide sequence encoding a signal peptide sequence.
  • the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding polypeptide or fusion protein.
  • Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve expression the therapeutic polypeptide when expressed from the vector, but is cleaved off by signal peptidase, e.g., upon secretion from the cell.
  • a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as the Ig heavy chain gamma signal peptide SPIgG or the Ig heavy chain epsilon signal peptide SPIgE.
  • Ig immunoglobulin
  • a vector such as a DNA plasmid
  • Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding a polypeptide or fusion protein, or in the opposite (reverse) orientation.
  • An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColEl, pMB l, pUC, pSClOl, R6K, and 15 A, preferably pUC.
  • Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene.
  • the promoter sequence operably linked to an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest.
  • the antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage.
  • Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kan r ), ampicillin resistance gene (Amp r ), and tetracycline resistance gene (Tet r ), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.
  • Kan r kanamycin resistance gene
  • Amicillin resistance gene Amicillin resistance gene
  • Tet r tetracycline resistance gene
  • An expression vector can include a tag sequence, such as those discussed above.
  • polypeptides, nucleic acids, or vectors encoding the disclosed polypeptides or fusion proteins are present within a host cells.
  • the cells include nucleic acids or vectors or genes that encode or express a CAR-mast.
  • the term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.
  • transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art.
  • Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation.
  • Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection.
  • Host cells e.g., a prokaryotic cell or a eukaryotic cell
  • the cell is from an established cell line, or a primary cell.
  • primary cell refers to cells and cell cultures derived from a subject and allowed to grow in vitro for a limited number of passages, i.e., splitting, of the culture.
  • cells are obtained from a human subject. Therefore, human cells expressing and/or including disclosed polypeptides and fusion proteins are described.
  • the human cells include or express a CAR-mast.
  • the cells are autologous cells, i.e., cells obtained from a subject prior to introduction of the disclosed polypeptides or fusion proteins such as a CAR-mast, and/or nucleic acids, or vectors encoding the same, and re-introduction to the same subject following modification.
  • the cells are heterologous cells, i.e., cells obtained from a different subject than the intended recipient.
  • the cells are frozen prior to or after introduction of the disclosed polypeptides or fusion proteins such as a CAR-mast, and/or nucleic acids, or vectors encoding the same. Methods and compositions for freezing and thawing viable eukaryotic cells are known in the art.
  • the cells are autologous immune cells, such as mast cells or progenitor cells/stem cells.
  • cells are obtained from a healthy subject. In other forms, cells are obtained from a subject identified as having or at risk of having a disease or disorder, such as cancer and/or an auto-immune disease.
  • the introduction of the disclosed polypeptides or fusion proteins such as a CAR-mast to the cells occurs through genetic modification of the cells.
  • genetic modification of the cell includes introduction of nucleic acids, or vectors encoding the disclosed polypeptides or fusion proteins such as a CAR-mast within the cell.
  • genetic modification of the cell includes transduction with a transposon encoding a disclosed polypeptide or fusion protein such as a CAR-mast.
  • a CAR-mast fusion peptide is introduced into a cell in vitro by transduction of the cell with a nucleic acid encoding a transposon including the CAR-mast. Therefore, genetically modified (transgenic) cells including CAR-mast and/or other mast intracellular signaling domain-fusion protein(s) according to the described compositions are described.
  • the cells are human immune cells, such as mast cells. Therefore, human mast cells that include or express the disclosed polypeptides or fusion proteins such as a CAR-mast are described. In some forms, prior to expansion and genetic modification, cells are obtained from a diseased or healthy subject.
  • Mast cells can be obtained from a number of samples, including peripheral blood and cord blood, adipose tissue, skin, and/or can be induced from progenitor cells.
  • mast cells are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan.
  • Mast cells are derived from CD34+ hematopoietic progenitor cells that are defined as KIT (CD 117)+ and CD13+, but FcsRI- cells (Kirshenbaum et al. Blood. 1999;94:2333-2342).
  • Undifferentiated mast cell progenitors leave the bone marrow and undergo their terminal differentiation in tissues; consequently, very few mature mast cells are found circulating in the peripheral blood. Even in their resident tissues, mast cells are only found in limited numbers, thus, primary human mast cells are difficult to isolate.
  • An alternative means of obtaining reasonable populations of primary human mast cells has been by in vitro differentiation of CD34+ progenitors.
  • CD34+ peripheral blood- derived human mast cells have the appearance of mature human mast cells having a well condensed non-lobate nucleus and abundant granules in their cytosol. They express FcsRI, KIT, and various G protein-coupled receptors (GPCRs), and respond, through these receptors to promote degranulation and cytokine production or, in the case of KIT or specific GPCRs in conjunction with FcsRI, to synergistically enhance these responses (Gilfillan et al, Nat.Rev.Immunol. 2006;6:218-230, Kuehn et al., Immunol. Lett. 2007;113:59-69). A number of toll-like receptors (TLRs) are also expressed on mast cells and, when activated, have the capacity to enhance antigen-mediated cytokine production (Qiao et al., Blood. 2006;107:610-618).
  • TLRs toll-like receptors
  • Cord blood contains a higher concentration of progenitors than does peripheral blood, which makes it a convenient progenitor source for mast cell generation. Both mononuclear cells and purified CD34+ and CD 133+ progenitors have been used.
  • compositions including, but not limited to the provided polypeptides, fusion proteins, and/or nucleic acids encoding the same, can be delivered to target cells using a delivery vehicle.
  • the delivery vehicles can be, for example, polymeric particles, inorganic particles, silica particles, liposomes, micelles, multilamellar vesicles, etc.
  • Delivery vehicles may be microparticles or nanoparticles. Nanoparticles are often utilized for intertissue application, penetration of cells, and certain routes of administration. The nanoparticles may have any desired size for the intended use. The nanoparticles may have any diameter from 10 nm up to about 1,000 nm.
  • the nanoparticle can have a diameter from 10 nm to 900 nm, from 10 nm to 800 nm, from 10 nm to 700 nm, from 10 nm to 600 nm, from 10 nm to 500 nm, from 20 nm from 500 nm, from 30 nm to 500 nm, from 40 nm to 500 nm, from 50 nm to 500 nm, from 50 nm to 400 nm, from 50 nm to 350 nm, from 50 nm to 300 nm, or from 50 nm to 200 nm.
  • the nanoparticles can have a diameter less than 400 nm, less than 300 nm, or less than 200 nm. The range can be between 50 nm and 300 nm.
  • the delivery vehicles are nanoscale compositions, for example, 10 nm up to, but not including, about 1 micron.
  • the particles can be smaller, or larger (e.g., microparticles, etc.).
  • nanoparticle or nanocarrier compositions it will be appreciated that in some embodiments and for some uses the carrier can be somewhat larger than nanoparticles.
  • Such compositions can be referred to as microparticulate compositions.
  • a nanocarriers according to the present disclosure may be a microparticle. Microparticles can a diameter between, for example, 0.1 and 100 pm in size.
  • compositions containing nucleic acids encoding the provided polypeptides and fusions, or a genetically modified cell, or a population of genetically modified cells expressing the disclosed polypeptides and fusion proteins such as a CAR- mast are also provided.
  • the pharmaceutical compositions include one or more of a pharmaceutically acceptable buffer, carrier, diluent, or excipients.
  • the pharmaceutical compositions include a specific number or population of cells, for example, expanded by culturing and expanding an isolated genetically modified cell (e.g. , CAR-mast cell), e.g., a homogenous population. Therefore, in some embodiments, pharmaceutical compositions include a homogenous population of modified cells, e.g., mast cells, including and/or expressing a disclosed polypeptide or fusion protein, such as a CAR-mast. In other forms, the pharmaceutical compositions include populations of cells that contain variable or different genetically modified cells, e.g., a heterogeneous population. In some forms, the pharmaceutical compositions include cells that are bispecific or multi-specific. In some embodiments, the cells have been isolated from a diseased or healthy subject prior to genetic modification to express the polypeptide or fusion protein, such as a CAR-mast.
  • pharmaceutically acceptable carrier describes a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier is a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
  • Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • Ringer of administration can refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and/or injections.
  • the pharmaceutical compositions are preferably formulated for intravenous administration.
  • the disclosed pharmaceutical compositions are administered in a manner appropriate to a disease to be treated (or prevented).
  • the quantity and frequency of administration is typically determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.
  • the disclosed pharmaceutical compositions can be delivered in a therapeutically effective amount.
  • the precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • compositions including, but not limited to, polypeptides and fusions proteins such as CAR-mast and cells expressing the same, e.g., CAR-mast cells, are provided.
  • the methods enhance the efficacy of cell receptor- mediated functions are provided.
  • the methods provide enhanced anti-tumor activity through administration of CAR-mast cells including CAR- mast fusion peptides.
  • the disclosed CAR-mast constructs can be used to maintain or enhance cytotoxicity of a host mast cell towards target cells, maintain or enhance expression one or more inflammatory cytokines and/or chemokines such as those discussed in the experiments below, to recruit or attract T and/or NK cells e.g., into tumor tissues, or a combination thereof.
  • mast intracellular signaling domains can be combined with other engineering strategies for improving antigen sensitivity, including selecting specific transmembrane or cosignaling domains (Majzner, R. G. et al., Cancer Discov, doi: 10.1158/2159-8290.CD-19- 0945 (2020); Heitzeneder, S.
  • the methods include Adoptive Cell Therapy (ACT) employing cells, e.g., mast cells, expressing recombinant CAR-mast fusion proteins.
  • ACT Adoptive Cell Therapy
  • the CAR cells including CAR-mast fusion proteins can proinflammatory and anti-tumor activity.
  • the CAR cells including CAR-mast fusion proteins show enhanced cytotoxicity of a host mast cell towards target cells, increased expression one or more inflammatory cytokines and/or chemokines such as those discussed in the experiments below, recruitment or attraction T and/or NK cells, e.g., into tumor tissues, or a combination thereof.
  • An exemplary method involves treating a subject (e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including cells, e.g., genetically modified cells, including mast intracellular signaling domain fusion polypeptides such as CAR-mast fusion proteins.
  • the methods administer the modified cells, e.g., CAR- mast cells, to a subject (e.g., a human) having a disease, disorder, or condition in an amount effective to treat the disease, disorder, or condition.
  • the methods treat a disease or disorder associated with an elevated expression or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition including cells modified to express recombinant CAR-mast fusion proteins.
  • the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition including cells modified to contain a CAR-mast that targets the antigen.
  • Methods of using mast intracellular fusion proteins, such as a CAR-mast, to treat a disease or disorder are provided.
  • the methods enhance ACT, for example, by providing CAR-mast-bearing cells with enhanced therapeutic efficacy in vivo.
  • the CAR- mast cells have prolonged survival/serum residency time in vivo relative to CAR cells lacking the CAR-mast fusion protein or CAR lacking a mast intracellular signaling domain.
  • Methods of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of a pharmaceutical composition including live, viable cells engineered to express a CAR-mast and/or another mast intracellular signaling domain fusion protein are provided.
  • the methods when the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen, include administering to the subject an effective amount of a cell, e.g., a mast cell, modified to express a CAR-mast that targets the antigen.
  • a cell e.g., a mast cell
  • the methods treat a subject having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition having a genetically modified cell, where the cell is modified by introducing to the cell:
  • a vector optionally including a transposon encoding a CAR-mast and/or other mast intracellular signaling domain fusion protein
  • the cell can have been isolated from the subject having the disease, disorder, or condition, or from a healthy donor, prior to genetic modification.
  • CAR-mast-expressing cells can be created in vivo in a subject in need thereof by introducing an effective amount of nucleic acids (e.g., RNA, viral vectors, etc.) encoding the CAR-mast into the subject.
  • nucleic acids e.g., RNA, viral vectors, etc.
  • the constructs are targeted to the tumor microenvironment using, e.g., targeted delivery vehicles. See, e.g., Xin, et al., Front. Oncol., 10 February 2022, Sec. Cancer Immunity and Immunotherapy, Volume 12 - 2022 I doi.org/10.3389/fonc.2022.809754.
  • the subject to be treated can have a disease, disorder, or condition such as but not limited to, cancer, an immune system disorder such autoimmune disease, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, or combinations thereof.
  • a disease, disorder, or condition such as but not limited to, cancer, an immune system disorder such autoimmune disease, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, or combinations thereof.
  • the disease, disorder, or condition can be associated with an elevated expression or specific expression of an antigen.
  • the methods treat or prevent cancer.
  • the methods treat or prevent cancer or other proliferative disease or disorder in a subject identified as having, or at risk of having cancer or other proliferative disease or disorder.
  • Cancer is a disease of genetic instability, allowing a cancer cell to acquire the hallmarks proposed by Hanahan and Weinberg, including (i) self-sufficiency in growth signals; (ii) insensitivity to anti-growth signals; (iii) evading apoptosis; (iv) sustained angiogenesis; (v) tissue invasion and metastasis; (vi) limitless replicative potential; (vii) reprogramming of energy metabolism; and (viii) evading immune destruction (Cell., 144:646-674, (2011)).
  • Tumors which can be treated in accordance with the disclosed methods, are classified according to the embryonic origin of the tissue from which the tumor is derived.
  • Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands.
  • Sarcomas which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage.
  • the leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.
  • compositions and methods of treatment thereof are generally suited for treatment of carcinomas, sarcomas, lymphomas and leukemias.
  • the described compositions and methods are useful for treating, or alleviating subjects having benign or malignant tumors by delaying or inhibiting the growth/proliferation or viability of tumor cells in a subject, reducing the number, growth, or size of tumors, inhibiting, or reducing metastasis of the tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth.
  • the types of cancer that can be treated with the provided compositions and methods include, but are not limited to, cancers such as vascular cancer such as multiple myeloma, adenocarcinomas, and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, and uterine.
  • cancers such as vascular cancer such as multiple myeloma, adenocarcinomas, and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, and uterine.
  • the compositions are used to treat multiple cancer types concurrently.
  • the compositions can also be used to treat metastases or tumors at multiple locations.
  • tumor cells include, but are not limited to, tumor cells of cancers, including leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin’s disease, non-Hodgkin’s disease; multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedul
  • the methods administer modified cells including CAR- mast and/or other mast intracellular signaling domain-fusion protein(s) to treat or prevent one or more immune system disorders, including autoimmune diseases.
  • Autoimmune diseases include over 100 types of diseases, with varied etiology and prognoses based on factors such as the affected region, the age of onset, response to the therapeutic agents and clinical manifestation may vary among different people (Muhammad, et al., Chimeric Antigen Receptor Based Therapy as a Potential Approach in Autoimmune Diseases: How Close Are We to the Treatment, Frontiers in Immunology, 11 (2020)).
  • autoimmunity is classified into two general categories, including organ-specific and systemic autoimmune.
  • the former involves a specific area of the body such as type I diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA), inflammatory bowel diseases (IBDs), and myasthenia gravis (MG), while the latter affects multiple regions of the body, causing systemic lupus erythematosus (SLE) and Sjogren’s syndrome (SS).
  • T1D type I diabetes
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • IBDs inflammatory bowel diseases
  • MG myasthenia gravis
  • SLE systemic lupus erythematosus
  • SS Sjogren’s syndrome
  • the methods reduce or prevent one or more physiological processes associated with the development or progression of autoimmune disease in a subject.
  • the methods reduce or prevent one or more of epitope spreading, for example, where infections alter the primary epitope into the secondary epitope or form several neoepitopes on antigen-presenting cells; bystander activation or pre-primed autoreactive T cell activation in a T cell receptor (TCR)- independent manner; persistent virus infection, or the constant presence of viral antigens that prompt immune responses; or immunological cross-reactivity between a host and pathogen, for example, due to shared immunologic epitopes or sequence similarities.
  • TCR T cell receptor
  • Non-limiting examples of immune system disorders that can be treated or prevented by the methods include 22ql l.2 deletion syndrome, Achondroplasia and severe combined immunodeficiency, Adenosine Deaminase 2 deficiency, Adenosine deaminase deficiency, Adult-onset immunodeficiency with anti-interferon-gamma autoantibodies, Agammaglobulinemia, non-Bruton type, Aicardi-Goutieres syndrome, Aicardi-Goutieres syndrome type 5, Allergic bronchopulmonary aspergillosis, Alopecia, Alopecia totalis, Alopecia universalis, Amyloidosis AA, Amyloidosis familial visceral, Ataxia telangiectasia, Autoimmune lymphoproliferative syndrome, Autoimmune lymphoproliferative syndrome due to CTLA4 haploinsuffiency, Autoimmune polyglandular syndrome type 1 , Autosomal dominant hyper IgE syndrome, Auto
  • compositions and methods can also be used to treat autoimmune diseases or disorders.
  • autoimmune diseases or disorders which are not mutually exclusive with the immune system disorders described above, include Achalasia, Addison’s disease, Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticarial, Axonal & neuronal neuropathy (AMAN), Bald disease, Behcet’s disease, Benign mucosal pe
  • the methods administer modified cells including CAR-mast and/or other mast intracellular signaling domain-fusion protein(s) to treat one or more additional disease or disorder in a subject in need thereof.
  • the methods treat one or more genetic disease or disorders in a subject, such as a hereditary genetic disease or disorder, or a somatic genetic disease or disorder in a subject.
  • any of the methods can include treating a subject having an underlying disease or disorder.
  • the methods treat a disease or disorder, such as a cancer or auto-immune disease in a patient having another disease or disorder, such as diabetes, a bacterial infection (e.g., Tuberculosis), viral infection (e.g., Hepatitis, HIV, HPV infection, etc.), or a drug-associated disease or disorder.
  • the methods treat an immunocompromised subject.
  • the methods treat a subject having a disease of the kidney, liver, heart, lung, brain, bladder, reproductive system, bowel/intestines, stomach, bones, or skin.
  • the methods administer modified cells including CAR-mast and/or other mast intracellular signaling domain-fusion protein(s) in an effective amount.
  • the effective amount or therapeutically effective amount of a pharmaceutical compositions including modified cells, such as therapeutic mast cells can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, such as a cancer or autoimmune disease, or to otherwise provide a desired pharmacologic and/or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying a disease or disorder, such as cancer or autoimmune disease.
  • the amount administered can be expressed as the amount effective to achieve a desired anti-cancer effect in the recipient.
  • the amount of the pharmaceutical compositions including modified cells, such as therapeutic cells, e.g., mast cells is effective to inhibit the viability or proliferation of cancer cells in the recipient.
  • the amount of the pharmaceutical composition including modified cells, such as therapeutic mast cells is effective to reduce the tumor burden in the recipient, or reduce the total number of cancer cells, and combinations thereof.
  • the amount of the pharmaceutical compositions including modified cells, such as therapeutic mast cells is effective to reduce one or more symptoms or signs of cancer in a cancer patient, or signs of an autoimmune disease in a patient having an autoimmune disease or disorder.
  • Signs of cancer can include cancer markers, such as PSMA levels in the blood of a patient.
  • the effective amount of the pharmaceutical compositions including modified cells, such as therapeutic mast cells, that is required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, and its mode of administration. Thus, it is not possible to specify an exact amount for every pharmaceutical composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the pharmaceutical compositions including therapeutic mast cells can be determined empirically, and making such determinations is within the skill in the art. In some forms, the dosage ranges for the administration of the compositions including therapeutic mast cells are those large enough to effect reduction in cancer cell proliferation or viability, or to reduce tumor burden for example.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, and sex of the patient, route of administration, whether other drugs are included in the regimen, and the type, stage, and location of the disease to be treated.
  • the dosage can be adjusted by the individual physician in the event of any counter-indications.
  • the effective dosage of the composition including therapeutic mast cells used for treatment can increase or decrease over the course of a particular treatment. Changes in dosage can result and become apparent from the results of diagnostic assays.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject or patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual pharmaceutical compositions and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models.
  • a pharmaceutical composition containing CAR- mast cells described herein can be administered at a dosage of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 7 cells/kg body weight, including all integer values within those ranges.
  • patients can be treated by infusing a disclosed pharmaceutical composition containing CAR-mast construct expressing cells (e.g., mast cells) in the range of about 10 4 to 10 12 or more cells per square meter of body surface (cells/m).
  • the infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved.
  • CAR-mast cell compositions can also be administered once or multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. I. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the unit dosage is in a unit dosage form for intravenous injection.
  • the unit dosage is in a unit dosage form for oral administration.
  • the unit dosage is in a unit dosage form for inhalation.
  • the unit dosage is in a unit dosage form for intra-tumoral injection.
  • Treatment can be continued for an amount of time sufficient to achieve one or more desired therapeutic goals, for example, a reduction of the amount of cancer cells relative to the start of treatment, or complete absence of cancer cells in the recipient. Treatment can be continued for a desired period of time, and the progression of treatment can be monitored using any means known for monitoring the progression of anti-cancer treatment in a patient.
  • administration is carried out every day of treatment, or every week, or every fraction of a week.
  • treatment regimens are carried out over the course of up to two, three, four or five days, weeks, or months, or for up to 6 months, or for more than 6 months, for example, up to one year, two years, three years, or up to five years.
  • the efficacy of administration of a particular dose of the pharmaceutical compositions including modified cells, such as therapeutic mast cells, according to the methods described herein can be determined by evaluating the aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of cancer or other diseases and/or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and/or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g.
  • a tumor has partially or fully regressed), (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.
  • efficacy is assessed as a measure of the reduction in tumor volume and/or tumor mass at a specific time point (e.g., 1-5 days, weeks, or months) following treatment.
  • the methods administer modified cells including CAR- mast and/or other mast intracellular signaling domain-fusion protein(s) in combination with a pharmaceutically acceptable carrier.
  • the compositions described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the therapeutics described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans.
  • these include solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • Other therapeutics can be administered according to standard procedures used by those skilled in the art.
  • the pharmaceutical compositions including modified cells, such as therapeutic mast cells, described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the therapeutic(s) of choice.
  • compositions containing one or more modified cells can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • a pharmaceutical composition including modified cells, such as therapeutic mast cells can be administered as an intravenous infusion, or directly injected into a specific site, for example, into or surrounding a tumor.
  • a pharmaceutical composition can be administered to a subject as an ophthalmic solution and/or ointment to the surface of the eye, vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes.
  • the compositions are administered directly into a tumor or tissue, e.g., stereotactically.
  • Parenteral administration if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. Suitable parenteral administration routes include intravascular administration (e.g.
  • intravenous bolus injection intravenous infusion
  • intra-arterial bolus injection intra-arterial infusion and catheter instillation into the vasculature
  • peri- and intra-tissue injection e.g., intraocular injection, intra-retinal injection, or sub-retinal injection
  • subcutaneous injection or deposition including subcutaneous infusion such as by osmotic pumps
  • direct application by a catheter or other placement device e.g., an implant including a porous, non-porous, or gelatinous material.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents, and other suitable additives.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • compositions containing one or more genetically modified cells can be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic.
  • the methods administer modified cells including CAR- mast and/or other mast intracellular signaling domain-fusion protein(s) in combination with other therapeutic agents or treatment modalities.
  • modified cells such as therapeutic mast cells (e.g. , containing a population of CAR-mast cells)
  • therapeutic mast cells e.g. , containing a population of CAR-mast cells
  • other therapeutic agents or treatment modalities for example, chemotherapy or stem-cell transplantation.
  • “combination” or “combined” refer to either concomitant, simultaneous, or sequential administration of the therapeutics.
  • the pharmaceutical compositions and other therapeutic agents are administered separately through the same route of administration. In other forms, the pharmaceutical compositions and other therapeutic agents are administered separately through different routes of administration.
  • the combinations can be administered either concomitantly (e.g. , as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject; one agent is given orally while the other agent is given by infusion or injection, etc.,), or sequentially (e.g., one agent is given first followed by the second).
  • preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder, or condition.
  • the therapeutic agent is one or more other targeted therapies (e.g. , a targeted cancer therapy) and/or immune-checkpoint blockage agents e.g. , anti-CTLA-4, anti-PDl, and/or anti-PDLl agents such as antibodies).
  • the CAR-mast cells can also be used in combination with other forms of adaptive cell therapy including, but not limited to, CAR T cell therapy, CAR NK cell therapy, CAR macrophage therapy, e.g., against the same of a different antigen.
  • compositions and methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.
  • therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.
  • the disclosed pharmaceutical compositions and/or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of remission or less active disease.
  • the pharmaceutical compositions can be administered before the additional treatment, concurrently with the treatment, posttreatment, or during remission of the disease or disorder.
  • the disclosed pharmaceutical compositions, and the additional therapeutic agents e.g., second or third agent
  • the disclosed pharmaceutical compositions, and the additional therapeutic agents can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy.
  • the administered amount or dosage of the disclosed pharmaceutical composition, the additional therapeutic agent (e.g., second or third agent), or all is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy (e.g., required to achieve the same therapeutic effect).
  • the methods administer one or more additional anti-cancer agents to a subject.
  • targeted therapies are therapeutic agents that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer.
  • molecules molecules that are involved in the growth, progression, and spread of cancer.
  • Many different targeted therapies have been approved for use in cancer treatment. These therapies include hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, and toxin delivery molecules.
  • Numerous antineoplastic drugs can be used in combination with the disclosed pharmaceutical compositions.
  • the additional therapeutic agent is a chemotherapeutic or antineoplastic drug. The majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other anti-tumor agents.
  • the methods also include administering one or more conventional therapies for autoimmune diseases to the subject.
  • Exemplary therapies for autoimmune diseases include immunosuppressive agents, such as steroids or cytostatic drugs, analgesics, non-steroidal anti-inflammatory drugs, glucocorticoids, immunosuppressive and immunomodulatory agents, such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine.
  • the methods administer one or more disease-modifying antirheumatic drugs (DMARDs).
  • DMARDs disease-modifying antirheumatic drugs
  • the methods administer one or more biologic agents for localized treatment (i.e., agents that do not affect the entire immune system), such as TNF-a inhibitors, belimumab and rituximab depleting B cells, T-cell co-stimulation blocker, antiinterleukin 6 (IL-6), anti-IL-1, and protein kinase inhibitors.
  • the methods also administer one or more monoclonal antibodies (mAbs), such as anti-TNFa, anti-CD19, anti-CD20, anti-CD22, and anti-IL6R, or other mAbs that target multiple B cell subtypes, and other aberrant cells in autoimmune diseases.
  • mAbs monoclonal antibodies
  • kits with one or more compositions for administration to a subject may include a pre-measured dosage of the composition in a sterile needle, ampule, tube, container, or other suitable vessel.
  • the kits may include instructions for dosages and dosing regimens.
  • kits containing CAR-mast and/or other mast intracellular signaling domain-fusion protein(s) within a vector e.g., a viral vector
  • the kit includes a plurality of vectors, where each vector independently contains CAR-mast and/or other mast intracellular signaling domain-fusion protein(s) for insertion into a host cell genome, such as a CAR expression cassette.
  • the kit contains a population of cells (e.g., mast cells) optionally collectively containing the CAR-mast and/or other mast intracellular signaling domain-fusion protein(s).
  • the instructional material can include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the kit.
  • the instructional material may provide instructions for methods using the kit components, such as performing transfections, transductions, infections, and conducting screens.
  • kits include a transposon that includes a promoter and/or polyadenylation signal operationally linked to a reporter gene and/or a CAR; in some forms, the kit includes a transposon including a CAR that is specific for an antigen selected from a cancer antigen, an inflammatory disease antigen, a neuronal disorder antigen, HIV/AIDS, a diabetes antigen, a cardiovascular disease antigen, an infectious disease antigen (including a viral antigen, a protozoan antigen, a bacterial antigen, and an allergen), an autoimmune disease antigen and an autoimmune disease antigen, or combinations thereof;
  • kits include a nucleic acid and/or a vector expressing or encoding the CAR-mast and/or other mast intracellular signaling domainfusion protein(s) and/or cells.
  • exemplary cells include a mast cell or mast progenitor cell, hematopoietic stem cell (HSC), etc.
  • amino acid sequence of (a) includes the intracellular domain of IgE receptor (FcsRI), a cytokine receptor (c- KIT), a G-protein-coupled receptor, or a toll-like receptor expressed in mast cells, or functional fragment or variant thereof.
  • FcsRI IgE receptor
  • c- KIT cytokine receptor
  • G-protein-coupled receptor G-protein-coupled receptor
  • the ITAM sequence includes DGVYTGLSTRNQETYETLKHE (SEQ ID NO: 11) or a functional variant thereof or a variant thereof having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 11.
  • amino acid sequence of (a) includes the amino acid sequence of any one of SEQ ID NOS: 11-21, or a functional fragment thereof, or a variant thereof having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOS: 11-21.
  • the CAR is specific for an antigen selected from the group consisting of a cancer antigen, an inflammatory disease antigen, a neuronal disorder antigen, HIV/AIDS, a diabetes antigen, a cardiovascular disease antigen, an infectious disease antigen (including a viral antigen, a protozoan antigen, a bacterial antigen, and an allergen), an autoimmune disease antigen and an autoimmune disease antigen, or combinations thereof.
  • an antigen selected from the group consisting of a cancer antigen, an inflammatory disease antigen, a neuronal disorder antigen, HIV/AIDS, a diabetes antigen, a cardiovascular disease antigen, an infectious disease antigen (including a viral antigen, a protozoan antigen, a bacterial antigen, and an allergen), an autoimmune disease antigen and an autoimmune disease antigen, or combinations thereof.
  • the CAR targets one or more antigens selected from the group consisting of B7H3, HER2, CD 19, GD2, AFP, AKAP 4, ALK, Androgen receptor, B7H3, BCMA, Bcr Abl, BORIS, Carbonic, CD123, CD138, CD174, CD20, CD22, CD30, CD33, CD38, CD80, CD86, CEA, CEACAM5, CEACAM6, Cyclin, CYP1B1, EBV, EGFR, EGFR806, EGFRvIII, EpCAM, EphA2, ERG, ETV6 AML, FAP, Fos related antigenl , Fucosyl, fusion, GD3, GloboH, GM3, gp 100, GPC3, HER 2/neu, HMWMAA, HPV E6/E7, hTERT, Idiotype, IL12, IL13RA2, IM 19, IX, LCK, Legumain, IgK, LMP2, MAD
  • the antigen is a cancer antigen selected from the group consisting of B7H3, HER2, CD19, GD2, 41BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C MET, CCR4, CD152, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI
  • a nucleic acid including a nucleic acid encoding the polypeptide of any one of paragraphs 1-13.
  • nucleic acid of paragraph 14 wherein the nucleic acid is RNA or DNA.
  • nucleic acid of paragraph 14 or 15 wherein the nucleic acid is mRNA.
  • nucleic acid includes an expression control sequence(s).
  • nucleic acid of any one of paragraphs 14-17 wherein the nucleic acid is, or is encoded by a vector or a transposon.
  • the viral vector is selected from the group consisting of a lentiviral vector, an Adeno-associated virus (AAV) vector, or an adenovirus vector, or a Herpes Simplex virus (HSV) vector, or a vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV), or a chimeric vector including a combination of any two or more of an Adeno-associated virus (AAV) vector, Herpes Simplex virus (HSV) vector, vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV).
  • AAV Adeno-associated virus
  • HSV Herpes Simplex virus
  • VSV vesicular stomatitis
  • nucleic acid of paragraph 20 wherein the vector is a nucleic acid expression vector selected from the group consisting of a plasmid, a cosmid, and a replicon.
  • HSC hematopoietic stem cell
  • a pharmaceutical composition including the population of cells of paragraph 29 and a pharmaceutically acceptable buffer, carrier, diluent, or excipient.
  • a method of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of the pharmaceutical composition of paragraph 30.
  • a method of treating a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen including administering to the subject an effective amount of a cell of any one of paragraphs 25-28, wherein the CAR targets the antigen.
  • the mast cell is isolated as a bone marrow mast cell, a spleen mast cell, or as a mast progenitor cell or hematopoietic stem cell and differentiated ex vivo into a mast cell.
  • Mature mast cells were generated according to standard protocols. Spleen and Bone marrow from C57BL/6 mice were dissected and cultured in vitro in the presence of mouse SCF and IL-3 (Figure 1A). Matured mast cells were obtained over 6-8 weeks, as indicated by the presence of intracellular granules (Figure IB) and the expression of mast cell marker c-kit and FceRI ( Figure 1C). These mast cells were further transduced with lentivirus encoding CARs as described below.
  • a mast cell CAR was constructed containing the immunoreceptor tyrosine-based activation motif (IT AM-) containing gamma chain of FceRI, the stalk and transmembrane domain from CD8a, and the single-chain variable fragment (FMC63) targeting CD19, a model B cell cancer-associated antigen for testing CAR function (Figure 2A).
  • IT AM- immunoreceptor tyrosine-based activation motif
  • FMC63 single-chain variable fragment
  • Figure 2A A monomer GFP is added at the C terminus of the structure for CAR labeling in later experiments.
  • a lentivirus encoding this CD 19 CAR was also generated.
  • the sequences for primary elements of the CD 19 CAR are provided below: a.
  • SP Signal Peptide
  • MALPVTALLLPLALLLHAARP (SEQ ID NO:1)
  • Human CD8a Stalk DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQ
  • TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:3)
  • Human CD8a transmembrane domain IYIWAPLAGTCGVLLLSLVITLYCR (SEQ ID NO:4)
  • e. Mouse FceRI gamma intracellular domain RLKIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQGSGS (SEQ ID NO:5)
  • mGFP
  • Mast cells are activated by receptors different than those for T cells.
  • a goal was to identify signaling domains that can be used for building a CAR to activate mast cells.
  • the native receptors that activate mast cells include the high affinity IgE receptor (FceRI), cytokine receptors (c- KIT), G-protein-coupled receptors, and toll-like receptors (Gilfillan, and Tkaczyk, Nat Rev Immunol 6:218-230 (2006)).
  • FcsRI is of particular interest because 1) the pathway by which FceRI triggers degranulation is well characterized and 2) FceRI contains the immunoreceptor tyrosine-based activation motif (IT AM). IT AM is important for the signal transduction in CAR-T cells.
  • a mast cell CAR was constructed which included the ITAM- containing gamma chain of FceRI, the stalk and transmembrane domain from CD8a, and the singlechain variable fragment (FMC63) targeting CD19, a model antigen for testing CAR function (Figure 2A).
  • a lentiviral construct encoding this CD19 CAR was generated. Lentivirus was produced from this construct and was found to infect mast cells with a high infection efficiency ( Figure 2B).
  • These CAR-mast cells were robustly activated by a mouse melanoma cell line B16 that ectopically expressed CD19, as indicated by the secretion of TNFa detected by ELISA.
  • Mature mast cells were generated as described in Example 1 and illustrated in Figures 1A-1C.
  • a CAR targeting GD2 (disialoganglioside) was generated ( Figure 3A).
  • This GD2 CAR was introduced into mast cells by lentiviral transduction ( Figure 3B).
  • These CAR-mast cells were co-cultured with GD2+B16 cells.
  • CAR-mast cells were co-cultured with GD2+ or GD2- B 16 cells, using plain mast cells as a control.
  • MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) b. HA tag:
  • IYIWAPLAGTCGVLLLSLVITLYCR (SEQ ID NO:4) f. Mouse FcsRl gamma intracellular domain: RLKIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQGSGS (SEQ ID NOG) g- mGFP
  • GD2 is a commonly overexpressed antigen in multiple solid tumors including melanoma, neuroblastoma, and small cell lung cancer (Suzuki & Cheung, Expert Opin Ther Targets 19:349-362 (2015); Nazha et al., Front Oncol., 10:1000 (2020)).
  • a CAR targeting GD2 (disialoganglioside) was generated and introduced into mast cells by lentiviral transduction ( Figures 3A and 3B). These CAR-mast cells were co-cultured with GD2+B16 cells.
  • CAR-mast cells were cocultured with GD2+ or GD2- B16 cells, using plain mast cells as a control. Luciferase assay revealed that CAR-mast cells showed much higher cytotoxicity towards GD2+B16 cells as compared to GD2-B16 cells ( Figure 3D). Furthermore, the cytokine and chemokine assay revealed effectors that were released by CAR-mast cells in responding to GD2 antigen. In addition to those secreted by CD 19 CAR-mast cells ( Figure 3E), GD2 CAR-mast cells also secreted CXCL1, CCL5/17/20, and CXCL5 ( Figure 3E).
  • CAR mast cells (CAR+) killing was also tested over several rounds. Killing of pre-seeded MC38-GD2-mCherry-Luciferase cells (GD2+) at 0.0 IM after 24 hr coculture at 37 °C at the E:T of 0.008:1, 0.04: 1, 0.2:1, 1:1, 5:1 respectively was assayed ( Figure 3F). At the end of each round the total supernatant is transferred to a new well preseeded with 0.0 IM fresh MC38-GD2-mCherry-Luciferase cells.
  • Killing rate was calculated by (mean luciferase signal in control - luciferase signal after coculture) / mean luciferase signal in control * 100%, where the control is 0.01M fresh MC38-GD2- mCherry-Luciferase cells treated with medium only.
  • Example 3 Generation and Characterization of Mast Cell CARs Targeting GD2 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSL
  • B7H3 (CD276 is another common antigen associated with a variety of solid tumors (Liu, S. et al. Front Oncol 11:654684, (2021)).
  • a CAR targeting B7H3(CD276) was constructed and characterized ( Figure 4A).
  • CAR-mast cells were activated in a B7H3 -depen dent manner, as reflected by the surface exposure of CD 107a and the secretion of TNFa ( Figure 4C). Consistently, CAR-mast cells displayed a higher cytotoxicity towards B7H3+MC38 cells, as compared to plain MC38 cells ( Figure 4D).
  • CAR-mast cells specifically secreted CCL2/3/4, TNFa, CCL17/20, and CXCL13 ( Figure 4E).
  • the common chemokines secreted by CD 19, GD2, or B7H3 CAR-mast cells include CCL2/3/4, which recruit monocytes, T cells and NK cells, and TNFa, which promotes cell death.
  • CAR-Mast cells were also investigated for and compared to allergic responses (e.g., IgE), B7-H3 CAR-mast cells (B7H3 CAR) or GD2 CAR-mast cells (GD2 CAR) were activated by either coculturing with MC38-B7H3-mCherry-Luciferase cells (B7H3- MC38) or MC38-GD2-mCherry-Luciferase cells (GD2-MC38) for 20 hr coculture at 37 °C at the E:T of 1:1 respectively, or by IgE where the CAR-mast cells are sensitized with lug/mL anti-DNP IgE for 1 hr then cocultured with 20ng/mL DNP for 20 hr at 37 °C ; 100/rL supernatant was taken for cytokine profiling using Multiplexed Mouse Proinflammatory Chemokine Panel (Biolegend) or Mouse CRS Panel (Biolegend) ( Figure 6A
  • MC38-B7H3-mCherry-Luciferase cells (B7-H3+) were treated with either B7-H3 CAR mast cells (CAR), wildtype mast cells (WT) or IgE-activated wildtype mast cells (WT+IgE) for 21 hr at 37 °C at the E:T of 1 : 1 , 3: 1 , 6:1 respectively with cancer cells total number at 0.02M.
  • the mast cells were sensitized with lug/mL anti-DNP IgE for 1 hr before cocultured with 20ng/mL DNP and the cancer cells.
  • Cancer cell killing rate is calculated by (mean luciferase signal in control - luciferase signal after coculture) / mean luciferase signal in control * 100% ( Figure 6B), where the control is cancer cells treated with medium only. Results demonstrated a necessity of CAR-mediated activation for mast cells to perform direct cancer killing.
  • Figure 6C is a diagram of mouse CAR-mast cell anaphylactic responses evaluation in mouse MC38-B7H3 tumor model. Timing of mast cell injections are indicated by the arrows.
  • MC38-B7H3ml are MC38-B7H3-mCherry-Luciferase cells.
  • mice were humanely killed by decapitation, and blood samples were obtained after a cardiac puncture to measure the plasma histamine concentration with a histamine enzyme immunoassay kit (Figure 6E).
  • a mast-cell CAR targeting HER2 was constructed containing the anti-HER2 single-chain variable fragment, the stalk and transmembrane domain from CD8a, cytoplasmic domain of FcsRI gamma chain, and a monomer GFP for CAR labelling.
  • This HER2 CAR was introduced into mature mouse mast cells by lentiviral transduction (Figure 7A).
  • the maturity of mast cells was validated by the expressions of mast cell maturation markers c-kit and FceRI ( Figure 7B).
  • HER2 CAR-mast cells were co-cultured with HER2-ectopically expressing EMT6 cells (HER2- EMT6), which are mouse syngeneic triple-negative breast cancer cells.
  • HER2 CAR-mast cells were specifically activated by HER2-EMT6 cells, as reflected by the increased surface exposure of CD107a (Figure 7C) and TNFa production (Figure 7D) after 48 hr of coculture with HER2-EMT6.
  • Figure 7E shows the cytokine and chemokine released by HER2 CAR-mast cells when cocultured with and without HER2- EMT6 cells for 48 hr. Similar with other CAR-mast cells introduced above, HER2 CAR- mast cells encountering HER2-EMT6 cells produced high amounts of CCL2, CCL3, CCL4, IL-6, TNFa and GM-CSF.
  • FIGS. 8A and 8B Cytotoxicity of HER2 CAR-mast cells were also investigated.
  • Figures 8A show cytotoxicity of HER2 CAR-mast cells against HER2-EMT6 cells across a broad range of effector-to-target ratios (Figure 8A).
  • the cytotoxicity was constrained in the presence of varying concentrations of Soybean Trypsin Inhibitor (SBTI) ( Figure 8B), which shows that cell-killing activity is dependent on tryptase activity.
  • SBTI Soybean Trypsin Inhibitor
  • the Fc(epsilon)RIbeta subunit functions as an amplifier of Fc(epsilon)RIgamma-mediated cell activation signals.

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Abstract

L'invention concerne des protéines de fusion comprenant (a) un domaine de signalisation intracellulaire de mastocytes ; et (b) une séquence d'acides aminés qui est hétérologue au domaine de signalisation intracellulaire de mastocytes. Dans certains modes de réalisation, (a) comprend le domaine intracellulaire du récepteur IgE (FcεRI), un récepteur de cytokine (c-KIT), un récepteur couplé à la protéine G, ou un récepteur de type toll exprimé dans des mastocytes, ou un fragment fonctionnel ou un variant de celui-ci. Dans certains modes de réalisation, la séquence d'acides aminés de (a) comprend une séquence ITAM. Dans des modes de réalisation préférés, les protéines de fusion sont des récepteurs antigéniques chimériques (appelés "CAR-mast"), qui comprennent un domaine transmembranaire et un domaine extracellulaire ciblant un antigène. L'invention concerne également des acides nucléiques codant pour les protéines de fusion, et des mastocytes comprenant ou exprimant les acides nucléiques et/ou les protéines de fusion, ainsi que leurs procédés d'utilisation pour traiter des maladies et des troubles caractérisés par l'expression de l'antigène.
EP24731778.7A 2023-05-15 2024-05-15 Compositions de récepteurs antigéniques chimériques, cellules car-mast ainsi formées et procédés d'utilisation associés Pending EP4713352A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN115298223A (zh) * 2020-03-12 2022-11-04 南京传奇生物科技有限公司 磷脂酰肌醇蛋白聚糖-2结合部分、嵌合抗原受体及其用途

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1601438A (fr) 1968-10-17 1970-08-24
US5906936A (en) 1988-05-04 1999-05-25 Yeda Research And Development Co. Ltd. Endowing lymphocytes with antibody specificity
US6352694B1 (en) 1994-06-03 2002-03-05 Genetics Institute, Inc. Methods for inducing a population of T cells to proliferate using agents which recognize TCR/CD3 and ligands which stimulate an accessory molecule on the surface of the T cells
US6534055B1 (en) 1988-11-23 2003-03-18 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US5858358A (en) 1992-04-07 1999-01-12 The United States Of America As Represented By The Secretary Of The Navy Methods for selectively stimulating proliferation of T cells
US6905680B2 (en) 1988-11-23 2005-06-14 Genetics Institute, Inc. Methods of treating HIV infected subjects
US6004811A (en) 1991-03-07 1999-12-21 The Massachussetts General Hospital Redirection of cellular immunity by protein tyrosine kinase chimeras
US5851828A (en) 1991-03-07 1998-12-22 The General Hospital Corporation Targeted cytolysis of HIV-infected cells by chimeric CD4 receptor-bearing cells
US5843728A (en) 1991-03-07 1998-12-01 The General Hospital Corporation Redirection of cellular immunity by receptor chimeras
US6753162B1 (en) 1991-03-07 2004-06-22 The General Hospital Corporation Targeted cytolysis of HIV-infected cells by chimeric CD4 receptor-bearing cells
US5912170A (en) 1991-03-07 1999-06-15 The General Hospital Corporation Redirection of cellular immunity by protein-tyrosine kinase chimeras
IE920716A1 (en) 1991-03-07 1992-09-09 Gen Hospital Corp Redirection of cellular immunity by receptor chimeras
US8211422B2 (en) 1992-03-18 2012-07-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric receptor genes and cells transformed therewith
IL104570A0 (en) 1992-03-18 1993-05-13 Yeda Res & Dev Chimeric genes and cells transformed therewith
US7175843B2 (en) 1994-06-03 2007-02-13 Genetics Institute, Llc Methods for selectively stimulating proliferation of T cells
US5712149A (en) 1995-02-03 1998-01-27 Cell Genesys, Inc. Chimeric receptor molecules for delivery of co-stimulatory signals
US6692964B1 (en) 1995-05-04 2004-02-17 The United States Of America As Represented By The Secretary Of The Navy Methods for transfecting T cells
US7067318B2 (en) 1995-06-07 2006-06-27 The Regents Of The University Of Michigan Methods for transfecting T cells
AU2474700A (en) 1998-12-01 2000-06-19 Genentech Inc. Secreted amd transmembrane polypeptides and nucleic acids encoding the same
WO2000063374A1 (fr) 1999-04-16 2000-10-26 Celltech Therapeutics Limited Composants transmembranaires de synthese
US6867041B2 (en) 2000-02-24 2005-03-15 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US6797514B2 (en) 2000-02-24 2004-09-28 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
KR20030032922A (ko) 2000-02-24 2003-04-26 싸이트 테라피스 인코포레이티드 세포의 동시 자극 및 농축
CA2534639C (fr) 2003-07-31 2013-07-30 Immunomedics, Inc. Anticorps anti-cd19
US7435596B2 (en) 2004-11-04 2008-10-14 St. Jude Children's Research Hospital, Inc. Modified cell line and method for expansion of NK cell
PH12013501201A1 (en) 2010-12-09 2013-07-29 Univ Pennsylvania Use of chimeric antigen receptor-modified t cells to treat cancer
CN113684185A (zh) 2013-02-26 2021-11-23 纪念斯隆-凯特琳癌症中心 用于免疫疗法的组合物和方法
US11026973B2 (en) * 2019-04-30 2021-06-08 Myeloid Therapeutics, Inc. Engineered phagocytic receptor compositions and methods of use thereof
US20220001031A1 (en) * 2019-04-30 2022-01-06 Myeloid Therapeutics, Inc. Engineered chimeric fusion protein compositions and methods of use thereof
EP3962497A4 (fr) * 2019-04-30 2023-01-18 Myeloid Therapeutics, Inc. Compositions de protéines de fusion chimériques modifiées et leurs procédés d'utilisation
AU2021347807A1 (en) * 2020-09-23 2023-05-04 Myeloid Therapeutics, Inc. Improved methods and compositions for expression of nucleic acids in cells

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