[go: up one dir, main page]

WO2024236547A1 - Cellules phagocytaires modifiées exprimant des récepteurs antigéniques chimériques comprenant un domaine co-stimulateur de médiateur d'entrée du virus de l'herpès (hvem) et utilisations associées - Google Patents

Cellules phagocytaires modifiées exprimant des récepteurs antigéniques chimériques comprenant un domaine co-stimulateur de médiateur d'entrée du virus de l'herpès (hvem) et utilisations associées Download PDF

Info

Publication number
WO2024236547A1
WO2024236547A1 PCT/IB2024/054836 IB2024054836W WO2024236547A1 WO 2024236547 A1 WO2024236547 A1 WO 2024236547A1 IB 2024054836 W IB2024054836 W IB 2024054836W WO 2024236547 A1 WO2024236547 A1 WO 2024236547A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
seq
amino acid
acid sequence
car
Prior art date
Application number
PCT/IB2024/054836
Other languages
English (en)
Inventor
Kevin CARBAJAL
Grace REYNOLDS
Kilsoo Jeon
Warren Anderson
Original Assignee
Inceptor Bio, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Inceptor Bio, Llc filed Critical Inceptor Bio, Llc
Publication of WO2024236547A1 publication Critical patent/WO2024236547A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • 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
    • A61K40/17—Monocytes; Macrophages
    • 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
    • A61K40/421—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
    • A61K40/4231—Cytokines
    • A61K40/4232—Tumor necrosis factors [TNF] or CD70
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/21—Transmembrane domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/55—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present disclosure relates to phagocytic cells genetically modified to express a chimeric antigen receptor (CAR) having a herpes virus entry mediator (HVEM) co-stimulatory domain and related compositions, vectors, methods, and systems.
  • CAR chimeric antigen receptor
  • HVEM herpes virus entry mediator
  • CARs Chimeric antigen receptors
  • I1158141020WO 0012.2
  • Chimeric antigen receptors are engineered receptors that direct a desired specificity with the functionality of an immune cell to target cancer cells.
  • these receptors graft the specificity of a monoclonal antibody onto a T cell.
  • the receptors are called chimeric because they are fusions of parts from different sources.
  • CAR-T cell therapy refers to a treatment that uses such transformed cells primarily for cancer therapy.
  • CAR-engineered immune cells The general premise of CAR-engineered immune cells is to endow such cells with the ability to target markers found on diseased cells, e.g., cancer cells.
  • diseased cells e.g., cancer cells.
  • CAR-T cells scientists can remove T cells from a person, genetically alter them to express a CAR, and put them back into the patient for them to attack the diseased cells.
  • CAR-T cells can offer better tumor targeting, stronger killing activity, longer persistence in vivo, and improvement in therapeutic effect, as compared to non-modified T cells.
  • challenges in engineering CAR-T cells for immunotherapy include low vector transformation efficiency, low gene editing efficiency, and/or the inability of expanded CAR-T cells to meet the clinically required cell dose.
  • Tumors include not only tumor cells and T cells, but also macrophages, fibroblasts, and other types of cells.
  • the complex tumor microenvironment can limit contact between CAR-T cells and tumor cells, or other cells can inhibit CAR-T cell cytotoxicity, leading to decreased effectiveness of immunotherapy.
  • the phagocytic cells are genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR comprises: an antigen binding domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 1, 2, or 7, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • CAR chimeric antigen receptor
  • HVEM herpes virus entry mediator
  • the CAR-modified phagocytic cells have increased phagocytosis and/or trogocytosis, increased cytokine secretion, increased secretion of proinflammatory factors, increased M1 proinflammatory 2 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) phenotype, increased M2 immunosuppressive phenotype, increased proliferation, and/or increased antigen presentation to T cells, as compared to a relevant control.
  • the CAR-modified phagocytic cells can have increased expression of a pro-inflammatory cytokine as compared to a relevant control. In some embodiments, the increase in expression of a pro-inflammatory cytokine is 1.1- to 20-fold as compared to a relevant control.
  • the CAR-modified phagocytic cells have increased phagocytosis and/or trogocytosis. In some embodiments, the increase in phagocytosis and/or trogocytosis is 5% to 100%. In some embodiments, the increase in phagocytosis is at least 20%. The increase in phagocytosis and/or trogocytosis of the CAR-modified phagocytic cells of the disclosure occurs even in the presence of a functional CD47 signaling in target cells.
  • the increased expression of pro-inflammatory cytokines, increased phagocytosis, and/or increased trogocytosis of modified phagocytic cells expressing a CAR having an intracellular HVEM co-stimulatory signaling domain are hallmarks of an increased M1 pro-inflammatory phenotype of the CAR-modified phagocytic cells and can change the environment in which their target cells are found.
  • CAR-modified phagocytic cells of the disclosure having an M1 phenotype change the tumor microenvironment such that anti-tumor immune responses are enhanced.
  • the environment is changed to promote defense mechanisms against infectious cells.
  • the present disclosure provides a genetically modified phagocytic cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: an antigen binding domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co- stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co- stimulatory activity.
  • CAR chimeric antigen receptor
  • HVEM herpes virus entry mediator
  • the HVEM co-stimulatory signaling domain has an amino acid sequence set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity. In some embodiments, the HVEM co- stimulatory signaling domain has an amino acid sequence set forth as SEQ ID NO: 7.
  • the co-stimulatory signaling domain further comprises a CD28 co-stimulatory signaling domain, a 4-1BB co-stimulatory signaling domain, an OX-40 co-stimulatory signaling domain, an ICOS co-stimulatory signaling domain, a functional fragment or variant of any of the co-stimulatory signaling domains having at least 90% identity 3 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) thereto, or any combination thereof.
  • the intracellular signaling domain comprises a CD3z, a 4-1BB, or a )F ⁇ 5 ⁇ signaling domain.
  • the intracellular signaling domain comprises an amino acid sequence set forth as any one of SEQ ID NOs: 10 or 14, or a functional fragment or variant thereof that retains signaling activity having an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as any one of SEQ ID NOs: 10 or 14.
  • the transmembrane domain comprises a CD8 transmembrane domain, such as a CD8a transmembrane domain, or a HVEM transmembrane domain, or functional fragment or variant thereof.
  • the transmembrane domain comprises an amino acid sequence set forth as SEQ ID NO: 12, or a functional fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NOs: 12
  • the CAR further comprises a hinge region.
  • the hinge region comprises a CD8 hinge region, such as a CD8a hinge region, or a HVEM hinge region, or a fragment thereof.
  • the hinge region comprises an amino acid sequence set forth as SEQ ID NO: 13 or 15, or a functional fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NOs: 13 or 15.
  • the hinge region is located between said antigen binding domain and said transmembrane domain.
  • the genetically modified phagocytic cell has increased phagocytosis and/or trogocytosis as compared to a control. In some embodiments, the genetically modified phagocytic cell has increased phagocytosis and/or trogocytosis of 5% to 100%. In some embodiments, the genetically modified phagocytic cell has increased phagocytosis and/or trogocytosis of at least 20%. In some embodiments of the above aspect, the increased phagocytosis and/or trogocytosis occurs in the presence of a cell expressing CD47.
  • the genetically modified phagocytic cell has increased expression of a pro-inflammatory cytokine as compared to a control.
  • the control comprises a phagocytic cell expressing a CAR that does not comprise the HVEM co-stimulatory signaling domain and/or does not comprise the antigen binding domain.
  • the antigen binding domain comprises a monovalent antibody fragment.
  • the antigen binding domain targets an antigen present on the surface of a viral particle and/or cancer cell.
  • the phagocytic cell is a monocyte, macrophage, a dendritic cell, a neutrophil, or a precursor thereof.
  • the phagocytic cell is a macrophage.
  • the phagocytic cell is a precursor.
  • the precursor comprises a bone marrow-derived cell or a stem cell.
  • the present disclosure provides a pharmaceutical composition comprising the genetically modified phagocytic cell as described hereinabove, and a pharmaceutically acceptable carrier.
  • the present disclosure provides use of the genetically modified phagocytic cell as described hereinabove in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.
  • the present disclosure provides use of the genetically modified phagocytic cell as described hereinabove in the manufacture of a medicament for the treatment of a tumor or cancer in a subject in need thereof.
  • the present disclosure provides use of the genetically modified phagocytic cell as described hereinabove in the manufacture of a medicament for the treatment of an infection in a subject in need thereof.
  • the present disclosure provides a method of treating a disease or condition associated with a tumor or cancer in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the genetically modified phagocytic cell as described hereinabove.
  • the present disclosure provides a method of treating a tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the genetically modified phagocytic cell as described hereinabove.
  • the present disclosure provides a method of treating an infection in a subject in need thereof, comprising administering to the subject an effective amount of the genetically modified phagocytic cell as described hereinabove.
  • the present disclosure provides a method for stimulating an immune response to a target tumor cell or tumor tissue in a subject comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the genetically modified phagocytic cell as described hereinabove.
  • the present disclosure provides a method of treating a subject by engulfment and/or trogocytosis of a target cell in the subject, the method comprising administering to the subject a therapeutically effective amount of the genetically modified phagocytic cell or the 5 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) composition as described hereinabove.
  • administering the genetically modified phagocytic cell is performed by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In some embodiments, administering the genetically modified phagocytic cell is performed by injecting the genetically modified phagocytic cell directly into a target region, a local disease site in the subject, a lymph node, an organ, and/or a tumor of the subject. In some embodiments, administering the composition is performed transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous injection, or intraperitoneally.
  • the administering is performed to treat a solid tumor or a hematologic malignancy.
  • the solid tumor comprises lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, neuroblastoma, rhabdomyosarcoma, or brain tumor.
  • the hematologic malignancy comprise acute myeloid leukemia, chronic myelogenous leukemia, myelodysplasia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
  • the administering is performed to treat bacteria infection, virus-infected cells, virions, defective neurons, or senescent cells.
  • the present disclosure provides a method of modifying a phagocytic cell, the method comprising: introducing a chimeric antigen receptor (CAR) into the phagocytic cell, wherein the CAR comprises: an antigen binding domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity, wherein the modified phagocytic cell expresses the CAR and has targeted effector activity.
  • CAR chimeric antigen receptor
  • HVEM herpes virus entry mediator
  • introducing the CAR into the phagocytic cell comprises introducing a nucleic acid molecule comprising a polynucleotide sequence encoding the CAR. In some embodiments, introducing the nucleic acid molecule comprises transducing the phagocytic cell with a viral vector comprising the nucleic acid sequence encoding the CAR. In some embodiments of the above method of modifying a phagocytic cell aspect, the targeted effector activity is directed against an antigen on a target cell that specifically binds the antigen binding domain of the CAR.
  • the targeted effector activity is 6 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) selected from the group consisting of phagocytosis, targeted cellular cytotoxicity, antigen presentation, and cytokine secretion.
  • the HVEM co-stimulatory signaling domain has an amino acid sequence set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • the HVEM co-stimulatory signaling domain has an amino acid sequence set forth as SEQ ID NO: 7.
  • the intracellular signaling domain comprises a CD3z, a 4-1BB, or a )F ⁇ 5 ⁇ signaling domain.
  • the intracellular signaling domain comprises an amino acid sequence set forth as any one of SEQ ID NOs: 10 or 14, or a functional fragment or variant thereof that retains signaling activity having an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as any one of SEQ ID NOs: 10 or 14.
  • the transmembrane domain comprises a CD8 transmembrane domain, such as a CD8a transmembrane domain, or a HVEM transmembrane domain, or a functional fragment or variant thereof.
  • the transmembrane domain comprises an amino acid sequence set forth as SEQ ID NO: 12, or a functional fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 12.
  • the CAR further comprises a hinge region.
  • the hinge region comprises a CD8 hinge region, such as a CD8a hinge region, or a HVEM hinge region, or a fragment thereof.
  • the hinge region comprises an amino acid sequence set forth as SEQ ID NO: 13 or 15, or a functional fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 13 or 15.
  • the hinge region is located between said antigen binding domain and said transmembrane domain.
  • the modified phagocytic cell has increased phagocytosis and/or trogocytosis as compared to a control.
  • the modified phagocytic cell has increased phagocytosis and/or trogocytosis of 5% to 100%. In some embodiments, the modified phagocytic cell has increased phagocytosis and/or trogocytosis of at least 20%. In some embodiments of the above method of modifying a phagocytic cell aspect, the increased phagocytosis and/or trogocytosis occurs in the presence of a cell expressing CD47.
  • the modified phagocytic cell has increased activation of NF- ⁇ % (Nuclear factor kappa B) pathway.
  • the modified phagocytic cell has increased expression of a pro-inflammatory cytokine as compared to a control. In some embodiments, the increased expression of the pro-inflammatory cytokine is 1.1- fold to 20-fold.
  • the control comprises a phagocytic cell expressing a CAR that does not comprise the HVEM co- stimulatory signaling domain and/or does not comprise the antigen binding domain.
  • the phagocytic cell is a monocyte, macrophage, dendritic cell, neutrophil, or a precursor thereof.
  • the phagocytic cell is a macrophage.
  • the phagocytic cell is a precursor.
  • the precursor comprises a bone marrow-derived cell or a stem cell.
  • the method further comprises culturing modified phagocytic cells expressing the CAR.
  • a composition comprising the cultured modified phagocytic cells expressing the CAR is administered to a subject in need thereof.
  • the composition provides an immune response against a target in the subject in need thereof.
  • the subject in need thereof has cancer and/or an infection.
  • the target is a cancer cell or an infectious agent.
  • the cancer is a solid tumor or a hematologic malignancy.
  • the solid tumor comprises lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, neuroblastoma, rhabdomyosarcoma, or brain tumor.
  • the hematologic malignancy comprises acute myeloid leukemia, chronic myelogenous leukemia, myelodysplasia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma.
  • the composition is administered by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation.
  • the composition is administered by injecting the composition directly into a target region, a local disease site in the subject, a lymph node, an organ, and/or a tumor of the subject.
  • the composition is administered transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous injection, or intraperitoneally.
  • I1158141020WO 0012.2
  • the present disclosure provides a composition comprising the phagocytic cell modified by the method as described hereinabove.
  • FIGS.1A-1F show that macrophages expressing a CAR (CAR-M) with an HVEM co- stimulatory signaling domain (termed “M83”) have increased phagocytosis and superior control of target cell expansion as compared to CAR-M having CD3z signaling domain only, having CD3z signaling domain (first-generation CAR) and 4-1BB co-stimulatory signaling domain (second- generation CAR), or as compared to control macrophages.
  • CAR-M CAR-M
  • M83 HVEM co- stimulatory signaling domain
  • FIG.1A shows increased phagocytosis in primary macrophages having a CAR having an HVEM costimulatory domain (“CD19.M83costim.CD3z”) compared to controls (“CD19.41BB.CD3z”;“CD19.CD3z”; and “Non-targeting”).
  • FIG.1B shows superior control of target cell expansion in THP-1 cells having an anti-CD19 CAR having an HVEM domain (“CD19.M83costim.CD3z”) compared to controls.
  • FIG.1C shows area under the curve analysis of the data depicted in the graph of FIG.1B.
  • FIG. 1D is a schematic that shows CAR constructs and domains used in the experiment depicted in FIG.1E.
  • FIG.1E shows superior control of target cell expansion in THP-1 cells having a CAR having an HVEM co-stimulatory signaling domain, a CD3z or )F ⁇ 5 ⁇ signaling domain, a CD8a or HVEM transmembrane domain, and a CD8a or HVEM hinge domain compared to controls, as depicted in FIG.1D.
  • FIG.1F shows superior control of target cell expansion in THP-1 cells having an anti-CD70 CAR having an HVEM costimulatory domain (“CD70.M83costim.CD3z”) compared to controls without an HVEM costimulatory domain.
  • CD70.M83costim.CD3z HVEM costimulatory domain
  • FIG.2A shows that phagocytosis by CAR-M with an M83 co-stimulatory signaling domain (“CD19.M83costim.CD3z”) is unaffected by CD47-SIRPa signaling (compare “Targets” and “+aCD47”).
  • FIG.2B shows that presence of an M83 costimulatory domains results in CAR cells with a higher level of resistance to CD47 signaling compared to a first-generation CAR without an M83 costimulatory domain.
  • FIG.3 shows a heat map depicting expression of M1-related marker genes and pro- inflammatory cytokine genes in CD19/M83 CAR-M cells (“CD19.M83costim.CD3z”) after co- culture with CD19-H1299 tumor cells compared to spheroid or non-targeting CAR-M cells. Expression is depicted by shading, with darker shading representing higher expression. R1: replicate 1; R2: replicate 2; R3: replicate 3.
  • FIG.4 shows the efficacy of CD19/M83 CAR-M cells (“CD19.M83costim.CD3z”) against CD19-positive spheroids using an in vitro three-dimensional (3D) spheroid model.
  • FIGS.5A-5C show efficacy of CD19/M83 CAR-M cells (“CD19.M83costim.CD3z”) against xenografted CD19-positive tumor cells in vivo compared to first generation CD19 CAR M cells lacking M83 (“CD19.CD3z”), non-targeting M83 CAR M cells and HBSS controls.
  • FIG.5A shows a schematic depicting the in vivo mouse model used to assess efficacy of CAR M cells.
  • FIG.5B shows a line graph depicting relative tumor volume at the indicated day following CAR M cell administration for the indicated treatment using the model depicted in FIG.5A.
  • D4 day 4;
  • D12 day 12;
  • D15 day 15;
  • D22 day 22;
  • D26 day 26.
  • FIG.5C shows a bar graph depicting total area under the curve analysis of the tumor volume data for each treatment presented in FIG.5B.
  • FIG.6 shows activation of the NF- ⁇ % ⁇ 1XFOHDU ⁇ IDFWRU ⁇ NDSSD ⁇ % ⁇ WUDQVFULSWLRQ ⁇ IDFWRU ⁇ pathway in THP1-DualTM cells having a CAR having a HVEM co-stimulatory signaling domain and a CD3z (‘CD19.M83costim.CD3z’) or )F ⁇ 5 ⁇ (‘CD19.M83costim.FcGr’) intracellular signaling domain.
  • CD3z ‘CD19.M83costim.CD3z’
  • )F ⁇ 5 ⁇ ‘CD19.M83costim.FcGr’
  • a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • description of a range such as from 1-10 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 2 to 4, from 2 to 6, from 2 to 8, from 2 to 10, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.
  • the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range.
  • the variable can be equal to any integer value within the numerical range, including the end-points of the range.
  • the variable can be equal to any real value within the numerical range, including the end-points of the range.
  • a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ⁇ 0 and ⁇ 2 if the variable is inherently continuous.
  • the term “gene” or “coding sequence”, herein used interchangeably refers to a functional nucleic acid unit encoding a protein, polypeptide, or peptide. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • nucleic acid As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g, chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • fragment will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical to the reference nucleic acid or nucleotide sequence.
  • a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • such fragments can comprise, consist essentially or and/or consist of, oligonucleotides having a length of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.
  • a “mutation” is any change in a nucleic acid sequence. Nonlimiting examples comprise insertions, deletions, duplications, substitutions, inversions, and translocations of any nucleic acid sequence, regardless of how the mutation is brought about and regardless of how or whether the mutation alters the functions or interactions of the nucleic acid.
  • a mutation may produce altered enzymatic activity of a ribozyme, altered base pairing between nucleic acids (e.g. RNA interference interactions, DNA-RNA binding, etc.), altered mRNA folding stability, and/or how a nucleic acid interacts with polypeptides (e.g. DNA- transcription factor interactions, RNA-ribosome interactions, gRNA-endonuclease reactions, etc.).
  • a mutation might result in the production of proteins with altered amino acid sequences (e.g. missense mutations, nonsense mutations, frameshift mutations, etc.) and/or the production of proteins with the same amino acid sequence (e.g. silent mutations).
  • Mutations may create no observed change in a cell while others that encode for an identical protein sequence nevertheless result in an altered cell phenotype (e.g. due to codon usage bias, altered secondary protein structures, etc.). Mutations may occur within coding regions (e.g., open reading frames) or outside of coding regions (e.g., within promoters, terminators, untranslated elements, or enhancers), and may affect, for example and without limitation, gene expression levels, gene expression profiles, protein sequences, and/or sequences encoding RNA elements such as tRNAs, ribozymes, ribosome components, and microRNAs.
  • RNA elements such as tRNAs, ribozymes, ribosome components, and microRNAs.
  • recombinant DNA construct As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded 12 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) polynucleotides.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature.
  • a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature.
  • Such a construct may be used by itself or may be used in conjunction with a vector.
  • the terms “engineered” or “recombinant” in reference to a phagocyte, gene, nucleic acid and/or protein as used herein refer to a phagocyte, gene, nucleic acid and/or protein that has been altered through human intervention.
  • the term “naturally occurring” as used herein in reference to a phagocyte, gene, nucleic acid and/or protein as used herein refer to a phagocyte, gene, nucleic acid and/or protein existing in nature and without any human intervention.
  • Exemplary human interventions comprise transfection with a heterologous polynucleotide, molecular cloning resulting in a deletion, insertion, modification and/or rearrangement with respect to a naturally occurring sequence such as a naturally occurring sequence in a phagocyte, gene, nucleic acid and/or protein herein described.
  • expression or “expressing” refers to the transcription and/or translation of a particular nucleic acid sequence driven by a promoter.
  • An expression construct or expression vector can permit transcription of a particular nucleic acid sequence in a cell (e.g., a phagocytic cell).
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • An expression cassette typically comprises at least three components: a promoter sequence, an open reading frame encoding gene(s) of interest, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site.
  • An expression cassette can be formed by manipulable fragment of DNA carrying and capable of expressing, one or more genes of interest optionally located between one or more sets of restriction sites.
  • Expression cassettes typically comprise further regulatory sequences additional to the promoter to regulate the expression of the gene or genes within the open reading frame herein also indicated as a coding region of the expression cassette.
  • "Operably linked" is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a promoter of the present invention and a heterologous nucleotide is a functional link that allows for expression of the heterologous nucleic acid molecule.
  • Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be co-transformed into a cell. Alternatively, the additional gene(s) can be provided on multiple expression cassettes or DNA constructs.
  • the expression cassette may 13 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) additionally contain selectable marker genes.
  • polypeptide indicates an organic linear, circular, or branched polymer composed of two or more amino acid monomers and/or analogs thereof.
  • polypeptide includes amino acid polymers of any length including full-length proteins and peptides, as well as analogs and fragments thereof.
  • a polypeptide of three or more amino acids is also called a protein oligomer, peptide, or oligopeptide.
  • the terms “peptide” and “oligopeptide” usually indicate a polypeptide with less than 100 amino acid monomers.
  • the polypeptide provides the primary structure of the protein, wherein the term “primary structure” of a protein refers to the sequence of amino acids in the polypeptide chain covalently linked to form the polypeptide polymer.
  • a protein “sequence” indicates the order of the amino acids that form the primary structure. Covalent bonds between amino acids within the primary structure can include peptide bonds or disulfide bonds, and additional bonds identifiable by a skilled person.
  • Polypeptides in the sense of the present disclosure are usually composed of a linear chain of alpha-amino acid residues covalently linked by peptide bond or a synthetic covalent linkage.
  • the two ends of the linear polypeptide chain encompassing the terminal residues and the adjacent segment are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity.
  • counting of residues in a polypeptide is performed from the N- terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond to the C-terminal end (-COOH group) which is the end where a COOH group is not involved in a peptide bond.
  • Proteins and polypeptides can be identified by x-ray crystallography, direct sequencing, immunoprecipitation, and a variety of other methods as understood by a person skilled in the art. Proteins can be provided in vitro or in vivo by several methods identifiable by a skilled person. In some instances where the proteins are synthetic proteins in at least a portion of the polymer two or more amino acid monomers and/or analogs thereof are joined through chemically-mediated condensation of an organic acid (-COOH) and an amine (-NH 2 ) to form an amide bond or a “peptide” bond.
  • -COOH organic acid
  • -NH 2 an amine
  • amino acid refers to a compound having a free carboxyl group and a free unsubstituted amino group on the a carbon, which may be joined by peptide bonds to form a peptide active agent as described herein.
  • a “basic amino acid” refers to any amino acid that is positively charged at a pH of 6.0, including but not limited to R, K, and H.
  • An "aromatic amino acid” refers to any amino acid that has an aromatic group in the side-chain coupled to the alpha carbon, including but not limited to F, Y, W, and H.
  • “function” of a gene, a peptide, a protein, or a molecule refers to activity of a gene, a peptide, a protein, or a molecule.
  • “Introducing,” “introduce,” and “introduced” in the context of a polynucleotide and/or polypeptide of interest means presenting a nucleotide sequence of interest (e.g., polynucleotide, a nucleic acid construct, and/or a guide nucleic acid) and/or polypeptide of interest to a host organism or cell of said organism (e.g., a mammalian cell) in such a manner that the nucleotide sequence and/or polypeptide gains access to the interior of a cell.
  • a nucleotide sequence of interest e.g., polynucleotide, a nucleic acid construct, and/or a guide nucleic acid
  • a host organism or cell of said organism e.g., a mammalian cell
  • “introducing” includes inserting a nucleic acid molecule (e.g., a recombinant DNA construct) into a cell, by means of transformation, transfection, or transduction.
  • the nucleic acid molecule may be incorporated into the genome of the cell (e.g., nuclear chromosome or mitochondrial chromosome), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a “subject” that may be treated by methods of the present disclosure include both human subjects for medical and/or therapeutic purposes and animal subjects for veterinary and drug screening and development purposes.
  • suitable animal subjects are, in general, mammalian subjects such as primates, bovines, ovines, caprines, porcines, equines, felines, canines, lagomorphs, rodents ( e.g ., rats and mice), etc.
  • Human subjects are the most preferred. Human subjects include fetal, neonatal, infant, juvenile, adult and geriatric subjects.
  • anti-tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the proliferation rate, a decrease in the number of metastases, an increase in life expectancy, and/or amelioration of various physiological symptoms associated with the cancerous condition.
  • an "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to delay the occurrence of tumor in the first place.
  • autologous is meant to refer to any material derived from the same individual to whom it is later to be re-introduced.
  • Allogeneic refers to a graft derived from a different animal of the same species.
  • Xenogeneic refers to a graft derived from an animal of a different species.
  • antibody refers to full-length immunoglobulins as well as to fragments thereof.
  • Such full-length immunoglobulins may be monoclonal, polyclonal, chimeric, humanized, veneered or human antibodies.
  • antibody fragments comprises portions of a full-length immunoglobulin retaining the targeting specificity of said immunoglobulin. Many but not all antibody fragments lack at least partially the constant region (Fc region) of the full-length immunoglobulin. In some embodiments, antibody fragments are produced by digestion of the full-length immunoglobulin.
  • An antibody fragment may also be a synthetic or recombinant construct comprising parts of the immunoglobulin or immunoglobulin chains (see e.g. Holliger, P. and Hudson, J. Engineered antibody fragments and the rise of single domains.
  • antibody fragments include, without being limited to, include scFv, Fab, Fv, Fab ⁇ , F(ab ⁇ )2 fragments, dAb, VHH, nanobodies, V(NAR) or minimal recognition units.
  • Single chain variable fragments or “single chain antibodies” or “scFv” are one type of antibody fragment. scFv are fusion proteins comprising the variable heavy (VH) and variable light (VL) of immunoglobulins connected by a linker. They thus lack the constant Fc region present in full- length immunoglobulins, but retain the specificity of the original immunoglobulin.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody 16 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Fc immunoglobulin constant region
  • the numbering system to identify amino acid residue positions in the VH and VL of an antibody can follow a system known to one of skill in the art, including Kabat (Wu and Kabat (1970) J Exp Med.132(2):211-50; Borden and Kabat (1987) PNAS, 84:2440-2443; Kabat et al. U.S. Department of Health and Human Services, 1991), Chothia (Chothia and Lesk (1987) J Mol. Biol., 196(4): 901-917; Chothia et al. (1989) Nature 342:877-883), and the "AHo" system described by Honegger & Pluckthun (2001) Journal of Molecular Biology 309:657-670.
  • antigen-binding portion or "antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments, portions or domains of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen binding function of an antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL1 and CH1 domains; (ii) an F(ab') 2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al.
  • VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single contiguous chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883).
  • scFv single chain Fv
  • single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of 17 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) an antibody.
  • Other forms of single chain antibodies, such as diabodies, are also encompassed (see e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448).
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope. Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of one (or more) linear polypeptide chain(s).
  • a linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include other moieties, such as saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • An "antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • an "antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, K and l light chains refer to the two major antibody light chain isotypes.
  • the term "antigen" or "Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may 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 comprises 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. It is readily apparent that the present invention 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 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.
  • specifically binds is meant an antibody or antigen binding domain which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody or antigen binding domain that specifically binds to an antigen from one species may also bind to that antigen from one or more species.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, antigen binding domain, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody or antigen binding domain recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • an antibody or antigen binding domain recognizes and binds to a specific protein structure rather than to proteins generally.
  • an “immune response” refers to the reaction of a subject to the presence of an antigen, which may include at least one of the following: making antibodies, developing immunity, developing hypersensitivity to the antigen, and developing tolerance.
  • the term “enhance an immune response” as used herein implies that the reaction of a subject to the presence of an antigen is increased and/or amplified in the presence of a CAR-modified phagocytic cell of the disclosure as compared to the reaction of a subject to the presence of an antigen in the absence of a CAR-modified phagocytic cell of the disclosure.
  • treat indicates that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.
  • therapeutic means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
  • An “effective” amount as used herein is an amount that provides a desired effect.
  • a “therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject.
  • modulating is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a 19 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • a “target site” or “target sequence” refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
  • target is meant a cell, organ, or site within the body that is in need of treatment.
  • detectable moiety includes any suitable detectable group, such as radiolabels (e.g. 35 S, 125 I, 131 I, etc.), enzyme labels (e.g, horseradish peroxidase, alkaline phosphatase, etc.), fluorescence labels (e.g., fluorescein, green fluorescent protein, etc.), etc., as are well known in the art and used in accordance with known techniques.
  • agent refers to a molecule that may be expressed, released, secreted or delivered to a target by the modified cell described herein.
  • the agent includes, but is not limited to, a nucleic acid, an antibiotic, an anti- inflammatory agent, an antibody or antibody fragments thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate or the like, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combination thereof.
  • the agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell.
  • the agent may diffuse or be transported into the cell, where it may act intracellularly.
  • expand refers to increasing in number, as in an increase in the number of phagocytic cells.
  • the phagocytic cells that are expanded ex vivo increase in number relative to the number originally present in the culture.
  • the phagocytic cells that are expanded ex vivo increase in number relative to other cell types in the culture.
  • ex vivo refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).
  • modified as used herein, is meant a changed state or structure of a molecule or cell of the disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.
  • the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” or “loss” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an 20 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) established standard control.
  • a comparison control e.g., an established normal or reference level of the parameter, or an 20 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) established standard control.
  • the terms “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control.
  • the term “increased” or “increasing” or “increase” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, or more) positive change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control.
  • a comparison control e.g., an established normal or reference level of the parameter, or an established standard control.
  • fold change indicates a measure describing how much a quantity changes between an original and a subsequent measurement.
  • fold change is defined as the ratio between two quantities. For example, for quantities A and B, the fold change of B with respect to A is B/A. For example, a change from 30 to 60 is defined as a fold-change of 2.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • isolated refers to at least partially separated from the natural environment e.g., from a cell.
  • 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.
  • heterologous nucleic acid sequence in reference to a nucleic acid sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a heterologous nucleic acid sequence may not be naturally expressed within a cell or may have altered expression when compared to the corresponding wild type cell.
  • a 21 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) heterologous polynucleotide encoding a CAR described herein can be a nucleic acid sequence that is not naturally present in a phagocytic cell in which it is present.
  • An exogenous polynucleotide may be introduced into the cell in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule.
  • RNA ribonucleic acid
  • the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the cell.
  • endogenous in reference to a gene or nucleic acid sequence or protein is intended a gene or nucleic acid sequence or protein that is naturally comprised within or expressed by a cell.
  • Endogenous genes can include genes that naturally occur in a cell (e.g., phagocytic cell), but that have been modified in the genome of the cell without insertion or replacement of a heterologous gene that is from another species or another location within the genome of the modified cell.
  • “Homolog” or “homologous sequence” may refer to both orthologous and paralogous sequences. Paralogous sequence relates to gene-duplications within the genome of a species. Orthologous sequence relates to homologous genes in different organisms due to ancestral relationship.
  • orthologs are evolutionary counterparts derived from a single ancestral gene in the last common ancestor of given two species and therefore have great likelihood of having the same function.
  • the term “homolog” as used herein refers to functional homologs of genes.
  • a functional homolog is a gene encoding a polypeptide that has sequence similarity to a polypeptide encoded by a reference gene, and the polypeptide encoded by the homolog carries out one or more of the biochemical or physiological function(s) of the polypeptide encoded by the reference gene.
  • Homology can be determined using any homology comparison software computing a pairwise sequence alignment.
  • "Homologous” can refer to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are 22 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • the term “homology” or “homologous” refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequence.
  • the homology is a global homology, e.g., a homology over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof. The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools which are described in WO2014/102774.
  • "homologous" as used herein refers to a sequence that has about 50% sequence identity.
  • the homologous sequence has about 75% sequence identity, even more preferably, has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • sequence identity refers to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment.
  • sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet.3:266-272; Madden et al. (1996), Meth. Enzymol.266:131-141; Altschul et al.
  • percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word VL]H ⁇ JDS ⁇ RSHQLQJ ⁇ SHQDOW ⁇ ⁇ JDS ⁇ H[WHQVLRQ ⁇ SHQDOW ⁇ ⁇ DQG ⁇ VFRULQJ ⁇ PDWUL[ %/2680 ⁇
  • percent similarity of two nucleic acid sequences is the score based upon the following parameters for thH ⁇ %/$67Q ⁇ DOJRULWKP ⁇ ZRUG ⁇ VL]H ⁇ JDS ⁇ RSHQLQJ ⁇ SHQDOW ⁇ ⁇ JDS ⁇ H[WHQVLRQ ⁇ SHQDOW ⁇ ⁇ PDWFK ⁇ UHZDUG ⁇ DQG ⁇ PLVPDWFK ⁇ SHQDOW ⁇ ⁇ :KHQ
  • sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff J G. (Proc Natl Acad Sci 89:10915-9 (1992)).
  • Identity e.g., percent homology
  • NCBI National Center of Biotechnology Information
  • the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art.
  • the issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety. II.
  • phagocytic cells genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 1, 2, or 7, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • CAR chimeric antigen receptor
  • HVEM herpes virus entry mediator
  • the CAR-modified phagocytic cells have increased phagocytosis and/or trogocytosis, increased cytokine secretion, increased secretion of proinflammatory factors, increased M1 proinflammatory phenotype, increased M2 immunosuppressive phenotype, increased proliferation, and/or increased antigen 24 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) presentation to T cells, as compared to a relevant control.
  • the CAR- modified phagocytic cells have increased expression of a pro-inflammatory cytokine as compared to a relevant control.
  • the increase in expression of the pro-inflammatory cytokine can be 1.1- to 20-fold.
  • the CAR-modified phagocytic cells have increased phagocytosis and/or trogocytosis.
  • the increase in phagocytosis and/or trogocytosis can be 5% to 100%. In some embodiments, the increase in phagocytosis is at least 20%.
  • the increase in phagocytosis and/or trogocytosis of the CAR-modified phagocytic cells of the disclosure occurs even in the presence of a functional CD47 signaling pathway, a pathway by which tumor cells can evade the immune system.
  • CAR-modified phagocytic cells of the disclosure having an M1 phenotype change the tumor microenvironment such that anti-tumor immune responses are enhanced.
  • the environment is changed to promote defense mechanisms against infectious cells.
  • CAR-modified phagocytic cells of the disclosure can lead to increased engulfment or destruction of tumor or infectious cells.
  • the CAR-modified phagocytic cells of the disclosure are particularly effective at limiting cell expansion of a target cell, or a plurality of target cells, within a cell population.
  • CAR-modified phagocytic cells having an M1 phenotype include antigen presentation in an adaptive immune response of captured antigens from a tumor or pathogenic agent, recruitment of other immune cells (e.g., cytotoxic T cells, natural killer (NK) cells, dendritic cells) to a tumor or infection site, release of reactive oxygen species or reactive nitrogen species toxic to tumor or infectious cells, and/or promotion of CD4+ T helper cell differentiation into a Th1 subtype that can further enhance anti-tumor responses, including secretion of interferon- ⁇ DQG ⁇ subsequent activation of macrophages, NK cells, and CD8+ cells.
  • immune cells e.g., cytotoxic T cells, natural killer (NK) cells, dendritic cells
  • CARs Chimeric Antigen Receptors
  • the present disclosure provides a phagocytic cell genetically modified to express a chimeric antigen receptor.
  • a chimeric antigen receptor is an artificial cell surface receptor that is engineered to be expressed on an immune effector cell and specifically bind an antigen.
  • a 25 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) CAR typically comprises an antigen binding domain and a signal transduction domain capable of mimicking the T cell receptor-mediated signaling pathway.
  • a “signaling pathway” or “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • the phrase "cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting the signal across the plasma membrane of a cell.
  • Antigen binding domains that are used in CARs include a natural ligand and the single- chain variable region of an antibody to a target molecule.
  • CAR-T cells T cells expressing CAR
  • MHC major histocompatibility complex
  • CARs that have one and two co-stimulatory signaling domains derived from CD28, 4-1BB or ICOS have been developed (Dotti et al., Immunol. Rev.257(1): 107 (2014)). These CARs have been shown to successfully mimic T cell receptor-mediated signal transduction upon antigen stimulation, leading to proliferation and activation of CAR-T cells (Maus et al, Blood 123(17):2625 (2014)).
  • the CAR can help immune cells such as macrophages find and kill cancer cells that have the specific protein the receptor is designed to bind.
  • phagocytic cells such as macrophages or monocytes can be removed from a blood, tumor, or ascites fluid of a patient and modified so that they express a CAR specific to a particular form of antigen on tumor cells. Therefore, a CAR can target cancers by redirecting a phagocyte such as a monocyte or macrophage expressing the CAR specific for tumor associated antigens.
  • CARs can comprise an antigen binding domain, a transmembrane domain, and an intracellular domain comprising a co-stimulatory signaling domain of a herpes virus entry mediator (HVEM) protein as described herein.
  • HVEM herpes virus entry mediator
  • Phagocytic cells modified to express a CAR comprising an intracellular co-stimulatory signaling domain of a HVEM protein described herein can have increased phagocytosis, increased cytokine secretion, increased secretion of proinflammatory factors, increased M1 proinflammatory phenotype, increased M2 immunosuppressive phenotype, increased proliferation, and/or increased antigen presentation to T cells, as compared to a relevant control.
  • 26 62059467v1 Attorney Docket No.: I1158141020WO (0012.2)
  • a CAR expressed by a modified phagocytic cell of the disclosure can comprise an antigen binding domain that binds to an antigen on a target cell.
  • Examples of cell surface markers that may act as an antigen that binds to the antigen binding domain of the CAR include those associated with viral, bacterial and parasitic infections, autoimmune disease, degenerative disease, and cancer cells.
  • the antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof.
  • the antigen binding domain portion comprises a mammalian antibody or a fragment thereof.
  • the antigen binding domain comprises a monovalent antibody fragment.
  • the monovalent antibody fragment can comprise a single chain variable fragment (scFv) or a Fab fragment.
  • the monovalent antibody fragment has a molecular weight of about 25 to about 30 kDa (or about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, or 30 kDa).
  • the monovalent antibody fragment can have a VH and VL domain connected in either orientation by a flexible linker (e.g., VL-linker-VH or VH-linker- VL).
  • the flexible linker typically comprises 10 to about 25 amino acids (e.g., glycine to confer flexibility and/or serines and/or threonines for improved solubility).
  • the antigen binding domain is derived from the same species in which the CAR will ultimately be used in.
  • the antigen binding domain of the CAR can comprise a human antibody, a humanized antibody, or a fragment thereof.
  • the antigen binding domain is operably linked to another domain of the CAR, such as the transmembrane domain or the intracellular domain, for expression in the cell.
  • a polynucleotide sequence encoding the antigen binding domain is operably linked at its 3’ end to a polynucleotide sequence encoding a transmembrane domain and a polynucleotide sequence encoding an intracellular domain.
  • the choice of antigen binding domain depends upon the type and number of antigens that are present on the surface of a target cell.
  • the antigen binding domain may be chosen to recognize an antigen that acts as a cell surface marker on a target cell associated with a particular disease state.
  • the antigen binding domain binds to a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest.
  • the tumor antigen can comprise one or more antigenic cancer epitopes.
  • tumor associated antigens include: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38;
  • the antigen binding domain targets an antigen present on the surface of a viral particle.
  • viral particles include influenza virus, equine infectious anemia virus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), lassa fever virus, herpes simplex virus, varicella zoster virus, cytomegalovirus, epstein-barr virus, variola virus, adeno virus, papilloma virus, parvo virus, measles virus, mumps virus, respiratory syncytial virus, para influenza virus, corona virus, rubella 29 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) virus, rabies virus, human T-cell lymphotropic virus, picoma virus, hepa DNA virus, flavivirus, deltavirus, calicivirus, polio virus, zika virus, west nile virus, SARS, rubella, norovirus, human papillomavirus,
  • Exemplary viral antigens include any surface protein and/or polypeptide present on the surface of the above listed viral particles.
  • surface proteins and/or polypeptides include: Zika capsid protein (C), Zika envelope protein (E), Zika precursor membrane protein (PrM), WNV glycoprotein E, WNV small membrane protein M, VZV glycoprotein E (gE), VZV gB, VZV gH, CoV nucleocapsid (N), CoV envelope (E), CoV membrane (M), Rubella El, Rubella E2, Norovirus Group VP1, HPV 11, HPV capsid protein L1, HPV capsid protein L2, Lassa Fever Virus GP1, Lassa Fever Virus GP2, influenza neuraminidase (NA, N1 to N11), influenza hemagglutinin (HA, H1 to H18), HTLV-1 Envelope, HTLV-1 gp21, HTLV-1 mosaic, HIV gp120, HIV gp41, hepatit
  • a phagocytic cell of the disclosure can also be modified to express additional targeting ligands on the surface of the phagocytic cell in addition to the antigen binding domain of a CAR described herein.
  • Additional targeting ligands comprise molecules configured to associate with any molecule presented on a target cell of interest, such as a target associated with an organ, a tissue, or an extracellular matrix of a target cell of interest.
  • additional targeting ligands bind to molecules associated with a particular state of a target cell, such as a cancerous condition.
  • An additional targeting ligand can be specific to one target or be configured to bind multiple target molecules.
  • Suitable target molecules recognized by an additional target ligand can include a protein (e.g., a receptor, a tumor marker, a transmembrane protein), a nucleic acid (e.g., DNA, RNA), and/or a carbohydrate (e.g., a monosaccharide, disaccharide, or polysaccharide) that is present on the surface of a target cell.
  • exemplary targeting ligands include an RGD-containing peptide, a small molecule (e.g., a peptide) mimetic ligand, and an antibody or antibody fragment specific for a particular target.
  • a CAR expressed by a modified phagocytic cell of the disclosure can comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain.
  • the transmembrane domain can function to stabilize the CAR as a whole.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of 30 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other molecules.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.
  • TCR T- cell receptor
  • the transmembrane domain can be from any type I transmembrane protein such as CD4, CD28 or HVEM or a functional fragment or variant thereof that retains co-stimulatory activity.
  • the transmembrane domain is a CD8 transmembrane domain or a functional fragment or variant thereof.
  • the transmembrane domain is a CD8a transmembrane domain or a functional fragment or variant thereof.
  • the transmembrane domain is a CD28 transmembrane domain or a functional fragment or variant thereof.
  • the transmembrane domain is a HVEM transmembrane domain or a functional fragment or variant thereof.
  • the transmembrane domain is a CD8a transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 16.
  • a functional fragment or variant of a CD8a transmembrane domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 16.
  • a functional fragment of a CD8a transmembrane domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 16.
  • the transmembrane domain is a CD8a transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 62.
  • a functional fragment or variant of a CD8a transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 62.
  • the CD8a transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID 31 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) NO: 16, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 62.
  • the functional fragment or variant of a CD8a transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 16, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 62, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 62.
  • the transmembrane domain is a HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 12.
  • a functional fragment or variant of a HVEM transmembrane domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12.
  • a functional fragment of a HVEM transmembrane domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 12.
  • the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 60.
  • a functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 60.
  • the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 12 is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 60.
  • the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 60, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 60.
  • the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 106.
  • a functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 106.
  • the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 12 is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 106.
  • the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 106, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 106.
  • the transmembrane domain is a HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 21.
  • a functional fragment or variant of a HVEM transmembrane domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 21.
  • a functional fragment of a HVEM transmembrane domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 21.
  • the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 61.
  • a functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 61.
  • the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 21, is encoded by a polynucleotide 33 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) sequence comprising a nucleotide sequence set forth as SEQ ID NO: 61.
  • the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 21, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 61, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 61.
  • the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 104.
  • a functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 104.
  • the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 21, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 104.
  • the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 21, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 104, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 104.
  • the transmembrane domain may be a hydrophobic alpha helix that spans across the membrane of the cell (e.g., macrophage).
  • the transmembrane domain can be naturally associated with one or more of the domains in the CAR.
  • the transmembrane domain may be synthetic, in which case it can comprise predominantly hydrophobic residues such as leucine and valine. A triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane domain.
  • Intracellular Domain A CAR expressed by a modified phagocytic cell of the disclosure can comprise an intracellular domain.
  • the intracellular (i.e., cytoplasmic) domain of a CAR is a signaling domain that transduces the event of receptor antigen binding to an intracellular signal that contributes to activation and/or transduction of signals in a cell in which the CAR is expressed. In some embodiments, absent appropriate co-stimulatory signals, this event is insufficient for useful cell activation and proliferation.
  • Cell activation can include cytokine production, clonal proliferation, differentiation, and survival.
  • an intracellular domain for use in the disclosure include the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in a phagocytic cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.
  • an intracellular domain examples include a fragment or domain from one or more molecules or receptors including: T cell receptor (TCR), CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon R1b), )F ⁇ 5 ⁇ )F ⁇ (SVLORQ ⁇ 5 ⁇ gamma), CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD
  • the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof. In some embodiments, the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof having an amino acid sequence set forth as SEQ ID NO: 9 or 10.
  • a functional fragment or variant of a CD3 zeta intracellular signaling domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 35 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 9 or 10.
  • a functional fragment or variant of a CD3 zeta intracellular signaling domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 9 or 10.
  • the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 11.
  • the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 11, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11.
  • CD3 zeta signaling domains are described in U.S. Patent No.7,446,190 and U.S. Patent No.8,911,993.
  • the intracellular signaling domain includes a )F ⁇ 5 ⁇ signaling domain or a functional variant thereof. In some embodiments, the intracellular signaling domain includes a )F ⁇ 5 ⁇ signaling domain or a functional variant thereof having an amino acid sequence set forth as SEQ ID NO: 14.
  • a functional fragment or variant of a )F ⁇ 5 ⁇ signaling domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 14.
  • a functional fragment or variant of a )F ⁇ 5 ⁇ VLJQDOLQJ ⁇ domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 14.
  • the intracellular signaling domain includes a )F ⁇ 5 ⁇ VLJQDOLQJ ⁇ domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 59.
  • the intracellular signaling domain includes a )F ⁇ 5 ⁇ VLJQDOLQJ ⁇ domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 59, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 59.
  • a "co-stimulatory molecule” refers to a molecule on an immune cell that is used to heighten or dampen the initial stimulus.
  • the intracellular signaling domain of the CAR includes at least 1, at least 2, at least 3, at least 4, or at least 5 immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • any intracellular signaling domain including an ITAM can be suitably used for the construction of CAR.
  • An “ITAM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells.
  • the motif may include two repeats of the amino acid sequence YXXL/I separated by 6-8 amino acids, wherein each X is independently any amino acid, producing the conserved motif YXXL/IX (6-8) YXXL/I (SEQ ID NO: 3).
  • ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways.
  • the intracellular signaling domain includes at least 1, at least 2, at least 3, at least 4, or at least 5 ITAMs derived from CD3z ⁇ )F5 ⁇ 0HJI ⁇ )F5 ⁇ DQG ⁇ FRPELQDWLRQV ⁇ WKHUHRI ⁇
  • the intracellular domain of a CAR useful in a modified phagocytic cell of the disclosure includes any portion of one or more co-stimulatory molecules. Co- stimulation is a key event for T cells to exhibit effective effector functions, and is mediated by co- stimulatory molecules. In some embodiments, the co-stimulatory domain of a CAR is essential for promoting the intracellular signal of the T-cell receptor domain to initiate T cell activation and proliferation.
  • Second generation CARs incorporate an intracellular co-stimulatory signaling domain in addition to an intracellular signaling domain (e.g., CD3z, or )F ⁇ 5 ⁇ ) and can enhance CAR T cell activation.
  • Co-stimulatory molecules are divided into two major families; the CD28 family which includes CD28 and ICOS, and the tumor necrosis factor receptor superfamily (TNFRSF) which includes 4-1BB (TNFRSF9), CD27, CD30, DR3, GITR, OX40, TNFR2 and herpes virus entry mediator (HVEM, TNFRSF14).
  • 4- 1BB-mediated co-stimulation averts T cell exhaustion induced by tonic signaling (Long et al, Nat. 37 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) Med.21(6):581 (2015)). Therefore, the co-stimulatory signaling domain derived from the TNFRSF appears to function better than the one from the CD28 family in the context of second generation CAR.
  • Third generation CARs incorporate two co-stimulatory domains and can promote CAR-T cell proliferation, reduce CAR-T cell apoptosis, and/or increase the the NF- ⁇ B pathway (Dai et al. (2020) Frontiers in Immunology 11:539654).
  • HVEM another member of the TNFRSF, is the main receptor targeted by the Herpes simplex virus to enter host cells (Sedy et al. (2005) Nature Immunology 6(1):90-98). It was previously described as ATAR (Another TRAF-Associated Receptor) (Hsu et al. (1997) Journal of Biological Chemistry 272(21):13471-13474) and is also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14).
  • ATAR Another TRAF-Associated Receptor
  • TNFRSF14 tumor necrosis factor receptor superfamily member 14
  • a human HVEM is 283 amino acids in length (UniProt ID Q92956-1; SEQ ID NO: 4), comprising: an extracellular region that includes amino acid residues 39-202; a transmembrane region that includes amino acid residues 203-223; and a cytoplasmic region that includes amino acid residues 224-283.
  • HVEM plays a role in effector CD8 + T cell effector function and memory T cell development. HVEM deficiency in CD8 + T cells is shown to profoundly impair effector CD8 + T cell survival and development of protective immune memory (Flynn et al, PLoS One 8(10):e77991 (2013)).
  • HVEM T lymphocyte attenuator
  • HVEM is constitutively expressed in na ⁇ ve T cells.
  • the HVEM receptor has both stimulatory and inhibitory effects depending on its ligand, and at least five different ligands have been described (Cai & Freeman (2009) Immunological Reviews 229(1):244-258; Del Rio et al. (2010) Journal of Leukocyte Biology 87(2):223-235; Granger & Rickert (2003) Cytokine Growth Factor Rev 14(3-4):289-296; Pasero & Olive (2013) Immunology Letters 151(1-2):71-75; Sorobetea & Brodsky (2016) Cell Host & Microbe 24(2):187-188).
  • HVEM binding to the /,*+7 ⁇ OLJDQG ⁇ RI ⁇ WKH ⁇ 71) ⁇ IDPLO ⁇ DQG ⁇ O ⁇ PSKRWR[LQ ⁇ DOSKD ⁇ /7 ⁇ produces a co-stimulatory signal, leading to increased T cell proliferation in vitro and formation of effector and memory T cells (Del Rio et al. (2010) Journal of Leukocyte Biology 87(2):223-235).
  • HVEM can generate an inhibitory signal when bound to BTLA or CD160 (Pasero & Olive (2013) Immunology Letters 38 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) 151(1-2):71-75).
  • BTLA/HVEM overexpression of BTLA/HVEM on T cells can contribute to T cell exhaustion (Ning et al. (2021) Frontiers in Immunology 12:654960; Shui et al. (2011) Journal of Leukocyte Biology, 89(4), 517-523).
  • the cytoplasmic domain of HVEM is 60 amino acids in length and includes an ⁇ -helix, a TRAF domain, and a tail (amino acid residues 224-283 of SEQ ID NO: 4; Hennecke, ever.2022.
  • TRAFs (TRAF5 and TRAF2) (Hsu et al. (1997) Journal of Biological Chemistry 272(21):13471-13474), which are important signaling molecules downstream of TNF receptors, connecting receptor signaling with kinase (e.g., , ⁇ % ⁇ NLQDVH ⁇ activation, which leads to transcription factor (e.g., AP-1, NFAT, NF- ⁇ % ⁇ activation (Zarnegar et al. (2008) Nature Immunology 9(12):1371-1378). Trancription factor activation is important, for example, in T cell priming and control of tumors in vivo (Barnes et al.
  • the term M83 refers to a co-stimulatory signaling domain from an HVEM protein.
  • the co-stimulatory signaling domain from an HVEM protein can include all or part of the transmembrane domain along with all or part of the intracellular region, or can include only all or part of the intracellular region of the HVEM protein.
  • a CAR comprising an intracellular co-stimulatory signaling domain from an HVEM protein, or a functional fragment or variant thereof that retains co-stimulatory activity can promote greater phagocytosis as compared to a CAR comprising a different co-stimulatory signaling domain (e.g., 4-1BB) or as compared to a CAR lacking a co-stimulatory signaling domain.
  • a CAR comprising a different co-stimulatory signaling domain e.g., 4-1BB
  • the ‘VEET’ domain of the HVEM protein (corresponding with amino acid positions 269- 272 of SEQ ID NO: 4) is thought to be important for co-stimulatory activity of the HVEM protein (see Ye et al., “The Structural Basis for the Recognition of Diverse Receptor Sequences by TRAF2, Molecular Cell, Vol.4, p.321-330; and Hsu et al, “ATAR, A Novel Tumor Necrosis Factor Receptor Family Member, Signals through TRAF2 and TRAF5, The Journal of Biological Chemistry, Vol.272, No.21, 1997, p.13471-13474, the contents of which are herein incorporated by reference in their entirety).
  • VEET domain within the HVEM co-stimulatory signaling domains disclosed herein is important, if not required, to promote co-stimulatory activity.
  • An HVEM protein intracellular co-stimulatory signaling domain, or a functional fragment or variant thereof is thought to retain its co-stimulatory activity so long as it functions to enhance or heighten the response of a phagocytic cell having a 39 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) CAR comprising said co-stimulatory domain, or functional fragment or variant thereof, compared to a CAR lacking an intracellular co-stimulatory signaling domain from an HVEM protein.
  • the CARs disclosed herein can comprise a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, wherein the encoded domain retains co-stimulatory activity.
  • the CAR comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, can comprise at least one residue corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least one residue corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof can comprise at least one residue selected from the following residues corresponding with SEQ ID NO: 4: V269, E270, E271, or T272.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, can comprise at least two residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof can comprise at least two residues selected from the following residues corresponding with SEQ ID NO: 4: V269, E270, E271, or T272.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • 40 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least three residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270,
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof can comprise at least three residues selected from the following residues corresponding with SEQ ID NO: 4: V269, E270, E271, or T272.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof comprises the following residues corresponding to SEQ ID NO: 4: V269, E270, E271, or T272.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof comprises a V269, corresponding with residue 269 of SEQ ID NO: 4.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof comprises a E270, corresponding with residue 270 of SEQ ID NO: 4
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 41 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof comprises a E271, corresponding with residue 271 of SEQ ID NO: 4.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof comprises a E272, corresponding with residue 272 of SEQ ID NO: 4.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least one, at least two, at least three, or at least four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain comprises a V269, corresponding with residue 269 of SEQ ID NO: 4; a E270, corresponding with residue 270 of SEQ ID NO: 4; a E271, corresponding with residue 271 of SEQ ID NO: 4; and a E272, corresponding with residue 272 of SEQ ID NO: 4.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, said HVEM co-stimulatory 42 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain comprises at least of one of the following: a V269, corresponding with residue 269 of SEQ ID NO: 4; a E270, corresponding with residue 270 of SEQ ID NO: 4; a E271, corresponding with residue 271 of SEQ ID NO: 4; and a E272, corresponding with residue 272 of SEQ ID NO: 4.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, 1, or 2, said HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain comprises at least of one of the following: a V269, corresponding with residue 269 of SEQ ID NO: 4; a E270, corresponding with residue 270 of SEQ ID NO: 4; a E271, corresponding with residue 271 of SEQ ID NO: 4; and a E272, corresponding with residue 272 of SEQ ID NO: 4.
  • the CAR comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 7, 1, or 2, said HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4 (i.e., V269, E270, E271, T272).
  • the HVEM co-stimulatory signaling domain comprises a V269, corresponding with residue 269 of SEQ ID NO: 4; a E270, corresponding with residue 270 of SEQ ID NO: 4; a E271, corresponding with residue 271 of SEQ ID NO: 4; and a E272, corresponding with residue 272 of SEQ ID NO: 4.
  • the CAR comprising a co-stimulatory domain having at least 90% sequence identity to an HVEM co-stimulatory protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity will have a domain comprising amino acid residues VEET corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 4.
  • the HVEM co-stimulatory protein of the present disclosure comprises a valine, or functional variant thereof, at an amino acid residue corresponding with position 269 of SEQ ID NO: 4.
  • the HVEM co- 43 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) stimulatory protein of the present disclosure comprises a glutamic acid, or functional variant thereof, at an amino acid residue corresponding with position 270 of SEQ ID NO: 4.
  • the HVEM co-stimulatory protein of the present disclosure comprises a glutamic acid, or functional variant thereof, at an amino acid residue corresponding with position 271 of SEQ ID NO: 4.
  • the HVEM co-stimulatory protein of the present disclosure comprises a threonine, or functional variant thereof, at an amino acid residue corresponding with position 272 of SEQ ID NO: 4.
  • a nucleic acid sequence disclosed herein can encode a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure, can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 7, wherein the encoded domain retains co-stimulatory activity.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has at least 90% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 7, wherein the encoded domain retains co-stimulatory activity. In some embodiments, a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure, has at least 95% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 7, wherein the encoded domain retains co-stimulatory activity.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain of the disclosure has 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 7.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 8.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 8, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 8.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 102.
  • the HVEM co-stimulatory signaling domain is encoded by the nucleotide sequence set forth as SEQ ID NO: 102, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 102.
  • the HVEM co- stimulatory signaling domain is encoded by the 44 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) nucleotide sequence set forth as SEQ ID NO: 103.
  • the HVEM co- stimulatory signaling domain is encoded by the nucleotide sequence set forth as SEQ ID NO: 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 103.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure can have an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity. In some embodiments, a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure, has an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain of the disclosure has an amino acid sequence set forth as SEQ ID NO: 7.
  • a HVEM co-stimulatory signaling domain can include amino acid residues 269-272 of SEQ ID NO: 4.
  • a HVEM co-stimulatory signaling domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 can include amino acid residues 269-272 of SEQ ID NO: 4.
  • a HVEM co-stimulatory signaling domain having at least 80% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 can include amino acid residues 269-272 of SEQ ID NO: 4.
  • a HVEM co-stimulatory signaling domain having at least 85% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 can include amino acid residues 269-272 of SEQ ID NO: 4.
  • a HVEM co-stimulatory signaling domain having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 can include amino acid residues 269-272 of SEQ ID NO: 4.
  • a HVEM co-stimulatory signaling domain having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 can include amino acid residues 269-272 of SEQ ID NO: 4.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure can have an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain of the disclosure has an amino acid sequence set forth as SEQ ID NO: 7.
  • a HVEM co-stimulatory signaling domain can include amino acid residues 224-283 of SEQ ID NO: 4.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 1, wherein the encoded domain retains co-stimulatory activity.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has at least 90% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 1, wherein the encoded domain retains co-stimulatory activity. In some embodiments, a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure, has at least 95% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 1, wherein the encoded domain retains co-stimulatory activity.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain 46 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) of the disclosure has 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 1.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 54.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 54, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 54.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 56.
  • the HVEM co-stimulatory signaling domain is encoded by the nucleotide sequence set forth as SEQ ID NO: 56, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 56.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure can have an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain of the disclosure has an amino acid sequence set forth as SEQ ID NO: 1.
  • a HVEM co-stimulatory signaling domain can include amino acid residues 201-283 of SEQ ID NO: 4.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 2, wherein the encoded domain retains co-stimulatory activity.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has at least 90% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 47 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) 2, wherein the encoded domain retains co-stimulatory activity.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has at least 95% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 2, wherein the encoded domain retains co-stimulatory activity.
  • a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain of the disclosure has 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 2.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 55.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 55, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 55.
  • the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof is encoded by the nucleotide sequence set forth as SEQ ID NO: 57.
  • the HVEM co-stimulatory signaling domain is encoded by the nucleotide sequence set forth as SEQ ID NO: 57, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 57.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure can have an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure has an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2 and retains co-stimulatory activity.
  • a HVEM co-stimulatory signaling domain of the disclosure has an amino acid sequence set forth as SEQ ID NO: 2.
  • a HVEM co-stimulatory signaling domain can include amino acid residues 210-277 of SEQ ID NO: 4.
  • a HVEM co-stimulatory signaling domain having co-stimulatory activity can promote the following in a phagocytic cell: increased phagocytosis, increased cytokine secretion, increased 48 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) secretion of proinflammatory factors, increased M1 proinflammatory phenotype, increased M2 immunosuppressive phenotype, increased proliferation, and/or increased antigen presentation to T cells, as compared to a relevant control.
  • a HVEM co-stimulatory signaling domain having co-stimulatory activity can bind a TRAF1, a TRAF2, a TRAF3, and/or a TRAF5 intracellular signaling adapters.
  • a HVEM co-stimulatory signaling domain having co-stimulatory activity does not bind a TRAF6 intracellular signaling adapter.
  • the HVEM co-stimulatory signaling domain having co- stimulatory activity promotes phagocytosis and/or trogocytosis in phagocytic cells expressing a CAR comprising the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, as compared to phagocytic cells that do not express a CAR comprising the HVEM co-stimulatory signaling domain having co-stimulatory activity.
  • the HVEM co-stimulatory signaling domain having co- stimulatory activity increases the ability of the phagocytic cells expressing a CAR comprising the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, to limit target cell expansion, or proliferation, by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, as compared to phagocytic cells that do not express a CAR comprising the HVEM co-stimulatory signaling domain having co-stimulatory activity.
  • the phagocytic cells expressing a CAR comprising the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof limit expansion or proliferation of plurality of target cells within a population of cells, by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, as compared to phagocytic cells that do not express a CAR comprising the HVEM co-stimulatory signaling domain having co- stimulatory activity.
  • the HVEM co-stimulatory signaling domain having co-stimulatory activity can increase pro-inflammatory cytokine expression in phagocytic cells expressing a CAR comprising the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, by 2-fold to 100-fold, or by 2-fold to 50-fold, or by 5-fold to 20-fold, or by 1.1-fold to 20-fold, or by at least 2- fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5- fold, at least 5.5-fold, at least 6-fold, at least 6.5 fold, at least 7-fold, at least 7.5 fold, at least 8- fold, at least 8.5 fold, at least 9-fold, at least 9.5 fold, at least 10-fold, at least 10.5 fold, at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17- 49 62059467v
  • the HVEM co-stimulatory signaling domain having co-stimulatory activity can increase NF- ⁇ % activation in phagocytic cells expressing a CAR comprising the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, by 2-fold to 100-fold, or by 2-fold to 50-fold, or by 5-fold to 20-fold, or by 1.1-fold to 20-fold, or by at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5 fold, at least 7-fold, at least 7.5 fold, at least 8-fold, at least 8.5 fold, at least 9-fold, at least 9.5 fold, at least 10-fold, at least 10.5 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold
  • a functional fragment or variant of a HVEM co-stimulatory signaling domain having co- stimulatory activity can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the full- length amino acid sequence set forth as any one of SEQ ID NOs: 7, 1, or 2.
  • a functional fragment of a HVEM co-stimulatory signaling domain having co-stimulatory activity can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the full-length amino acid sequence set forth as any one of SEQ ID NOs: 7, 1, or 2.
  • the co-stimulatory signaling domain further comprises one or more additional co-stimulatory signaling domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), or functional fragments or variants thereof.
  • additional co-stimulatory signaling domains e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.
  • Non-limiting examples include a CD28 co-stimulatory signaling domain, a 4-1BB co-stimulatory signaling domain, an OX-40 co-stimulatory signaling domain, an ICOS co-stimulatory signaling domain, or any other co-stimulatory signaling domain and/or functional fragment or variant thereof now known or later identified.
  • a linker may be present between two or more of the domains, e.g., a 3-12 amino acid linker, or a 5-8 amino acid linker.
  • An intracellular signaling domain of the present disclosure can include a CD3 zeta intracellular signaling domain and an HVEM co-stimulatory signaling domain as described herein.
  • the term "spacer” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the antigen binding domain or, the intracellular domain in the CAR polypeptide.
  • the spacer domain may comprise up to 300 amino acids, 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, the spacer is a short spacer which comprises less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 amino acids.
  • the spacer may include a hinge region. In some embodiments, the hinge region may be located between the antigen binding domain and the transmembrane domain of the CAR.
  • a hinge region can include at least a portion of a Fc region, for example, a hinge portion of a human Fc region of a CH3 domain or variants thereof.
  • the spacer includes all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e. the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., IgG4 Fc hinge or a CD8 hinge region.
  • an immunoglobulin e.g., IgG1, IgG2, IgG3, IgG4
  • IgG4 Fc hinge a CD8 hinge region.
  • Examples include CD8 hinge, CD28 hinge, IgG4 (HL-CH3), or IgG4 (L235E, N297Q).
  • cysteines in the hinge region may be replaced with serines.
  • Other examples of hinge regions are well known in the art.
  • the hinge region comprises a CD8 hinge region, or a fragment or functional variant of a CD8a hinge region.
  • the hinge region comprises a CD8a hinge comprising an amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the hinge region comprises a HVEM hinge region, or fragment or functional variant of a HVEM hinge region. In some embodiments, the hinge region comprises a HVEM hinge comprising an amino acid sequence set forth as SEQ ID NO: 13.
  • a hinge region or a fragment or functional variant of a hinge region can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 13 or 15.
  • a hinge region can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 13 or 15.
  • the hinge region or functional fragment or variant thereof is encoding by the nucleotide sequence set forth as SEQ ID NO: 63, 64, or 105.
  • the hinge region or functional fragment or variant thereof is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 63, 64, or 105. 51 62059467v1 Attorney Docket No.: I1158141020WO (0012.2)
  • a short oligo- or polypeptide linker between 2 and 10 amino acids in length, may connect two different domains of a CAR.
  • the linker may be located between the antigen binding domain and the transmembrane domain of the CAR, or between the transmembrane domain and the intracellular domain of the CAR.
  • the linker may be a Gly linker, a Gly-Ser linker, a EAAAK (SEQ ID NO: 5) linker, a PAPAP (SEQ ID NO: 6) linker, or an (Ala- Pro) n linker.
  • the length and amino acid composition of the linker peptide sequence can be optimized to vary the orientation and/or proximity of the polypeptide domains to one another to achieve a desired activity of the chimeric polypeptide.
  • the orientation and/or proximity of the polypeptide domains to one another can be varied as a “tuning” tool to achieve a tuning effect that would enhance or reduce the biological activity of the chimeric polypeptide.
  • the linker is a synthetic compound linker such as, for example, a chemical cross-linking agent.
  • Non-limiting examples of suitable cross-linking agents include N-hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidyl)uccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2 (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]
  • a CAR useful for expression in modified phagocytic cells of the disclosure can further comprise a detectable moiety as would be known in the art and/or an effector molecule, nonlimiting examples of which include a drug, a toxin, a small molecule, an antibody, and/or an antibody fragment, singly or in any combination.
  • a CAR may be glycosylated, pegylated, and/or otherwise post- translationally modified. Glycosylation, pegylation, and/or other post-translational modifications may occur in vivo or in vitro and/or may be performed using chemical techniques.
  • any glycosylation, pegylation and/or other post-translational modifications may be N-linked or O-linked.
  • Phagocytic cells genetically modified to express CARs The present disclosure provides a phagocytic cell genetically modified to express a CAR.
  • the phagocytic cell comprising a CAR recognizes and binds to an antigen present on the surface of a target cell, such as a cancer cell and/or viral particle.
  • the CAR includes a co-stimulatory 52 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) domain having at least 90% sequence identity to an HVEM co-stimulatory protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co- stimulatory activity.
  • A. Phagocytic cells Phagocytic cells are used in the compositions and methods described herein.
  • the terms “phagocytic cell” or “phagocyte” or plural forms thereof as used herein indicate a cell that is capable of phagocytosis, which is the process by which a cell uses its plasma membrane to engulf a large particle ( ⁇ P), giving rise to an internal compartment called the phagosome.
  • Phagocytosis is one type of endocytosis as will be understood by a skilled person. Phagocytes of an individual typically use their plasma membrane to engulf and remove cellular debris, foreign substances, microbes, and cells to protect the body of an individual. Phagocytes in the sense of the disclosure can typically also perform trogocytosis, a process whereby one cell contacts and quickly nibbles another cell. For example, trogocytosis occurs when lymphocytes (e.g., B, T, NK cells) conjugated to antigen-presenting cells (APCs) extract surface molecules from the APCs and express them on their own surface.
  • lymphocytes e.g., B, T, NK cells
  • APCs antigen-presenting cells
  • Exemplary phagocytic cells herein described include macrophages, monocytes, neutrophils, dendritic cells and precursors thereof as a person skilled in the art would understand, though singled celled organisms such as Dictyostelium amoebae are also phagocytes.
  • Phagocytic cells of the disclosure also include tissue-resident macrophages that not only sense and respond to invading pathogens but are important in tissue development, remodeling, and homeostasis. Tissue-resident macrophages originate from embryonic precursors and can self-renew.
  • tissue-resident macrophages can include Kupffer macrophages (forms the lining of sinusoids of the liver), alveolar macrophages (lung), microglia macrophages (brain), red pulp macrophages (spleen), and macrophages in the heart.
  • tissue-resident macrophages enter tissue during non-inflammatory conditions.
  • a monocyte is a type of white blood cell of the immune system that is capable of phagocytosis.
  • Macrophages are derived from blood monocytes that migrate into tissue. One of macrophages’ main functions is to phagocytose microbes and clear cellular debris. Macrophages also play an important role in both the initiation and resolution of inflammation.
  • Macrophages comprise cells typically diffusely scattered in the connective tissue and in liver (Kupffer cells), spleen and lymph nodes (sinus histiocytes), lungs (alveolar macrophages), and central nervous system (microglia) as will be understood by a skilled person. Macrophages can also display different responses, ranging from pro-inflammatory to anti- inflammatory, depending on the type of stimuli they receive from the surrounding microenvironment.
  • M1 and M2 are two major macrophage phenotypes that have been proposed to 53 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) correlate with extreme macrophage responses.
  • M1 pro-inflammatory macrophages are activated upon contact with certain molecules such as lipopolysaccharide (LPS), interferon-gamma (IFN- ⁇ ), interleukin (IL)- ⁇ tumor necrosis factor (TNF)- ⁇ DQG ⁇ 7ROO-like receptor engagement.
  • LPS lipopolysaccharide
  • IFN- ⁇ interferon-gamma
  • IL interleukin
  • TNF tumor necrosis factor- ⁇ DQG ⁇ 7ROO-like receptor engagement
  • M1 macrophages constitute a potent arm of the immune system deployed to fight infections. They are capable of either direct (pathogen pattern recognition receptors) or indirect (Fc receptors, complement receptors) recognition of the pathogen. They are also armed in their ability to produce reactive oxygen species (ROS) as means to help kill pathogens.
  • ROS reactive oxygen species
  • M1 macrophages secrete pro-inflammatory cytokines and chemokines attracting other types of immune cells and integrating/orchestrating the immune response. M1 activation is induced by IFN- ⁇ , TNF ⁇ , granulocyte macrophage colony-stimulating factor (GM-CSF), LPS, and other toll-like receptors (TLR) ligands.
  • M2 anti-inflammatory macrophages also known as alternatively activated macrophages, are activated by anti-inflammatory molecules such as IL-4, IL-13, and IL-10. M2 macrophages exhibit immunomodulatory, tissue repair, and angiogenesis properties which allow them to recruit regulatory T cells to sites of inflammation.
  • M2 macrophages do not constitute a uniform population and often are further subdivided into M2a, M2b and M2c categories.
  • the common denominator of all three subpopulations is high IL-10 production accompanied by low production of IL-12.
  • One of their signatures is production of enzyme arginase-1 that depletes L- arginine, thereby suppressing T cell responses and depriving iNOS of its substrate.
  • arginase-1 enzyme that depletes L- arginine, thereby suppressing T cell responses and depriving iNOS of its substrate.
  • the in vivo molecular mechanisms of macrophage polarization to M1 or M2 phenotype are poorly characterized because of the variety of signals macrophages experience in the cellular microenvironment.
  • IFN- ⁇ DQG ⁇ ,/-4 are two cytokines that can polarize macrophages to M1 and M2 phenotypes, respectively.
  • a monocyte is a type of leukocyte or white blood cell capable of phagocytosis and can differentiate into macrophages and myeloid lineage dendritic cells.
  • monocytes As a part of the vertebrate innate immune system, monocytes also influence the process of adaptive immunity. There are at least three subclasses of monocytes in human blood based on their phenotypic receptors including CD14++ CD16 ⁇ monocyte, CD14 + CD16 ++ monocyte and CD14 ++ CD16 + monocytes, as will be understood by a person skilled in the art. Monocytes serve as precursors for various tissue 54 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) macrophage and dendritic cell populations and contribute to both protective and pathological immune responses.
  • Dendritic cells are specialized antigen-presenting cells capable of phagocytosis that have long outgrowths called dendrites that help to engulf microbes and other invaders. Dendritic cells are present in the tissues that are in contact with the external environment, mainly the skin, the inner lining of the nose, the lungs, the stomach, and the intestines. Once activated, they mature and migrate to the lymphoid tissues where they interact with T cells and B cells to initiate and orchestrate the adaptive immune response. Mature dendritic cells activate T helper cells and cytotoxic T cells. The activated helper T cells interact with macrophages and B cells to activate them in turn.
  • dendritic cells can influence the type of immune response produced; when they travel to the lymphoid areas where T cells are held, they can activate T cells, which then differentiate into cytotoxic T cells or helper T cells.
  • Neutrophils are phagocytes that form the most abundant type of granulocytes and the most abundant type of white blood cells in most mammals as known to a person skilled in the art. Neutrophils are formed from stem cells in the bone marrow and differentiated into subpopulations of neutrophil-killers and neutrophil-cagers.
  • a phagocytic cell includes a precursor cell that can develop into and/or be differentiated into a phagocyte.
  • precursors when used in connection with macrophages, monocytes, dendritic cells, and/or neutrophils indicate parent cells in a cellular lineage resulting in phagocytic cells herein described.
  • exemplary precursor cells include bone marrow-derived cells, stem cells, and other precursor cells identifiable by a person skilled in the art.
  • precursor cells can be differentiated by culturing the precursor cells under conditions that promote development of phagocytic cells.
  • cytokines that are typically used for differentiation of bone-marrow derived monocytes or hematopoietic stem cells into macrophages include granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • M-CSF macrophage colony-stimulating factor
  • Macrophage differentiation protocols are described in, e.g., van Furth et al. (1972) Bulletin of the World Health Organization, 46(6), 845- 852; Mosser and Edwards (2008) Nature Reviews Immunology, 8(12), 958-969; and Lutz et al. (1999) Journal of Immunological Methods, 223(1), 77-92.
  • the precursor cells can comprise a bone marrow-derived cell or a stem cell.
  • the bone-marrow derived cell is a stem cell.
  • the stem cells of the present disclosure can be from any subject, such as an animal. In some instances, the animal can be a mammal, such as a human.
  • One type of stem cell that can be used is a hematopoietic stem cell (HSC).
  • HSCs hematopoietic stem cells
  • HSCs are multipotent precursors that have a unique ability to self- 55 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) renew. HSCs produce hematopoietic progenitor cells that differentiate into every type of mature blood cell within a well-defined hierarchy (Bonnet, 2002; McCulloch and Till, 2005).
  • HSCs can be found in various tissue, including bone marrow, mobilized peripheral blood, peripheral blood, and umbilical cord blood. HSCs and other blood cell progenitors can be isolated from fetal and embryonic tissues. Specifically, umbilical cord blood (UCB) and placenta are rich sources of HSCs (Abdulrazzak, Hassan et al. “Biological characteristics of stem cells from foetal, cord blood and extraembryonic tissues.” Journal of the Royal Society, Interface vol.7 Suppl 6, Suppl 6 (2010): S689-706). Somatic cells can also be a source of HSCs, by conversion of these cells into induced pluripotent stem cells (iPSCs) (see Vo, Linda T, and George Q Daley.
  • iPSCs induced pluripotent stem cells
  • induced pluripotent stem cells can be generated by any technique known to the skilled artisan.
  • stems cells used in the present disclosure can include hematopoietic stem cells or induced pluripotent stem cells, or a precursor, or derivative, thereof.
  • myeloid cells include, but are not limited to, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets.
  • lymphoid cells include T cells, B cells, natural killer cells, and innate lymphoid cells.
  • CD34 is a glycoprotein predominantly regarded as a marker of hematopoietic stem cells (HSC) and hematopoietic progenitor cells. (Civin et al., 1996b; Shizuru et al., 2005; Shpall et al., 1994).
  • CD34 is expressed on ⁇ 0.2–3% of the nucleated cells in cord blood, bone marrow and mobilized peripheral blood (Civin et al., 1984; Krause et al., 1996; Sutherland et al., 1996). CD34 has commonly been used to identify and select for populations of HSCs. For example, in clinical practice, CD34 expression is evaluated to ensure rapid engraftment in BM transplants; it can also be used as a selective marker in cell sorting to enrich a population of immature hematopoietic cells (Berardi, A C et al.
  • phagocytic cells such as macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof is obtained from a subject.
  • the cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen 56 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) tissue, umbilical cord, and tumors.
  • I1158141020WO 0012.2 tissue
  • the cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation.
  • Cells can be isolated from peripheral blood by lysing the red blood cells and depleting the lymphocytes and red blood cells, for example, by centrifugation through a PERCOLLTM gradient. Alternatively, cells can be isolated from umbilical cord. Cells from the circulating blood of an individual can be obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or a wash solution that lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps.
  • PBS phosphate buffered saline
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • a specific subpopulation of the monocytes, macrophages and/or dendritic cells can be further isolated by positive or negative selection techniques.
  • the isolated mononuclear cells can be depleted of cells expressing certain antigens, including CD34, CD3, CD4, CD8, CD14, CD19, or CD20. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites fluid, an antibody bound to a physical support, and a cell bound antibody. Enrichment of a macrophage, monocyte, dendritic cell, neutrophil, and/or precursor cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • enrichment of a cell population for macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof by negative selection can be accomplished using a monoclonal antibody cocktail that typically includes antibodies to CD34, CD3, CD4, CD8, CD14, CD19 or CD20.
  • concentration of cells and surface e.g., particles such as beads
  • a concentration of 2 billion cells/ml, 1 billion cells/ml, or greater than 100 million cells/ml can be used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 million cells/ml can be used.
  • concentrations of 125 to 150 million cells/ml can be used.
  • the use of high concentrations of cells can result in increased cell yield, cell activation, and cell expansion.
  • a population of cells of the present disclosure can include macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof.
  • Examples of a population of cells include: peripheral blood mononuclear cells; cord blood cells; a purified population of macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof; and a cell line.
  • peripheral blood mononuclear cells comprise the population of macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof.
  • purified cells comprise the population of macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof.
  • the phagocytic cells have upregulated M1 markers and downregulated M2 markers.
  • At least one M1 marker such as HLA DR, CD86, CD80, and PDL1 is upregulated in the phagocytic cell.
  • at least one M2 marker such as CD206 or CD163, is downregulated in the phagocytic cell.
  • the phagocytic cell has at least one upregulated M1 marker and at least one downregulated M2 marker.
  • targeted effector activity in the phagocytic cell is enhanced by inhibition of either CD47 or SIRPa activity.
  • CD47 is expressed on the surface to engage the SIRPa receptor on macrophages to suppress engulfment of healthy cells.
  • a cell expressing CD47 refers to a cell that has a functional CD47 signaling pathway, a pathway by which a cell (e.g., tumor cell) can evade the immune system by inhibiting or decreasing phagocytosis and/or trogocytosis function of a phagocytic cell.
  • CD47 and/or SIRPa activity may be inhibited by treating the phagocytic cell with an anti-SIRPa antibody and/or treating a target cell with an anti-CD47 antibody.
  • CD47 or SIRPa activity may be inhibited by any method known to those skilled in the art.
  • the cells or population of cells comprising macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof can be cultured for expansion prior to and/or after genetic modification to express a CAR.
  • the cells or population of cells comprising 58 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) precursor cells are cultured for differentiation and expansion of macrophages, monocytes, dendritic cells, and/or neutrophils.
  • the present disclosure comprises expanding a population of macrophages, monocytes, dendritic cells, neutrophils, and/or precursors thereof comprising a CAR as described herein.
  • Expansion of phagocytic cells can increase the amount of cells by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater.
  • the cells are expanded in the range of about 20 fold to about 50 fold.
  • the cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus.
  • the culturing apparatus can be any culture apparatus commonly used for culturing cells in vitro.
  • the level of confluence is 70% or greater before passing the cells to another culture apparatus.
  • the level of confluence is 90% or greater.
  • a period of time can be any time suitable for the culture of cells in vitro.
  • the culture medium may be replaced during the culture of the cells at any time. In some embodiments, the culture medium is replaced about every 2 to 3 days.
  • the cells are then harvested from the culture apparatus whereupon the cells can be used immediately or stored for use at a later time.
  • the culturing step can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
  • the culturing step can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.
  • the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between.
  • Conditions appropriate for cell culture include an appropriate media (e.g., macrophage complete medium, DMEM/F12, DMEM/F 12-10 (Invitrogen)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), L-glutamine, insulin, M-CSF, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and 71) ⁇ , or any other additives for the growth of cells known to the skilled artisan).
  • Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N- acetyl-cysteine and 2-mercaptoethanol.
  • culture media can include RPMI 1640, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, X-Vivo 20, and Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of 59 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) cytokine(s) sufficient for the growth and expansion of the cells.
  • Antibiotics e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).
  • the medium used to culture the cells may include an agent that can activate the cells.
  • an agent that is known in the art to activate a macrophage, monocyte, dendritic cell, and/or a neutrophil is included in the culture medium.
  • a vector may be used to introduce a CAR described herein into a phagocytic cell (e.g., macrophage, monocyte, dendritic cell, neutrophil, and/or precursor thereof).
  • the present disclosure provides a vector comprising a nucleic acid sequence encoding a CAR as described herein.
  • the vector comprises a plasmid vector, viral vector, retrotransposon (e.g. piggyback, sleeping beauty), site directed insertion vector (e.g. CRISPR, Zn finger nucleases, TALEN), suicide expression vector, or other known vector in the art.
  • site directed insertion vector e.g. CRISPR, Zn finger nucleases, TALEN
  • suicide expression vector or other known vector in the art.
  • the expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vector is one generally capable of replication in a mammalian cell, and/or also capable of integration into the cellular genome of the mammal.
  • Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the nucleic acid can be cloned into any number of different types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • a vector is used as a gene delivery vehicle to transfer a gene into a cell.
  • Expression vectors can also include gene delivery nanomaterial such as polymeric nanoparticles or liposomes, and others identifiable by a person skilled in the art.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.
  • Vectors including those derived from retroviruses such as 60 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self- inactivating lentiviral vector as described in, e.g., Milone et al. (2009) Molecular therapy 17(8):1453-1464.
  • lentivirus vectors include the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen and the like.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of resulting in low immunogenicity in the subject into which they are introduced.
  • adeno-associated viral vectors can be used to deliver a nucleic acid (e.g., encoding a CAR of the disclosure) to a phagocytic cell.
  • AAVs are nonenveloped, single-stranded DNA viruses of the Dependoparvovirus genus of the Parvoviridae family. AAVs are innately nonpathogenic, poorly immunogenic, and broadly tropic, making them attractive gene delivery candidates.
  • AAV vectors have shown to stably transfect mammalian cells without integration into the target genome.
  • Exemplary suitable AAVs comprise AAVs of various serotypes that can be used as vectors for carrying genes.
  • AAV serotypes are identified based on their interacting glycan moieties that mediate the initial attachment of AAVs to the cell surface.
  • AAV serotypes examples include AAV serotype 1 (“AAV1”), AAV2, AAV3, AAV5, AAV6, AAV9 and other serotypes identifiable to a person skilled in the art such as AAV7, AAV8, AAV11, and AAV-DJ.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193). Additional regulatory regions, e.g., enhancers, regulate the frequency of transcriptional initiation.
  • promoters typically contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • regulatory sequence or “regulatory regions” as described herein indicate a segment of a nucleic acid molecule which is capable of increasing or decreasing transcription or translation of a gene within an organism either in vitro or in vivo.
  • nucleic acid molecules of the present disclosure includes phagocyte regulatory regions that control expression of a gene in a phagocyte.
  • a phagocyte promoter includes a nucleotide sequence that drives or regulates expression in phagocytes.
  • Promoters specific to the mononuclear phagocyte system including macrophages, neutrophils, dendritic cells, and osteoclasts will constitute phagocyte promoters.
  • Examples of such promoters include CSF-1 promoter, CD68, CD11c, DC-SIGN, DC-STAMP, langerin, human neutrophil elastase, and any synthetic promoter containing elements of the phagocyte system designed to achieve high level of expression in phagocytic cells.
  • a constitutive promoter includes an unregulated promoter that allows for continual transcription of its associated genes.
  • An example of a strong constitutive promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • This promoter is capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences may also be used, including a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an elongation factor-1a promoter, PGK1 promoter from a phosphoglycerate kinase gene, as well as human gene promoters including an actin promoter, a ubiquitin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • conditional or inducible promoters include a promoter with activity regulatable or controlled by endogenous transcription factors or exogenous inputs such as chemical compounds, thermal inducers, or optical induction.
  • inducible promoters include a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, a tetracycline promoter, and a Lac promoter.
  • the expression vector to be introduced into a phagocytic cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of phagocytic cells expressing a nucleic acid sequence of interest (e.g., a CAR described herein) from the population of phagocytic cells transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure.
  • Both selectable marker genes and reporter 62 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neomycin and the like. Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • an expression vector of the disclosure comprises a gene encoding a CAR as described herein and appropriate regulatory elements such as promoters, enhancers, and post-transcriptional and post-translational regulatory sequences that are compatible with the phagocytic cell expressing the gene encoding a CAR as would be understood by a skilled person.
  • the CAR expression vector is configured for genomic insertion allowing long-term overexpression of the CAR in a phagocytic cell (e.g., macrophage, monocyte, dendritic cell, neutrophil, and/or precursors thereof) .
  • the genomic insertion can be achieved by stable transfection.
  • lentiviral transduction is preferred over physical or chemical transfections or adenoviral transduction. In some embodiments, lentiviral transduction is expected to be used effectively in vivo for delivery of a gene encoding a CAR of the disclosure and will permit stable expression in dividing and non-dividing cells.
  • a heterologous nucleic acid comprising a polynucleotide sequence encoding a CAR of the disclosure can be introduced into the genome of a phagocytic cell using transposases or targeted nucleases (e.g., Zinc finger nucleases (ZFN), meganucleases, or transcription activator-like effector (TALE) nucleases (TALENs), clustered regularly-interspaced short palindromic repeats associated nucleases (CRISPR/Cas)).
  • ZFN Zinc finger nucleases
  • TALE transcription activator-like effector
  • TALENs transcription activator-like effector
  • CRISPR/Cas clustered regularly-interspaced short palindromic repeats associated nucleases
  • the system When utilized for genome editing, the system includes a CRISPR associated (Cas) protein (e.g., Cas9) capable of modify nucleic acids when complexed with a guide RNA, a guide RNA comprising a CRISPR RNA (crRNA) that binds and directs the Cas protein to the target nucleic acid, optionally 63 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) a trans-activating CRISPR RNA (tracrRNA, that hybridizes to the crRNA and forms an active complex with the Cas protein), and optionally a homology dependent repair (HDR) template that guides the cellular repair process, allowing insertion of a specific nucleic acid sequence.
  • Cas CRISPR associated protein
  • an HDR template comprising a polynucleotide sequence encoding a CAR of the disclosure is included.
  • a CRISPR/Cas system is typically transfected into a target cell (e.g., a phagocytic cell to be genetically modified) by means of one or more plasmids.
  • CRISPR/Cas systems are described in, e.g., US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014/018423, WO2014/093595, WO2014/093622, WO2014/093635, WO2014/093655, WO2014/093661, WO2014/093694, WO2014/093701, WO2014/093709, WO2014/093712, WO2014/093718, WO2014/145599, WO2014/204723, WO2014/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO2014/204729, WO2015/065964, WO2015/089351, WO2015
  • a ZFN is an artificial restriction enzyme which is generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain.
  • a zinc finger domain can be engineered to target specific DNA sequences which allows a zinc-finger nuclease to target desired sequences within genomes.
  • the DNA-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of base pairs.
  • the most common method to generate new zinc-finger domains is to combine smaller zinc-finger "modules" of known specificity.
  • the most common cleavage domain in ZFNs is the non-specific cleavage domain from the type II restriction endonuclease FokI.
  • a cell e.g., a phagocytic cell
  • ZFNs can be used to insert the CAR expression cassette into the cell genome.
  • the HR machinery searches for homology between the damaged chromosome and the DNA template, and then copies the sequence of the template between the two broken ends of the chromosome, whereby the DNA template is integrated at the target nucleic acid sequence.
  • ZFNs are described in, e.g., US 6,534,261; US 6,607,882; US 6,746,838; US 6,794,136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241,573; US 7,241,574; US 7,585,849; US 7,595,376; US 64 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) 6,903,185; US 6,479,626; US 2003/0232410 and US 2009/0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11
  • TALENs are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN system operates on almost the same principle as ZFNs. They are generated by combining a TALE DNA-binding domain with a DNA cleavage domain.
  • TALEs are composed of 33-34 amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome.
  • TALENs are described in, e.g., US 8,440,431; US 8,440,432; US 8,450,471; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al.
  • Meganucleases are rare-cutting endonucleases that generate double-strand breaks (DSB) in a nucleic acid and recognize sequences larger than 12 base pairs. In the wild, such endonucleases are essentially represented by homing endonucleases (Chevalier and Stoddard (2001) Nucleic Acids Research 29:3757-3774). Homing endonucleases are found in fungi, algae, eubacteria and archae, and are often encoded in mobile genetic elements. Their cleavage activities initiate the spreading of these mobile elements by homologous recombination.
  • HO and I-Scel have been used to induce homologous gene targeting in yeast, in cultured mammalian cells, and in plants. See, e.g., US 5,792,632; US 6,238,924; US 5,792,632; US 5,830,729; US 6,238,924; US 5,792,632; US 6,238,924.
  • the disclosure includes a method of modifying a phagocytic cell, the method comprising: introducing a CAR into the phagocytic cell, wherein the CAR comprises: an antigen binding 65 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co- stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co- stimulatory activity, wherein the modified phagocytic cell expresses the CAR and has targeted effector activity.
  • HVEM herpes virus entry mediator
  • introducing the CAR into the phagocytic cell comprises introducing a nucleic acid sequence comprising a polynucleotide sequence encoding the CAR.
  • the polynucleotide sequence comprises a nucleotide sequence set forth as 8, 54, 55, 56, 57, 102, or 103.
  • the polynucleotide sequence comprises a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 54, 55, 56, 57, 102, or 103.
  • introducing the nucleic acid molecule comprises transducing the phagocytic cell with a viral vector comprising the nucleic acid sequence encoding the CAR.
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • HVEM herpes virus entry mediator
  • the intracellular co-stimulatory signaling domain comprises an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7.
  • the intracellular co-stimulatory signaling domain comprises an amino acid sequence set for as SEQ ID NO: 7.
  • the disclosure includes a method of modifying a phagocytic cell, the method comprising: introducing a CAR into the phagocytic cell, wherein the CAR comprises: an antigen binding domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co- stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as SEQ ID NO: 7, or a functional fragment or variant thereof that retains co-stimulatory activity, wherein the modified phagocytic cell expresses the CAR and has targeted effector activity, wherein the polynucleotide sequence comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%,
  • the methods of modifying a phagocytic cell of the present disclosure can comprise introducing a nucleic acid sequence comprising a polynucleotide sequence encoding the CAR, wherein the polynucleotide sequence comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103.
  • the polynucleotide sequence can comprise a nucleotide sequence set for as any one of the SEQ ID NOs listed in Table 2 or Table 4.
  • the polynucleotide sequence can comprise a nucleotide sequence set for as any one of the SEQ ID NOs: 8, 11, 54-57, 59-65, 81, 83, 85-89, 92-99, 102-103, 104-106.
  • the methods of modifying a phagocytic cell of the present disclosure can comprise introducing a nucleic acid sequence comprising a polynucleotide sequence encoding the CAR, wherein the polynucleotide sequence comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, wherein the polynucleotide sequence comprises a nucleotide sequence set for as any one of SEQ ID NOs: 8, 11, 54-57, 59-65, 81, 83, 85-89, 92-99, 102-103, 104-106, or 109
  • the CAR further comprises a CD3 zeta intracellular signaling domain or a functional variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 9, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 9.
  • the CAR further comprises a CD3 zeta intracellular signaling domain or a functional variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 10, or an amino 67 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 10.
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding a CD3 zeta intracellular signaling domain or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 11, or a nucleotide sequence having at least 80%, 81%
  • the intracellular co-stimulatory signaling domain comprises an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7,
  • the CAR further comprises D ⁇ )F ⁇ 5 ⁇ VLJQDOLQJ ⁇ GRPDLQ ⁇ RU ⁇ D ⁇ IXQFWLRQDO ⁇ YDULDQW ⁇ WKHUHRI ⁇ FRPSULVLQJ ⁇ DQ ⁇ amino acid sequence set forth as SEQ ID NO: 14, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 14.
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as 8, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding an intracellular signaling domain comprising a FF ⁇ 5 ⁇ VLJQDOLQJ ⁇ GRPDLQ ⁇ RU ⁇ D ⁇ functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 59, or a nucleotide sequence having
  • the method of modifying a phagocytic cell comprising introducing a CAR into the phagocytic cell, wherein the intracellular co-stimulatory signaling domain comprises an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7, the CAR further comprises a CD8a transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 16.
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding a CD8a transmembrane domain or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 62, or a nucleotide sequence having at least 80%, 81%
  • the CAR further comprises a HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12.
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide 69 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) sequence set forth as SEQ ID NO: 8, 102, or 103, the polynucleotide sequence further comprises polynucleotide sequence encoding an HVEM protein transmembrane domain or a functional variant thereof comprising a nucleotide sequence set forth as
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding an HVEM protein transmembrane domain or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 106, or a nucleotide sequence having at least 80%, 81%
  • the CAR further comprises a HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 21, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 21.
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding an HVEM protein transmembrane domain or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 61, or a nucleotide 70 62059467v1 Attorney Dock
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding an HVEM protein transmembrane domain or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 104, or a nucleotide sequence having at least 80%, 81%
  • the CAR further comprises a CD8a hinge region or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 15, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 15.
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding CD8a hinge region or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 64, or a nucleotide sequence having at least 80%, 81%, 82%, 83%,
  • the polynucleotide sequence 71 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, the polynucleotide sequence further comprises polynucleotide sequence encoding CD8a hinge region or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO:
  • the CAR further comprises a HVEM protein hinge region or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 13, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 13.
  • the polynucleotide sequence encoding the CAR comprises a nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 8, 102, or 103
  • the polynucleotide sequence further comprises polynucleotide sequence encoding an HVEM protein hinge region or a functional variant thereof comprising a nucleotide sequence set forth as SEQ ID NO: 63, or a nucleotide sequence having at least 80%, 81%, 82%,
  • transformation or “transfection” may be used interchangeably and refer to the introduction of a nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, in some embodiments, a host cell or host organism may be stably transformed with a polynucleotide/nucleic acid molecule of the disclosure. In some embodiments, a host cell or host organism may be transiently transformed with a nucleic acid construct of the disclosure.
  • Transient 72 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) transformation in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
  • Stably introducing a polynucleotide introduced into a cell means that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.
  • Stable transformation of a cell means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Stable transformation can also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome or a plasmid.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well- known in the art.
  • Nucleic acids can be introduced into target cells using commercially available methods which include electroporation (e.g., Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), the Gene Pulser II (BioRad, Denver, Colo.), or Multiporator (Eppendort, Hamburg Germany)).
  • electroporation e.g., Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), the Gene Pulser II (BioRad, Denver, Colo.), or Multiporator (Eppendort, Hamburg Germany)).
  • Nucleic acids can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
  • Biolistic particle delivery systems such as "gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • RNA vectors include vectors having a RNA promoter and/or other relevant domains for production of a RNA transcript.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. 73 62059467v1 Attorney Docket No.: I1158141020WO (0012.2)
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g. , an artificial membrane vesicle).
  • a liposome e.g. , an artificial membrane vesicle
  • an exemplary delivery vehicle is a liposome.
  • the use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates.
  • Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium.
  • Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • the lipid components undergo self-rearrangement before the formation of closed 74 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • the nucleic acids introduced into a phagocytic cell can be RNA.
  • the RNA is mRNA that comprises in vitro transcribed RNA or synthetic RNA.
  • the RNA is produced by in vitro transcription using a PCR-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired template for in vitro transcription is a polynucleotide sequence encoding a CAR having an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 1, 2, or 7, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • HVEM herpes virus entry mediator
  • PCR can be used to generate a template for in vitro transcription of mRNA which is then introduced into cells. Methods for performing PCR are well known in the art.
  • Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
  • “Substantially complementary”, as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
  • the primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs.
  • the primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest.
  • the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs.
  • Primers useful for PCR are generated by synthetic methods that are well known in the art.
  • "Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
  • Upstream is used herein to refer to a location 5 ⁇ to the DNA sequence to be amplified relative to the coding strand.
  • Reverse primers are primers that contain a region of nucleotides that are 75 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. "Downstream” is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand. Chemical structures that have the ability to promote stability and/or translation efficiency of the RNA may also be used.
  • the RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length.
  • the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA.
  • 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • Various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter.
  • Other useful promoters include T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13 :6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003)).
  • the conventional method of integration of poly A/T stretches into a DNA template is molecular cloning.
  • poly A/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with poly A/T 3' stretch without cloning is highly desirable.
  • the poly A/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation.
  • the length of a poly(A) tail positively correlates with the stability of the transcribed RNA.
  • the poly(A) tail is between 100 and 5000 adenosines.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli poly A polymerase (E- PAP).
  • E- PAP E. coli poly A polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase mRNA stability.
  • Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • 5' caps also provide stability to RNA molecules. The 5' cap is provided using techniques known in the art and described in, e.g., Cougot et al. Trends in Biochem. Sci.29:436-444 (2001); Stepinski et al. RNA 7: 1468-95 (2001); and Elango et al. Biochim. Biophys. Res. Commun, 330:958-966 (2005).
  • RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • IVT-RNA vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in such a way that stabilized RNA transcripts are produced.
  • IVT-RNA vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in such a way that stabilized RNA transcripts are produced.
  • protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3' and/or 5' by untranslated regions (UTR), and a 3' polyadenyl cassette containing 50-70 A nucleotides.
  • nucleic acids Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II restriction enzymes (recognition sequence corresponds to cleavage site).
  • Nucleic acids can be delivered into cells by electroporation.
  • the nucleic acid includes an RNA. See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in, e.g., US 2004/0014645, US 2005/0052630A1, US 2005/0070841, US 2004/0059285, and US 2004/0092907.
  • the various parameters including electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in the field.
  • Electroporation may also be utilized to deliver nucleic acids into cells in vitro utilizing any of the many available devices and electroporation systems known to those of skill in the art. Electroporation of cells in vitro is described, e.g., in US 2007/0128708.
  • C. Modified phagocytic cells Regardless of the method used to introduce exogenous nucleic acids (i.e. transgenes) into a host cell or otherwise expose a cell to the molecules described herein, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed.
  • Such assays include, for example, molecular biological assays well known to those of skill in the art, such as sequencing, Southern and Northern blotting, reverse transcription-polymerase chain reaction (RT- PCR) and PCR; biochemical assays, such as detecting the presence or absence of a particular polypeptide, e.g., by immunological means (e.g., enzyme-linked immunosorbent assays (ELISAs), Western blots, immunoblots, immunohistochemistry, flow cytometry) or by assays described herein to identify agents falling within the scope of the disclosure.
  • immunological means e.g., enzyme-linked immunosorbent assays (ELISAs), Western blots, immunoblots, immunohistochemistry, flow cytometry
  • assays described herein to identify agents falling within the scope of the disclosure.
  • Transient transformation may be detected by, for example, an ELISA or Western blot, which can detect the presence of a peptide 78 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) or polypeptide encoded by one or more transgene introduced into a cell.
  • Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a cell (e.g., a phagocytic cell).
  • Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into the cell.
  • Stable transformation of a cell can also be detected by, e.g., PCR, or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods.
  • the phagocytic cells disclosed herein are genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 1, 2, or 7, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • CAR chimeric antigen receptor
  • HVEM herpes virus entry mediator
  • the phagocytic cells disclosed herein are genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence having an amino acid sequence set forth as any one of SEQ ID NOs: 1, 2, or 7, or an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 1, 2, or 7, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • CAR chimeric antigen receptor
  • HVEM herpes virus entry mediator
  • the CAR comprising an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 1, 2, or 7, or a functional fragment or variant thereof that retains co-stimulatory activity, can comprise an amino acid sequence set forth as any one of the SEQ ID NOs listed in Table 1, Table 3, or Table 5.
  • the CAR can comprise an amino acid sequence set forth as any one of SEQ ID NOs: 1, 2, 5-7, 9, 10, 12-33, 35- 41, 44-51.
  • the CAR comprising an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 1, 2, or 7, or a functional fragment or variant thereof that retains co-stimulatory activity, can comprise an amino acid sequence set forth as any one of SEQ ID NOs: 1, 2, 5-7, 9, 10, 12-33, 35-41, 44-51 or 107-108.
  • HVEM herpes virus entry mediator
  • the phagocytic cell comprises a CAR comprising an intracellular co-stimulatory signaling domain having an amino acid sequence comprising a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as SEQ ID NO: 7, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • HVEM herpes virus entry mediator
  • the phagocytic cell comprises a CAR comprising an intracellular co-stimulatory signaling domain having an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as SEQ ID NO: 7, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • HVEM herpes virus entry mediator
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7
  • said CAR further comprises an intracellular signaling domain comprising an intracellular signaling domain comprising a CD3 zeta intracellular signaling domain or a functional variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 9, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 9.
  • the CAR comprises an intracellular co- stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7
  • said CAR further comprises an intracellular signaling domain comprising an intracellular signaling domain comprising a CD3 zeta intracellular signaling domain or a functional variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 10.
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7, said CAR further comprises an intracellular signaling domain comprising an intracellular signaling domain comprising a )F ⁇ 5 ⁇ VLJQDOLQJ ⁇ domain or a functional variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 14, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 14.
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7, said CAR further comprises a transmembrane domain, wherein the transmembrane domain is a CD8a transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 16.
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7, said CAR further comprises a transmembrane domain, wherein the transmembrane domain is a HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12.
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7, said CAR further comprises a transmembrane domain, wherein the transmembrane domain is an HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 21, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 21.
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7, said CAR further comprises a CD8a hinge region or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 15, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 15.
  • the CAR comprises an intracellular co-stimulatory signaling domain having an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7, said CAR further comprises a HVEM protein hinge region or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 13, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 13.
  • genetically modified phagocytic cells expressing a CAR comprising a co-stimulatory domain having at least 90% sequence identity to an HVEM co-stimulatory protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity exhibit NF- ⁇ % ⁇ (nuclear factor kappa B) pathway activation.
  • the genetically modified phagocytic cells can comprise a hinge region, transmembrane domain, and/or intracellular signaling domain described herein.
  • the genetically modified phagocytic cells expressing a CAR comprising a co-stimulatory domain having at least 90% sequence identity to an HVEM co- stimulatory protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity exhibit increased expression of any gene that is positively associated with activation of NF- ⁇ %, such as a factor positively regulated by the NF- ⁇ % pathway.
  • the genetically modified phagocytic cells exhibit decreased expression of any gene that is negatively associated with activation of NF- ⁇ %, such as a factor negatively regulated by the NF- ⁇ % pathway.
  • genetically modified phagocytic cells expressing a CAR comprising a co-stimulatory domain having at least 90% sequence identity to an HVEM co-stimulatory protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity show increased expression of pro-inflammatory cytokines.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased expression of pro-inflammatory cytokines of 5-500%, 5-100%, 20-450%, 30-400%, 40-300%, 50-200%, 60-200%, 70-200%, 80-200%, 20-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, 90-100%, 100-120%, 120-140%, 140-160%, 160-180%, 180-200%, 200-220%, 220-240%, 240-260%, 260-280%, 280-300%, 300-320%, 320-340%, 340-360%, 360-380%, 380-400%, 400- 82 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) 420%, 420-440%, 440-460%, 460-480%, or
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased expression of pro-inflammatory cytokines of 2-fold to 100-fold, or 2-fold to 50-fold, or 5-fold to 20-fold, or 1.1-fold to 20-fold, or of at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6- fold, at least 6.5 fold, at least 7-fold, at least 7.5 fold, at least 8-fold, at least 8.5 fold, at least 9- fold, at least 9.5 fold, at least 10-fold, at least 10.5 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, or more, as compared to a control.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased expression of CD14, CD16, CD163, CD206, CD80, CD86, CXCL10, IL- 6, IL-10, IL-1beta, TGF-beta, or TNF-alpha, or any combination thereof, as compared to a control.
  • Cytokine expression either at the gene or protein level, can be measured with methods known to a skilled person. Cytokine gene expression can be measured by methods including real- time quantitative PCR and microarray.
  • Cytokine protein expression can be measured by assays including immunoassay (ELISA, automated ELISA, ELISpot), antibody arrays, microparticle multiplex assays (e.g., Luminex), flow cytometry, and nanoparticle-modified aptamers.
  • ELISA immunoassay
  • ELISpot automated ELISA
  • microparticle multiplex assays e.g., Luminex
  • flow cytometry e.g., Luminex
  • nanoparticle-modified aptamers e.g., Luminex
  • genetically modified phagocytic cells expressing a CAR comprising a co-stimulatory domain having at least 90% sequence identity to an HVEM co-stimulatory protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity show increased phagocytosis capability and increased ability to engulf target cells and/or to increase trogocytosis of cells.
  • increased phagocytosis ability results in limitation of target cell expansion.
  • the genetically modified phagocytic cells show increased ability to limit target cell expansion.
  • 83 62059467v1 Attorney Docket No.: I1158141020WO (0012.2)
  • Increased phagocytosis as well as ability to engulf target cells, to limit target cell expansion, and/or to perform trogocytosis can be detected with methods known to a skilled person. For example, in some embodiments, engulfment in a co-culture experiment with macrophages and target cells can be detected in live and fixed imaging and quantified using flow cytometry.
  • Different fluorescently labelled genetically modified phagocytic cells e.g., labeled with a first label such as GFP and expressing a CAR of the disclosure
  • target cells e.g., tumor cells labeled with a second label such as HA-mCherry and expressing an antigen recognized by the antigen binding domain of the CAR
  • single cells e.g. either GFP or mCherry
  • dual positive cells e.g. GFP and mCherry-positive cells
  • the dual positive cells e.g. GFP and mCherry-positive cells
  • a detectable label for the genetically modified phagocytic cells is fused to the CAR that the phagocytic cells are expressing.
  • a detectable label for the target cells is fused to the antigen that the target cells are expressing.
  • the experiment can be set up with control macrophages.
  • Control macrophages can include macrophages not expressing a CAR, macrophages expressing a CAR that recognizes an antigen unrelated to the antigen expressed by the target cells, and macrophages modified to express a CAR of the disclosure that lacks the HVEM co-stimulatory domain described herein and/or that lacks intracellular signaling function.
  • Trogocytosis events can also be quantified in live imaging experiments. Trogocytosis events can be recorded, measured by dedicated algorithms/programs, and normalized to controls to measure the trogocytosis percentage.
  • Additional tests suitable to identify trogocytosis, engulfment, and/or increased phagocytosis of a phagocyte include Incucyte Live-Cell Analysis System which can be used to perform real-time, automated phagocytosis/trogocytosis analysis, and High Content Analysis (HCA) which can provide another high sensitivity/low background analysis technique to measure phagocytosis, as well as additional tests identifiable by a skilled person.
  • HCA High Content Analysis
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased phagocytosis, engulfment, and/or trogocytosis as compared to a control.
  • the control can include: phagocytic cells not expressing a CAR, phagocytic cells expressing a CAR that recognizes an antigen unrelated to the antigen expressed by the target cells, phagocytic cells modified to express a CAR of the disclosure that lacks the HVEM co-stimulatory domain described herein and/or that lacks intracellular signaling function, and a culture condition/medium/buffer/vehicle lacking any phagocytic cells.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased phagocytosis, engulfment, and/or trogocytosis of 5-500%, 5-100%, 20- 450%, 30-400%, 40-300%, 50-200%, 60-200%, 70-200%, 80-200%, 20-100%, 30-100%, 40- 100%, 50-100%, 60-100%, 70-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70- 80%, 80-90%, 90-100%, 100-120%, 120-140%, 140-160%, 160-180%, 180-200%, 200-220%, 220-240%, 240-260%, 260-280%, 280-300%, 300-320%, 320-340%, 340-360%, 360-380%, 380- 400%, 400-420%, 420-440%
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased phagocytosis, engulfment, and/or trogocytosis of 1.1- to 10-fold, 1.1- to 8-fold, 1.1- to 5-fold, 1.1- to 2-fold, or 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7- fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more as compared to a control.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure can have increased phagocytosis, engulfment, and/or trogocytosis as compared to a control within 6 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, 72 hrs, 84 hrs, or 96 hrs of co-culture.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure is able to engulf more than one target cell.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased ability to limit target cell expansion as compared to a control.
  • the control can include: phagocytic cells not expressing a CAR, phagocytic cells expressing a CAR that recognizes an antigen unrelated to the antigen expressed by the target cells, phagocytic cells 85 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) modified to express a CAR of the disclosure that lacks the HVEM co-stimulatory domain described herein and/or that lacks intracellular signaling function and/or the CD8a or HVEM hinge domain described herein and/or that lacks the CD8a or HVEM transmembrane domain described herein, and a culture condition/medium/buffer/vehicle lacking any phagocytic cells.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased ability to limit target cell expansion of 5-500%, 5-100%, 20-450%, 30- 400%, 40-300%, 50-200%, 60-200%, 70-200%, 80-200%, 20-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, 90-100%, 100-120%, 120-140%, 140-160%, 160-180%, 180-200%, 200-220%, 220-240%, 240-260%, 260-280%, 280-300%, 300-320%, 320-340%, 340-360%, 360-380%, 380-400%, 400- 420%, 420-440%, 440-460%, 460-480%, or 480-500%, or of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%
  • a genetically modified phagocytic cell expressing a CAR of the disclosure has increased ability to limit target cell expansion of 1.1- to 10-fold, 1.1- to 8-fold, 1.1- to 5-fold, 1.1- to 2-fold, or 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8- fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more as compared to a control.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure can have increased ability to limit target cell expansion as compared to a control within 6 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, 72 hrs, 84 hrs, or 96 hrs of co-culture.
  • a genetically modified phagocytic cell expressing a CAR of the disclosure is able to limit cell expansion of more than one target cell.
  • the control can include: phagocytic cells not expressing a CAR, phagocytic cells expressing a CAR that recognizes an antigen unrelated to the antigen expressed by the target cells, phagocytic cells modified to express a CAR of the disclosure that lacks the HVEM co-stimulatory 86 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) domain described herein and/or that lacks intracellular signaling function, and a culture condition/medium/buffer/vehicle lacking any phagocytic cells.
  • the genetically modified phagocytic cells expressing a CAR of the disclosure can be comprised in a composition together with a compatible vehicle.
  • vehicle indicates any of various media acting usually as solvents, carriers, binders, or diluents for the modified phagocytic cells expressing a CAR as described herein that are comprised in the composition as an active ingredient.
  • the vehicle is a pharmaceutically acceptable vehicle and the composition is a pharmaceutically acceptable composition.
  • Pharmaceutical compositions of the present disclosure can comprise modified phagocytic cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • compositions of the present disclosure are preferably formulated for intravenous administration.
  • Suitable vehicles for an injectable composition comprise a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Suitable vehicles for oral composition comprise inert diluent or an edible carrier and excipients which can be combined with the active ingredients in the form of tablets, pills, troches, or capsules, e.g., gelatin capsules.
  • Suitable binding agents, and/or adjuvant materials can be included as part of the composition, such as microcrystalline cellulose, gum tragacanth or gelatin and additional binding agents and/or adjuvant identifiable by a skilled person.
  • Suitable vehicles for aerosol spray used for inhalation from a pressured container or dispenser can contain a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer and are expected to be formulated and/or administered with methods such as the ones described in US 6,468,798, incorporated herein by reference in its entirety.
  • Suitable vehicles for transmucosal or transdermal administration comprising penetrants appropriate to the barrier to be permeated can be used in the formulation.
  • Such penetrants are 87 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Suitable vehicles for composition in the form of suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials. When “an immunologically effective amount”, “a therapeutically effective amount”, “an effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, immune response, and condition of the patient (subject).
  • a pharmaceutical composition comprising modified phagocytic cells expressing a CAR as described herein may be administered at a dosage of about 10 3 to about 10 10 cells/kg body weight, and in some embodiments, the dosage can be from about 10 5 to about 10 8 cells/kg body weight or from about 10 6 to about 10 8 cells/kg body weight, including all integer values (e.g., 10 4 , 10 5 , 10 6 , 10 7 ,10 8 , 10 9 ) within those ranges.
  • the cell compositions of the disclosure can be administered multiple times (e.g., hourly, four times daily, three times daily, two times daily, daily, twice weekly, three times weekly, weekly, monthly, bi-monthly, semi-annually, annually, etc.) 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. J. 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.
  • cells are modified using the methods described herein, or other methods known in the art where the cells are expanded to therapeutic levels, are administered to a patient in conjunction any number of relevant treatment modalities, including one or more of radiation therapy, genetically engineered cellular immunotherapy (e.g., T cell, natural killer cell, chimeric antigen receptor (CAR) therapy), antibody therapy, immune 88 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) checkpoint molecule inhibitor therapy, or a pharmaceutical therapy, such as a chemotherapeutic, a therapeutic peptide, antibiotic, anti-viral agent, anti-fungal agent, anti-inflammatory agent, or a small molecule therapy.
  • a pharmaceutical therapy such as a chemotherapeutic, a therapeutic peptide, antibiotic, anti-viral agent, anti-fungal agent, anti-inflammatory agent, or a small molecule therapy.
  • a composition comprising modified phagocytic cells expressing a CAR as described herein are administered in combination with one or more additional therapies
  • the one or more additional therapies may be administered at a subtherapeutic dose due to an additive or synergistic effect of the combination with the composition comprising modified phagocytic cells.
  • Combination therapy includes administration of a composition comprising modified phagocytic cells of the disclosure before an additional therapy (e.g., 1 day to 30 days or more before the additional therapy), concurrently with an additional therapy (on the same day), or after an additional therapy (e.g., 1 day to 30 days or more after the additional therapy).
  • a composition comprising modified phagocytic cells as described herein may be administered after the initial dose of the one or more additional therapies, after the final dose of the one or more additional therapies, or in between multiple doses of the one or more additional therapies.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of a composition comprising modified phagocytic cells of the present disclosure.
  • a composition comprising modified phagocytic cells of the present disclosure may be administered before or following surgery.
  • the dosage of one or more additional therapies to be administered to a subject will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • kits of parts for performing any one of the methods herein described, the expression vectors, genes encoding CARs, genetically modified phagocytic cells expressing a CAR of the disclosure, and compositions comprising the CAR-modified phagocytic cells can be included in the kit alone or in the presence of additional labels for detection of cells as well as additional components identifiable by a skilled person.
  • kits of parts the expression vectors, genes encoding CARs, genetically modified phagocytic cells expressing a CAR of the disclosure, compositions comprising the CAR-modified phagocytic cells, and additional reagents identifiable by a skilled person are comprised in the kit independently and possibly includes suitable vehicle carriers or auxiliary agents. Additional components can include labels, reference standards, and 89 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) additional components identifiable by a skilled person upon reading of the present disclosure.
  • label and “labeled molecule” as used herein refer to a molecule capable of detection, including radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like.
  • fluorophore refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image.
  • labeling signal indicates the signal emitted from the label that allows detection of the label, including radioactivity, fluorescence, chemoluminescence, production of a compound in an enzymatic reaction, and the like.
  • the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here disclosed.
  • the kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes, CD-ROMs, flash drives, or by indication of a Uniform Resource Locator (URL), which contains a pdf copy of the instructions for carrying out the assay, will usually be included in the kit.
  • URL Uniform Resource Locator
  • the kit can also contain, depending on the particular method used, other packaged reagents and materials (e.g., wash buffers and the like).
  • Other packaged reagents and materials e.g., wash buffers and the like.
  • the modified phagocytic cells expressing a CAR with a HVEM co-stimulatory signaling domain described herein may be included in a composition for treatment of a disease or disorder in a subject.
  • the composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier.
  • a therapeutically effective amount of the pharmaceutical composition comprising the modified phagocytic cells may be administered.
  • a "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a “disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • the disclosure includes a method of treating a disease or condition associated with a tumor or cancer in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a genetically modified phagocytic cell described herein.
  • the method of treating a disease or condition associated with a tumor or cancer in a subject comprises limiting and/or reducing target cell proliferation and/or expansion.
  • the target cell can be a cancer cell.
  • the method comprises limiting or 90 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) reducing proliferation and/or expansion of a plurality of target cells within a cell population.
  • limiting target cell proliferation reduces a sign or symptom of a disease, such as a tumor or cancer.
  • the disclosure further includes a method of treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a genetically modified phagocytic cell described herein.
  • the method of treating a tumor in a subject comprises limiting and/or reducing target cell proliferation and/or expansion.
  • the target cell can be a tumor cell or a cancer cell.
  • the method comprises limiting or reducing proliferation and/or expansion of a plurality of target cells, such as tumor or cancer cells, within a cell population.
  • limiting the tumor or cancer cell proliferation reduces the size of a tumor, prevents growth of a tumor, and/or reduces a sign or symptom associated with a tumor.
  • the disclosure further provides use of the modified cell described herein in the manufacture of a medicament for the treatment of a tumor or cancer in a subject in need thereof.
  • the disclosure provides for a method for stimulating an immune response to a target tumor cell or tumor tissue in a subject comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a genetically modified phagocytic cell described herein.
  • the method of stimulating an immune response to a target tumor cell or tumor tissue in a subject can comprise limiting and/or reducing target cell proliferation and/or expansion.
  • the target cell can be a tumor cell or a cancer cell.
  • the method comprises limiting or reducing proliferation and/or expansion of a plurality of target cells, such as tumor or cancer cells, within a cell population.
  • limiting the tumor or cancer cell proliferation reduces the size of a tumor, prevents growth of a tumor, and/or reduces a sign or symptom associated with a tumor.
  • Cancers include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • the cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • Types of cancers to be treated with CAR-modified cells of the disclosure include carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • sarcomas e.g., sarcomas, carcinomas, and melanomas.
  • Adult tumors/cancers and pediatric tumors/cancers are also included.
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant.
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma
  • Hematologic cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • Treatment can result in a reduction in tumor size.
  • the tumor size/volume in a subject treated with a composition comprising CAR-modified phagocytic cells described herein is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to untreated tumors.
  • treatment results in an increased survival rate of the subject.
  • the survival rate increases by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% relative to untreated subjects.
  • Treatment can result in a reduction the number of cancer cells present in the subject.
  • the number of cancer cells in subjects treated with a composition comprising CAR-modified phagocytic cells described herein is reduced by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold relative to untreated subjects.
  • 92 62059467v1 Attorney Docket No.: I1158141020WO (0012.2)
  • the disclosure provides for treating an infection in a subject in need thereof, comprising administering to the subject an effective amount of a genetically modified phagocytic cell described herein.
  • the method of treating an infection in a subject can comprise limiting and/or reducing target cell proliferation and/or expansion.
  • the method comprises limiting or reducing proliferation and/or expansion of a plurality of target cells, within a cell population.
  • limiting target cell proliferation reduces a sign or symptom associated with an infection.
  • the pathogen recognized by the CAR may be essentially any kind of pathogen, but in some embodiments the pathogen is a fungus, bacteria, or virus.
  • Exemplary viral pathogens include those of the families of Adenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HSV, HHV family of viruses, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae.
  • Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chicken pox, ebola, and rubella.
  • Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys.
  • Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella.
  • the pathogen receptor Dectin-1 may be used to generate a CAR that recognizes the carbohydrate structure on the cell wall of fungi such as Aspergillus.
  • CARs can be made based on an antibody recognizing viral determinants (e.g., the glycoproteins from CMV and Ebola) to interrupt viral infections and pathology.
  • the disclosure provides a method of providing an immune response against a target in a subject in need thereof, the method comprising: transfecting phagocytic cells obtained from the subject with a nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: an antigen binding domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co-stimulatory signaling domain having an amino acid sequence having at least at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity sequence identity to a cytoplasmic domain of a herpes
  • the subject has cancer and/or an infection.
  • the target is a cancer cell or an infectious agent.
  • the disclosure further includes use of the CAR- modified phagocytic cells described herein in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.
  • the disclosure further provides a method of treating a subject by engulfment and/or trogocytosis of a target cell in the subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a genetically modified phagocytic cell described herein.
  • methods of the disclosure can be used to treat individuals who have, who are suspected of having, or who may be at high risk for developing one or more health conditions or disorders for which trogocytosis and/or engulfment of a target cell is known or expected to have a therapeutic effect.
  • target cells to be recognized and eliminated by the CAR-modified phagocytic cells of the disclosure include tumor cells, bacteria, virus-infected cells, viral particles, senescent cells and other cells identifiable to a person skilled in the art.
  • target cells also include neurons that are non- functional or dying due to accumulation of abnormal forms of Tau or beta-amyloid.
  • the method of treating a subject by engulfment and/or trogocytosis of a target cell can comprise limiting and/or reducing target cell proliferation and/or expansion.
  • the method comprises limiting or reducing proliferation and/or expansion of a plurality of target cells, within a cell population.
  • the CAR-modified phagocytic cells described herein possess targeted effector activity.
  • the CAR-modified phagocytic cells have targeted effector activity directed against an antigen on a target cell, such as through specific binding to an antigen binding domain of a CAR.
  • the targeted effector activity includes, but is not limited to, phagocytosis, targeted cellular cytotoxicity, antigen presentation, and cytokine secretion.
  • the CAR-modified phagocytic cells described herein have the capacity to deliver an agent, a biological agent or a therapeutic agent to the target.
  • the cell may be modified or engineered to deliver an agent to a target, wherein the agent is selected from the group consisting of a nucleic acid, an antibiotic, an anti -inflammatory agent, an antibody or antibody fragments thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule, a carbohydrate or the like, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combination thereof.
  • a macrophage modified with a CAR of the disclosure is capable of secreting an agent, such as a cytokine or antibody, to aid in macrophage function.
  • Antibodies such as anti- CD47/antiSIRPa monoclonal antibody, may also aid in macrophage function.
  • the macrophage modified with a CAR of the disclosure is engineered to encode a siRNA that aids macrophage function by downregulating inhibitory genes (e.g., SIRPa).
  • the CAR-modified phagocytic cell is engineered to express a dominant negative (or otherwise mutated) version of a receptor or enzyme that aids in macrophage function.
  • the phagocytic cell (e.g., macrophage) can be modified with multiple genes, wherein at least one gene encodes a CAR and at least one other gene comprises a genetic element that enhances CAR macrophage function.
  • the phagocytic cell (e.g., macrophage) is modified with multiple genes, wherein at least one gene encodes a CAR and at least one other gene aids or reprograms the function of other immune cells (such as T cells within the tumor microenvironment).
  • the CAR-modified phagocytic cells can be administered to an animal, preferably a mammal, even more preferably a human, to suppress an immune reaction, such as those common to autoimmune diseases such as diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, graft versus host disease (GVHD), transplant rejection, and the like.
  • an immune reaction such as those common to autoimmune diseases such as diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, graft versus host disease (GVHD), transplant rejection, and the like.
  • the CAR-modified phagocytic cells of the present disclosure can be used for the treatment of any condition in which a diminished or otherwise inhibited immune response, especially a cell- mediated immune response, is desirable to treat or alleviate a disease.
  • methods of the disclosure includes treating a condition, such as an autoimmune disease, in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the CAR-modified phagocytic cells described herein.
  • autoimmune disease examples include Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis- juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia- fibromyositis, Grave
  • a composition comprising the CAR-modified phagocytic cells can also be used to treat inflammatory disorders.
  • inflammatory disorders include chronic and acute inflammatory disorders.
  • inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, GVHD, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy, and ventilator induced lung injury.
  • the CAR comprises an intracellular co- stimulatory signaling domain having an amino acid sequence having at least at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as any one of SEQ ID NOs: 7, 1, or 2, or a functional fragment or variant thereof that retains co-stimulatory activity.
  • HVEM herpes virus entry mediator
  • the genetically modified phagocytic cells comprising the CAR have increased phagocytosis and/or trogocytosis as compared to phagocytic cells that do not comprise the disclosed HVEM co-stimulatory signaling domain.
  • the genetically modified phagocytic cells have increased phagocytosis and/or trogocytosis of 5% to 100%.
  • the genetically modified phagocytic cells have increased phagocytosis and/or trogocytosis of at least 20%.
  • the phagocytic cells can include monocytes, macrophages, dendritic cells, neutrophils, and/or precursors thereof.
  • the phagocytic cells include macrophages.
  • CAR-modified phagocytic cells of the disclosure can be administered in dosages and by routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of CAR-modified phagocytic cells of the disclosure may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art. CAR-modified phagocytic cells of the disclosure to be administered may be autologous, allogeneic or xenogeneic with respect to the subject undergoing therapy.
  • compositions 96 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) described herein may be administered to a subject transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally.
  • modified phagocytic cells of the disclosure are injected directly into a target region, a local disease site in the subject, a site of inflammation, a site of infection, a lymph node, an organ and/or a tumor of the subject.
  • EXAMPLE 1 Cells expressing a CAR with an M83 co-stimulatory signaling domain have increased phagocytosis and superior control of target cell expansion Lentiviral constructs were generated to express CARs with: a CD3z intracellular signaling domain; a CD3z intracellular signaling domain and a 4-1BB co-stimulatory signaling domain; or a CD3z intracellular signaling domain and a M83 co-stimulatory signaling domain.
  • Primary monocytes were cultured with GMCSF for 5 days and transduced with lentiviral vectors to express test and control CARs with and without an anti-CD19 binder.
  • FIG.1A shows data taken following 3 hrs of co-culture.
  • CD47-blocking antibodies were added to Raji for 10 minutes prior to addition of Raji to the CAR-M.
  • CAR signaling with the M83 co- stimulatory signaling domain (‘CD19.M83costim.CD3z’) resulted in a significant increase in the percentage of CAR-M that engulfed Raji target cells as compared to the percentage of CAR-M having CD3z signaling domain only, having CD3z signaling domain and 4-1BB co- stimulatory signaling domain, or as compared to control macrophages.
  • Additional lentiviral constructs were generated to express CARs with: a CD3z intracellular signaling domain, a M83 co-stimulatory signaling domain, a CD8alpha transmembrane domain, and a CD8alpha hinge region (FIG.1D; ‘CD19.M83costim.CD3z’ and ‘Non-targeting’); a CD3z intracellular signaling domain, a CD8a transmembrane domain, and a CD8alpha hinge region (FIG.1D; ‘CD19.CD3z’); a CD32alpha intracellular signaling domain, transmembrane domain, and hinge region (FIG.1D; ‘SEQ ID NO: 37’); a CD3z intracellular signaling domain, a M83 co- stimulatory signaling domain, a M83 transmembrane domain, and a CD8alpha hinge region (FIG.1D; ‘CD19.M83costim.CD3z’ and ‘Non-targeting’); a CD3z
  • FIG.1D A schematic of these constructs is depicted in FIG.1D.
  • THP-1 monocyte cells were transduced with the lentiviral vectors to express test and control CARs with an anti-CD19 binder, or non-targeting binder (‘Non-targeting’), as shown in FIG.1D.
  • THP-1 monocyte cell lines transduced and stably expressing a non-targeting control CAR with a CD8a hinge domain, a CD8a transmembrane domain, an M83 costimulatory domain, and a CD3z intracellular signaling domain (FIG.1D) and anti-CD19 test CARs were co-cultured with CD19-positive Raji target cells.
  • the superior control of target cell expansion by a CAR containing an M83 co-stimulatory domain and a CD3z intracellular signaling domain occurred with a CAR comprising an M83 transmembrane domain and a CD8alpha hinge domain, and with a CAR comprising an M83 transmembrane domain and an M83 hinge domain.
  • a CAR containing an M83 co-stimulatory domain and a CD3z intracellular signaling domain demonstrate that the M83 co-stimulatory domain is functional in conjunction with different stimulatory domains (e.g., a CD3z intracellular signaling domain, and D ⁇ )F ⁇ 5 ⁇ LQWUDFHOOXODU ⁇ VLJQDOLQJ ⁇ GRPDLQ), and is functional when used in conjunction with the hinge domain and/or the M83 transmembrane domain.
  • a CAR containing an M83 co-stimulatory domain, a CD3z intracellular signaling domain, a CD8alpha transmembrane domain, and a CD8alpha hinge domain (“CD19.
  • FIG.1F shows a schematic of the tested anti-CD70 CARs.
  • THP-1 monocyte cell lines transduced and stably expressing a first-generation anti-CD70 CAR (CD70.CD3z) or an anti-CD70 CAR with an M83 costimulatory domain (CD70.M83costim.CD3z) were co-cultured with CD70-positive Raji target cells. Expansion of Raji target cells was assessed using live cell imaging over 72 hours (FIG.1F). Raji cells cocultured with parental THP-1 cells (THP-1 WT) were used as controls. THP-1 cells expressing CD70.M83costim.CD3z CAR exhibited a statistically significant reduction in target cell expansion compared to THP-1 WT and CD70.CD3z.
  • EXAMPLE 2 Cells expressing a CAR with an M83 co-stimulatory signaling domain can overcome CD47 signaling pathway-mediated inhibition of phagocytosis CD47 signaling through SIRPa receptor on phagocytes results in a "don't eat me” signal from the CD47+ cell to phagocytes. Many tumors are known to take advantage of this signaling axis by over-expressing CD47 (Morrissey and Vale (2019) bioRxiv: 752311). Fluorescently labeled macrophages expressing a CAR with a CD3z intracellular signaling domain or a CAR with a CD3z intracellular signaling domain and a M83 co-stimulatory signaling domain were generated as described in Example 1.
  • the fluorescently labeled CAR-Ms and fluorescently labeled CD47+ target cells were co-cultured and % phagocytosis determined by flow cytometry as described in Example 1.
  • CD47+ Raji target cells were readily phagocytosed by macrophages expressing a CAR with an M83 co-stimulatory signaling domain (‘targets’ and ‘+aCD47’ in CD3z CAR-M of FIG.2B).
  • Macrophages expressing a CAR with a CD3z intracellular signaling domain and a M83 co-stimulatory signaling domain have significantly greater phagocytosis of CD47+ target cells, regardless of whether the target cells had been pre-treated (“+aCD47”; FIG.2A and FIG.2B) or not pre-treated with a CD47-blocking antibody to macrophages expressing a first-generation CAR without an M83 co-stimulatory signaling domain (compare ‘CD19.M83costim.CD3z’ vs. ‘CD19.CD3z’ in FIG.2B).
  • EXAMPLE 3 Enhanced anti-tumor cell activity in vitro and in vivo of cells expressing a CAR with an M83 co-stimulatory signaling domain Generation of iCAR-M To obtain uniform embryoid bodies (EBs), induced pluripotent stem cells (iPSCs) were seeded into AggreWell 800 plates (StemCell Technologies).
  • EBs embryoid bodies
  • iPSCs induced pluripotent stem cells
  • Lentiviral constructs were generated to express CARs with: a CD3z intracellular signaling domain and a CD19 targeting domain (CD19.CD3z); a CD3z intracellular signaling domain and a M83 co-stimulatory signaling domain and a CD19 targeting domain (‘CD19.M83costim.CD3z’) and a CD3z intracellular signaling domain and a M83 co-stimulatory signaling domain and a non- CD19 targeting domain (‘Non-targeting’).
  • Cells were transduced with lentiviral particles containing antiCD19.M83.CD3z.P2A.GFP, antiCD19.CD3z.P2A.GFP, or antiVRCO.M83.CD3z.P2A.GFP constructs (each also containing a puromycin resistance cassette) and sorted by flow cytometry to identify CAR-iPSC. Sorted cells were expanded and banked as undifferentiated iPSCs.
  • mesoderm induction and subsequent hemogenic endothelium induction were initiated by replacing 75% (2 mL out of 2.5 mL in each well) of the mTeSRplus medium with fresh mTeSRplus medium supplemented with 50 ng/mL human Bone Morphogenetic Protein 4 (hBMP4), 50 ng/mL human Vascular Endothelial Growth Factor (hVEGF), and 20 ng/mL human Stem Cell Factor (hSCF), repeated over the following two days.
  • hBMP4 human Bone Morphogenetic Protein 4
  • hVEGF human Vascular Endothelial Growth Factor
  • hSCF Stem Cell Factor
  • the EBs were collected and transferred to Myeloid Induction Medium, composed of X-VIVO 15 medium (Lonza) or SFEM II supplemented with 2 mM Glutamax, 1% penicillin/streptomycin ⁇ RSWLRQDO ⁇ J ⁇ P/ ⁇ PHUFDSWRHWKDQRO ⁇ 0-CSF (100 ng/mL), and IL3 (25 ng/mL).
  • the EBs were plated at a density of 1 EB per cm 2 on cell culture vessels ranging from 2 to 1000 cm 2 or 6-well cell dishes. The dishes were pre-coated for 1 hour at room temperature (RT) with growth factor-reduced Matrigel (Corning), diluted in cold DMEM F12 GlutaMax.
  • iPSC-derived CD19/M83 CD19.M83costim.CD3z
  • CAR M cells exhibit robust expression of M1-related marker genes and pro-inflammatory cytokine genes after co-culture with CD19-H1299 tumor cells compared to spheroid and non-targeting CAR macrophage cells.
  • expression of CD14, CD16, CD163, CD80, CD86, and IL-1B was increased, with CXCL10 and IL-10 expression showing the largest expression increase, compared to controls.
  • Tumor spheroids are one of the most common and versatile scaffold-free methods for 3D cell culture. Spheroids are either self- assembling or are forced to grow as cell clusters starting from single cell suspensions. Compared to cells cultured on a flat surface, they more closely mimic the complex scenario of tissues and organs where each cell interacts with nearby cells through the formation of desmosomes and dermal junctions.
  • CD19/M83 CAR M cells (CD19.M83costim.CD3z; triangles) exhibited CAR-dependent efficacy against CD19-positive spheroids.
  • Non-targeting CAR cells (squares) did not reduce the size of spheroid cultures and showed a similar normalized spheroid area over 6 days of culture compared to spheroid only control.
  • CD19/M83 102 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) CAR M cells caused a noticeable decrease in the size of the spheroid starting by day 3 of culture, and extending throughout day 6 of culture.
  • H1299 is a human Non-Small Cell Lung Cancer (NSCLC) cell-line that was obtained from ATCC and cultured in RPMI-1640-Glutamax medium containing 10% Fetal Bovine Serum (FBS). H1299 cells were stably transduced with lentivirus particles containing a puromycin-resistance cassette and CD19-iRFP-mCherry. Following selection with puromycin, CD19 over-expression in H1299-CD19 was confirmed by Flow Cytometry.
  • NSCLC Non-Small Cell Lung Cancer
  • H1299-CD19 cells were harvested during log phase growth and resuspended in 50% Cultrex Type-3 (Trevigen) in HBSS.
  • xenografts were initiated by subcutaneously (s.c.) implanting 10 6 H1299-CD19 cells (in a 0.2 mL suspension) into the right flank of each mouse. Tumor volume was estimated using Digital calipers.
  • each group received HBSS, Non-targeting CAR, CD19.M83costim.CD3z, or CD19.CD3z iCAR-M administered in 200 uL of HBSS via intra tumoral (IT) injection. 10 7 cells were injection in each dose. Mice were sacrificed on Day 26.
  • FIG.5B shows tumor volume measurements in mice administered subcutaneous CD19+H1299 tumors and subsequently treated with HBSS or iPSC-generated non-targeting VRC01-based CAR-M (‘Non-targeting’), 1st Generation CAR-M (‘CD19.CD3z’) or M83-CAR- M (‘CD19.M83costim.CD3z’), as noted above.
  • HBSS non-targeting
  • CD19.CD3z 1st Generation CAR-M
  • M83-CAR- M ‘CD19.M83costim.CD3z’
  • EXAMPLE 4 Cells expressing a CAR with an M83 co-stimulatory signaling domain have enhanced pro-inflammatory signaling
  • Lentiviral constructs were generated to express CARs with: a M83 co-stimulatory domain and a CD3z intracellular signaling domain; or a M83 co-stimulatory signaling domain and a Fc ⁇ R1 ⁇ intracellular domain.
  • THP-1 DualTM cells were transduced with lentiviral vectors to stably express CARs with an anti-CD19 binder.
  • THP1-DualTM cells were derived from the human THP-1 monocyte cell line by stable integration of two inducible reporter constructs.
  • THP1- DualTM cells allow the simultaneous study of the NF- ⁇ B pathway, by monitoring the activity of SEAP, and the IRF pathway, by assessing the activity of a secreted luciferase, Lucia luciferase. Both reporter proteins are readily measurable in the cell culture supernatant via a detection reagent. Tissue culture plates were coated with human CD19 protein at multiple concentrations (0 QJ ⁇ PO ⁇ QJ ⁇ PO ⁇ J ⁇ PO ⁇ . THP-1 DualTM cells were added to tissue culture plates and the level of NF-kB reporter was measured in response to plate-bound sCD19 antigen. Unmodified THP-1 Dual cells were used as controls.
  • THP1-DualTM cells comprising a CAR with a M83 co-stimulatory domain and a CD3z intracellular signaling domain (‘CD19.M83costim.CD3z’) or a M83 co- stimulatory signaling domain and D ⁇ )F ⁇ 5 ⁇ LQWUDFHOOXODU ⁇ GRPDLQ (‘CD19.M83costim.FcGr’) induced the NF- ⁇ % ⁇ SDWKZD ⁇ .
  • the level of induction was positively correlated with the concentration of sCD19 present on the plate, with higher levels of induction occurring in the presence of ⁇ J ⁇ PO ⁇ compared to 100 ng/ml.
  • THP1-DualTM cells comprising a CAR with a M83 co-stimulatory domain and a CD3z intracellular signaling domain was moderately increased compared to cells comprising a M83 co-stimulatory signaling domain and D ⁇ )F ⁇ 5 ⁇ LQWUDFHOOXODU ⁇ GRPDLQ.
  • Table 1 CAR Domain Amino Acid Sequences of the Disclosure SEQ ID Description NO: 1 Amino acid sequence of an HVEM co-stimulatory domain 2 Amino acid sequence of an HVEM co-stimulatory domain 5 Amino acid linker 104 62059467v1 Attorney Docket No.: I1158141020WO (0012.2) 6 Amino acid linker 7 Amino acid sequence of an HVEM co-stimulatory domain (‘M83’) 9 Amino acid sequence of a CD3 zeta signaling domain (aa 52-164 of UniProt Accession no. P20963-1 10 Amino acid sequence of a CD3 zeta signaling domain (aa 52-163 of UniProt Accession no.
  • CAR Construct Amino Acid Domain Combinations of the Disclosure CAR C/S T/C/S T/C H/T/C H/T/C H/T/C/S Construct SEQ ID SEQ ID SEQ ID SEQ ID NO: SEQ ID NO: NO: NO: 33 17 19 23 24 27 36 17 19 23 24 27 38 17 20 1 25 29 39 18 22 1 25 30 40 17 20 1 26 31 41 18 22 1 26 32 42 17 19 23 24 27 C/S: costimulatory domain + intracellular signaling domain T/C/S: transmembrane domain + costimulatory domain + intracellular signaling domain T/C: transmembrane domain + costimulatory domain H/T/C: hinge region + transmembrane domain + costimulatory domain H/T/C/S: hinge region + transmembrane domain + costimulatory domain + costimulatory domain + signaling domain
  • the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.
  • any of the aspects disclosed herein may be combined with each other (e.g., the feature according to one aspect may be added to the features of another aspect or replace an equivalent feature of another aspect) or with features that are well known in the art, unless indicated otherwise by context.
  • 109 62059467v1 Attorney Docket No.: I1158141020WO (0012.2)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des cellules phagocytaires génétiquement modifiées pour exprimer un récepteur antigénique chimérique (CAR) ayant un domaine co-stimulateur provenant d'une protéine médiateur d'entrée du virus de l'herpès (HVEM, « herpes virus entry mediator »), ainsi que des vecteurs, des compositions et des procédés associés qui permettent une phagocytose améliorée de cellules cibles et le traitement d'états chez un patient.
PCT/IB2024/054836 2023-05-18 2024-05-18 Cellules phagocytaires modifiées exprimant des récepteurs antigéniques chimériques comprenant un domaine co-stimulateur de médiateur d'entrée du virus de l'herpès (hvem) et utilisations associées WO2024236547A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202363503058P 2023-05-18 2023-05-18
US63/503,058 2023-05-18
US202463575479P 2024-04-05 2024-04-05
US63/575,479 2024-04-05
US202463645382P 2024-05-10 2024-05-10
US63/645,382 2024-05-10

Publications (1)

Publication Number Publication Date
WO2024236547A1 true WO2024236547A1 (fr) 2024-11-21

Family

ID=91375728

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2024/054836 WO2024236547A1 (fr) 2023-05-18 2024-05-18 Cellules phagocytaires modifiées exprimant des récepteurs antigéniques chimériques comprenant un domaine co-stimulateur de médiateur d'entrée du virus de l'herpès (hvem) et utilisations associées

Country Status (1)

Country Link
WO (1) WO2024236547A1 (fr)

Citations (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350674A (en) 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
US5792632A (en) 1992-05-05 1998-08-11 Institut Pasteur Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
US5830729A (en) 1996-04-18 1998-11-03 Institut Pasteur I Sce I-induced gene replacement and gene conversion in embryonic stem cells
US5993434A (en) 1993-04-01 1999-11-30 Genetronics, Inc. Method of treatment using electroporation mediated delivery of drugs and genes
US6181964B1 (en) 1997-08-01 2001-01-30 Genetronics, Inc. Minimally invasive apparatus and method to electroporate drugs and genes into tissue
WO2001029058A1 (fr) 1999-10-15 2001-04-26 University Of Massachusetts Genes de voies d'interference d'arn en tant qu'outils d'interference genetique ciblee
US6238924B1 (en) 1992-05-05 2001-05-29 Institut Pasteur Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
US6241701B1 (en) 1997-08-01 2001-06-05 Genetronics, Inc. Apparatus for electroporation mediated delivery of drugs and genes
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
US6468798B1 (en) 1991-12-17 2002-10-22 The Regents Of The University Of California Expression of cloned genes in the lung by aerosol and liposome-based delivery
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20030232410A1 (en) 2002-03-21 2003-12-18 Monika Liljedahl Methods and compositions for using zinc finger endonucleases to enhance homologous recombination
US6678556B1 (en) 1998-07-13 2004-01-13 Genetronics, Inc. Electrical field therapy with reduced histopathological change in muscle
US20040014645A1 (en) 2002-05-28 2004-01-22 Advisys, Inc. Increased delivery of a nucleic acid construct in vivo by the poly-L-glutamate ("PLG") system
US20040059285A1 (en) 2002-07-04 2004-03-25 Inovio As Electroporation device and injection apparatus
US20040092907A1 (en) 1997-04-03 2004-05-13 Inovio As Method for muscle delivery of drugs, nucleic acids and other compounds
US6746838B1 (en) 1997-05-23 2004-06-08 Gendaq Limited Nucleic acid binding proteins
WO2004067753A2 (fr) 2003-01-28 2004-08-12 Cellectis Utilisation de meganucleases pour induire une recombinaison homologue ex vivo et in toto dans des tissus somatiques de vertebre et application de cette utilisation
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US20050052630A1 (en) 2002-03-07 2005-03-10 Advisys, Inc. Constant current electroporation device and methods of use
US20050070841A1 (en) 2002-07-04 2005-03-31 Inovio As Electroporation device and injection apparatus
US7013219B2 (en) 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7171264B1 (en) 1999-05-10 2007-01-30 Genetronics, Inc. Intradermal delivery of active agents by needle-free injection and electroporation
US7173116B2 (en) 2000-03-03 2007-02-06 Genetronics Biomedical Corporation Nucleic acid formulations for gene delivery and methods of use
US20070128708A1 (en) 2005-12-07 2007-06-07 Genetronics, Inc. Variable volume electroporation chamber and methods therefore
WO2008010009A1 (fr) 2006-07-18 2008-01-24 Cellectis Variants de méganucléase clivant une séquence d'adn cible provenant d'un gène rag et leurs utilisations
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
WO2008149176A1 (fr) 2007-06-06 2008-12-11 Cellectis Variants de méganucléase clivant une séquence cible d'adn issue du locus rosa26 de souris et leurs utilisations
WO2009013559A1 (fr) 2007-07-23 2009-01-29 Cellectis Variants de méganucléase clivant une séquence cible d'adn à partir du gène bêta de l'hémoglobine humaine et ses utilisations
WO2009059195A2 (fr) 2007-10-31 2009-05-07 Precision Biosciences Méganucléases monocaténaires conçues rationnellement contenant des séquences de reconnaissance non palindromiques
US20090203140A1 (en) 2007-09-27 2009-08-13 Sangamo Biosciences, Inc. Genomic editing in zebrafish using zinc finger nucleases
US7585849B2 (en) 1999-03-24 2009-09-08 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US8119381B2 (en) 2005-10-18 2012-02-21 Duke University Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
US8338157B2 (en) 2008-03-11 2012-12-25 Precision Biosciences, Inc. Rationally-designed meganuclease variants of lig-34 and I-crei for maize genome engineering
US8440431B2 (en) 2009-12-10 2013-05-14 Regents Of The University Of Minnesota TAL effector-mediated DNA modification
WO2014018423A2 (fr) 2012-07-25 2014-01-30 The Broad Institute, Inc. Protéines de liaison à l'adn inductibles et outils de perturbation du génome et leurs applications
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
WO2014093701A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Génomique fonctionnelle employant des systèmes crispr-cas, des compositions, des procédés, des banques d'inactivation et leurs applications
WO2014093709A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Procédés, modèles, systèmes et appareil pour identifier des séquences cibles pour les enzymes cas ou des systèmes crispr-cas pour des séquences cibles et transmettre les résultats associés
WO2014093718A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Procédés, systèmes et appareil pour identifier des séquences cibles pour les enzymes cas ou des systèmes crispr-cas pour des séquences cibles et transmettre les résultats associés
WO2014093622A2 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques
WO2014093595A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Systèmes de composants de crispr-cas, procédés et compositions pour la manipulation de séquences
WO2014093712A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Fabrication de systèmes, procédés et compositions de guide optimisées pour la manipulation de séquences
WO2014093694A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Systèmes, procédés et compositions de crispr-nickase cas pour la manipulation de séquences dans les eucaryotes
WO2014093635A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Fabrication et optimisation de systèmes, procédés et compositions d'enzyme améliorés pour la manipulation de séquences
WO2014102774A1 (fr) 2012-12-26 2014-07-03 Evogene Ltd. Polynucléotides et polypeptides isolés, constructions et plantes comprenant lesdits polynucléotides et polypeptides isolés et leurs procédés d'utilisation pour augmenter l'efficacité d'utilisation de l'azote
WO2014145599A2 (fr) 2013-03-15 2014-09-18 The Broad Institute, Inc. Virus recombinant et ses préparations
US8911993B2 (en) 2010-12-09 2014-12-16 The Trustees Of The University Of Pennsylvania Compositions for treatment of cancer
WO2014204725A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Systèmes, procédés et compositions à double nickase crispr-cas optimisés, pour la manipulation de séquences
WO2014204729A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Administration, utilisation et applications thérapeutiques de systèmes crispr-cas et compositions pour cibler les troubles et maladies en utilisant des éléments viraux
WO2014204728A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Délivrance, modification et optimisation de systèmes, procédés et compositions pour cibler et modéliser des maladies et des troubles liés aux cellules post-mitotiques
WO2014204723A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Modèles oncogènes basés sur la distribution et l'utilisation de systèmes crispr-cas, vecteurs et compositions
WO2014204724A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Administration, modification et optimisation de systèmes guides tandems, méthodes et compositions pour la manipulation de séquence
WO2014204726A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Administration et utilisation de systèmes crispr-cas, vecteurs et compositions pour le ciblage et le traitement du foie
WO2014204727A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Génomique fonctionnelle utilisant des systèmes crispr-cas, procédés de composition, cribles et applications de ces derniers
US8927247B2 (en) 2008-01-31 2015-01-06 Cellectis, S.A. I-CreI derived single-chain meganuclease and uses thereof
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
WO2015065964A1 (fr) 2013-10-28 2015-05-07 The Broad Institute Inc. Génomique fonctionnelle utilisant des systèmes crispr-cas, compositions, procédés, cribles et applications de ces systèmes
WO2015089462A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Distribution, utilisation et applications thérapeutiques des systèmes crispr-cas et compositions pour l'édition du génome
WO2015089354A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Compositions et procédés d'utilisation de systèmes crispr-cas dans les maladies dues à une répétition de nucléotides
WO2015089486A2 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Systèmes, procédés et compositions pour manipulation de séquences avec systèmes crispr-cas fonctionnels optimisés
WO2015089465A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Relargage, utilisation et applications thérapeutiques de systèmes crispr-cas et compositions pour maladies et troubles viraux et attribuables au vhb
WO2015089419A2 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Délivrance, utilisation et applications thérapeutiques des systèmes crispr-cas et compositions permettant de cibler des troubles et maladies au moyen de constituants de délivrance sous forme de particules
WO2015089364A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Structure cristalline d'un système crispr-cas, et ses utilisations
WO2015089427A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Systèmes crispr-cas et méthodes de modification de l'expression de produits géniques, informations structurales et enzymes cas modulaires inductibles
WO2015089473A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Ingénierie de systèmes, procédés et compositions guides optimisées avec de nouvelles architectures pour la manipulation de séquences
WO2016205711A1 (fr) 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes
WO2017062439A1 (fr) 2015-10-05 2017-04-13 Precision Biosciences, Inc. Méganucléases modifiées avec des séquences de reconnaissances rencontrées dans le gène de la région constante alpha du récepteur des lymphocytes t humains
WO2017106657A1 (fr) 2015-12-18 2017-06-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes associés
WO2017112859A1 (fr) 2015-12-23 2017-06-29 Precision Biosciences, Inc. Méganucléases génétiquement modifiées comportant des séquences de reconnaissance que l'on trouve dans le gène de la microglobuline bêta-2 humaine
WO2017127807A1 (fr) 2016-01-22 2017-07-27 The Broad Institute Inc. Structure cristalline de crispr cpf1
WO2020219843A1 (fr) * 2019-04-26 2020-10-29 The University Of North Carolina At Chapel Hill Constructions de récepteurs antigéniques chimériques et leur utilisation dans des cellules car-t
US11046775B2 (en) 2017-02-14 2021-06-29 Kite Pharma, Inc. CD70 binding molecules and methods of use thereof

Patent Citations (105)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
US6468798B1 (en) 1991-12-17 2002-10-22 The Regents Of The University Of California Expression of cloned genes in the lung by aerosol and liposome-based delivery
US5792632A (en) 1992-05-05 1998-08-11 Institut Pasteur Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
US6238924B1 (en) 1992-05-05 2001-05-29 Institut Pasteur Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
US5350674A (en) 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
US5993434A (en) 1993-04-01 1999-11-30 Genetronics, Inc. Method of treatment using electroporation mediated delivery of drugs and genes
US6567694B2 (en) 1993-04-01 2003-05-20 Genetronics, Inc. Needle electrodes for mediated delivery of drugs and genes
US5830729A (en) 1996-04-18 1998-11-03 Institut Pasteur I Sce I-induced gene replacement and gene conversion in embryonic stem cells
US20040092907A1 (en) 1997-04-03 2004-05-13 Inovio As Method for muscle delivery of drugs, nucleic acids and other compounds
US7241573B2 (en) 1997-05-23 2007-07-10 Gendaq Ltd. Nucleic acid binding proteins
US7241574B2 (en) 1997-05-23 2007-07-10 Gendaq Ltd. Nucleic acid binding proteins
US6866997B1 (en) 1997-05-23 2005-03-15 Gendaq Limited Nucleic acid binding proteins
US6746838B1 (en) 1997-05-23 2004-06-08 Gendaq Limited Nucleic acid binding proteins
US6241701B1 (en) 1997-08-01 2001-06-05 Genetronics, Inc. Apparatus for electroporation mediated delivery of drugs and genes
US6233482B1 (en) 1997-08-01 2001-05-15 Genetronics, Inc. Method of electroporation mediated delivery of drugs and genes
US6516223B2 (en) 1997-08-01 2003-02-04 Genetronics, Inc. Apparatus for electroporation mediated delivery for drugs and genes
US6181964B1 (en) 1997-08-01 2001-01-30 Genetronics, Inc. Minimally invasive apparatus and method to electroporate drugs and genes into tissue
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US7595376B2 (en) 1998-03-02 2009-09-29 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6903185B2 (en) 1998-03-02 2005-06-07 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6678556B1 (en) 1998-07-13 2004-01-13 Genetronics, Inc. Electrical field therapy with reduced histopathological change in muscle
US7220719B2 (en) 1999-01-12 2007-05-22 Sangamo Biosciences, Inc. Modulation of endogenous gene expression in cells
US6979539B2 (en) 1999-01-12 2005-12-27 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6607882B1 (en) 1999-01-12 2003-08-19 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6824978B1 (en) 1999-01-12 2004-11-30 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7013219B2 (en) 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7585849B2 (en) 1999-03-24 2009-09-08 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7171264B1 (en) 1999-05-10 2007-01-30 Genetronics, Inc. Intradermal delivery of active agents by needle-free injection and electroporation
WO2001029058A1 (fr) 1999-10-15 2001-04-26 University Of Massachusetts Genes de voies d'interference d'arn en tant qu'outils d'interference genetique ciblee
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
US7173116B2 (en) 2000-03-03 2007-02-06 Genetronics Biomedical Corporation Nucleic acid formulations for gene delivery and methods of use
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US20050052630A1 (en) 2002-03-07 2005-03-10 Advisys, Inc. Constant current electroporation device and methods of use
US20030232410A1 (en) 2002-03-21 2003-12-18 Monika Liljedahl Methods and compositions for using zinc finger endonucleases to enhance homologous recombination
US20040014645A1 (en) 2002-05-28 2004-01-22 Advisys, Inc. Increased delivery of a nucleic acid construct in vivo by the poly-L-glutamate ("PLG") system
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
US20050070841A1 (en) 2002-07-04 2005-03-31 Inovio As Electroporation device and injection apparatus
US20040059285A1 (en) 2002-07-04 2004-03-25 Inovio As Electroporation device and injection apparatus
WO2004067753A2 (fr) 2003-01-28 2004-08-12 Cellectis Utilisation de meganucleases pour induire une recombinaison homologue ex vivo et in toto dans des tissus somatiques de vertebre et application de cette utilisation
US8119381B2 (en) 2005-10-18 2012-02-21 Duke University Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
US20070128708A1 (en) 2005-12-07 2007-06-07 Genetronics, Inc. Variable volume electroporation chamber and methods therefore
WO2008010009A1 (fr) 2006-07-18 2008-01-24 Cellectis Variants de méganucléase clivant une séquence d'adn cible provenant d'un gène rag et leurs utilisations
WO2008149176A1 (fr) 2007-06-06 2008-12-11 Cellectis Variants de méganucléase clivant une séquence cible d'adn issue du locus rosa26 de souris et leurs utilisations
WO2009013559A1 (fr) 2007-07-23 2009-01-29 Cellectis Variants de méganucléase clivant une séquence cible d'adn à partir du gène bêta de l'hémoglobine humaine et ses utilisations
US20090203140A1 (en) 2007-09-27 2009-08-13 Sangamo Biosciences, Inc. Genomic editing in zebrafish using zinc finger nucleases
WO2009059195A2 (fr) 2007-10-31 2009-05-07 Precision Biosciences Méganucléases monocaténaires conçues rationnellement contenant des séquences de reconnaissance non palindromiques
US8927247B2 (en) 2008-01-31 2015-01-06 Cellectis, S.A. I-CreI derived single-chain meganuclease and uses thereof
US8338157B2 (en) 2008-03-11 2012-12-25 Precision Biosciences, Inc. Rationally-designed meganuclease variants of lig-34 and I-crei for maize genome engineering
US8586363B2 (en) 2009-12-10 2013-11-19 Regents Of The University Of Minnesota TAL effector-mediated DNA modification
US8440431B2 (en) 2009-12-10 2013-05-14 Regents Of The University Of Minnesota TAL effector-mediated DNA modification
US8440432B2 (en) 2009-12-10 2013-05-14 Regents Of The University Of Minnesota Tal effector-mediated DNA modification
US8450471B2 (en) 2009-12-10 2013-05-28 Regents Of The University Of Minnesota TAL effector-mediated DNA modification
US8697853B2 (en) 2009-12-10 2014-04-15 Regents Of The University Of Minnesota TAL effector-mediated DNA modification
US8911993B2 (en) 2010-12-09 2014-12-16 The Trustees Of The University Of Pennsylvania Compositions for treatment of cancer
WO2014018423A2 (fr) 2012-07-25 2014-01-30 The Broad Institute, Inc. Protéines de liaison à l'adn inductibles et outils de perturbation du génome et leurs applications
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8895308B1 (en) 2012-12-12 2014-11-25 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
WO2014093718A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Procédés, systèmes et appareil pour identifier des séquences cibles pour les enzymes cas ou des systèmes crispr-cas pour des séquences cibles et transmettre les résultats associés
WO2014093622A2 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques
WO2014093595A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Systèmes de composants de crispr-cas, procédés et compositions pour la manipulation de séquences
WO2014093712A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Fabrication de systèmes, procédés et compositions de guide optimisées pour la manipulation de séquences
WO2014093694A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Systèmes, procédés et compositions de crispr-nickase cas pour la manipulation de séquences dans les eucaryotes
WO2014093635A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Fabrication et optimisation de systèmes, procédés et compositions d'enzyme améliorés pour la manipulation de séquences
US8932814B2 (en) 2012-12-12 2015-01-13 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8771945B1 (en) 2012-12-12 2014-07-08 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8945839B2 (en) 2012-12-12 2015-02-03 The Broad Institute Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8871445B2 (en) 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8889418B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
WO2014093661A2 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Systèmes crispr-cas et procédés pour modifier l'expression de produits de gène
WO2014093709A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Procédés, modèles, systèmes et appareil pour identifier des séquences cibles pour les enzymes cas ou des systèmes crispr-cas pour des séquences cibles et transmettre les résultats associés
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
WO2014093701A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Génomique fonctionnelle employant des systèmes crispr-cas, des compositions, des procédés, des banques d'inactivation et leurs applications
US8999641B2 (en) 2012-12-12 2015-04-07 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
WO2014102774A1 (fr) 2012-12-26 2014-07-03 Evogene Ltd. Polynucléotides et polypeptides isolés, constructions et plantes comprenant lesdits polynucléotides et polypeptides isolés et leurs procédés d'utilisation pour augmenter l'efficacité d'utilisation de l'azote
WO2014145599A2 (fr) 2013-03-15 2014-09-18 The Broad Institute, Inc. Virus recombinant et ses préparations
WO2014204729A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Administration, utilisation et applications thérapeutiques de systèmes crispr-cas et compositions pour cibler les troubles et maladies en utilisant des éléments viraux
WO2014204727A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Génomique fonctionnelle utilisant des systèmes crispr-cas, procédés de composition, cribles et applications de ces derniers
WO2014204726A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Administration et utilisation de systèmes crispr-cas, vecteurs et compositions pour le ciblage et le traitement du foie
WO2014204724A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Administration, modification et optimisation de systèmes guides tandems, méthodes et compositions pour la manipulation de séquence
WO2014204723A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Modèles oncogènes basés sur la distribution et l'utilisation de systèmes crispr-cas, vecteurs et compositions
WO2014204728A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Délivrance, modification et optimisation de systèmes, procédés et compositions pour cibler et modéliser des maladies et des troubles liés aux cellules post-mitotiques
WO2014204725A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Systèmes, procédés et compositions à double nickase crispr-cas optimisés, pour la manipulation de séquences
WO2015065964A1 (fr) 2013-10-28 2015-05-07 The Broad Institute Inc. Génomique fonctionnelle utilisant des systèmes crispr-cas, compositions, procédés, cribles et applications de ces systèmes
WO2015089465A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Relargage, utilisation et applications thérapeutiques de systèmes crispr-cas et compositions pour maladies et troubles viraux et attribuables au vhb
WO2015089473A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Ingénierie de systèmes, procédés et compositions guides optimisées avec de nouvelles architectures pour la manipulation de séquences
WO2015089486A2 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Systèmes, procédés et compositions pour manipulation de séquences avec systèmes crispr-cas fonctionnels optimisés
WO2015089462A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Distribution, utilisation et applications thérapeutiques des systèmes crispr-cas et compositions pour l'édition du génome
WO2015089419A2 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Délivrance, utilisation et applications thérapeutiques des systèmes crispr-cas et compositions permettant de cibler des troubles et maladies au moyen de constituants de délivrance sous forme de particules
WO2015089364A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Structure cristalline d'un système crispr-cas, et ses utilisations
WO2015089427A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Systèmes crispr-cas et méthodes de modification de l'expression de produits géniques, informations structurales et enzymes cas modulaires inductibles
WO2015089354A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Compositions et procédés d'utilisation de systèmes crispr-cas dans les maladies dues à une répétition de nucléotides
WO2015089351A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Compositions et procédés d'utilisation de systèmes crispr-cas dans les maladies dues à une répétition de nucléotides
WO2016205711A1 (fr) 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes
WO2017062439A1 (fr) 2015-10-05 2017-04-13 Precision Biosciences, Inc. Méganucléases modifiées avec des séquences de reconnaissances rencontrées dans le gène de la région constante alpha du récepteur des lymphocytes t humains
WO2017106657A1 (fr) 2015-12-18 2017-06-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes associés
WO2017112859A1 (fr) 2015-12-23 2017-06-29 Precision Biosciences, Inc. Méganucléases génétiquement modifiées comportant des séquences de reconnaissance que l'on trouve dans le gène de la microglobuline bêta-2 humaine
WO2017127807A1 (fr) 2016-01-22 2017-07-27 The Broad Institute Inc. Structure cristalline de crispr cpf1
US11046775B2 (en) 2017-02-14 2021-06-29 Kite Pharma, Inc. CD70 binding molecules and methods of use thereof
WO2020219843A1 (fr) * 2019-04-26 2020-10-29 The University Of North Carolina At Chapel Hill Constructions de récepteurs antigéniques chimériques et leur utilisation dans des cellules car-t

Non-Patent Citations (83)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
BARNES ET AL., J FOR IMMUNOTHERAPY OF CANCER, vol. 3, no. 1, 2015, pages 1 - 10
BERARDI, A C ET AL.: "Functional isolation and characterization of human hematopoietic stem cells", SCIENCE, vol. 267, no. 5194, 1995, pages 04 - 8
BERENSON, R J ET AL.: "Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma", BLOOD, vol. 77, no. 8, 1991, pages 1717 - 22
BEURDELE ET AL., NAT COMMUN, no. 4, 2013, pages 1762
BIBIKOVA ET AL., GENETICS, vol. 161, 2002, pages 1169 - 1175
BIBIKOVA ET AL., SCIENCE, vol. 300, 2003, pages 764
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BORDENKABAT, PNAS, vol. 84, 1987, pages 2440 - 2443
CAIFREEMAN, INUNUNOLOGICAL REVIEWS, vol. 229, no. 1, 2009, pages 244 - 258
CHEVALIERSTODDARD, NUCLEIC ACIDS RESEARCH, vol. 29, 2001, pages 3757 - 3774
CHOTHIA ET AL., NATURE, vol. 241, 1989, pages 544 - 546
CHOTHIALESK, J MOL. BIOL., vol. 196, no. 4, 1987, pages 901 - 917
CHRISTIAN ET AL., GENETICS, vol. 186, 2010, pages 757 - 761
COUGOT ET AL., TRENDS IN BIOCHEM. SCI, vol. 29, 2001, pages 436 - 444
DAI ET AL., FRONTIERS IN IMMUNOLOGY, vol. 11, 2020, pages 539654
DEL RIO ET AL., JOURNAL OF LEUKOCYTE BIOLOGY, vol. 87, no. 2, 2010, pages 223 - 235
DOTTI ET AL., IMMUNOL. REV, vol. 257, no. 1, 2014, pages 107
ELANGO: " Biochim.Biophys. Res. Commun", BIOPHYS. RES. COMMUN, vol. 330, 2005, pages 958 - 966
GAJ ET AL., NAT METHODS, vol. 1 -4, no. 8, 2012, pages 805 - 7
GHOSH ET AL., GLYCOBIOLOGY, vol. 5, 1991, pages 505 - 10
GISHSTATES, NATURE GENET., vol. 3, 1993, pages 266 - 272
GRANGERRICKERT, CYTOKINE GROWTH FACTOR REV, vol. 14, no. 3-4, 2003, pages 289 - 296
HASSAN ET AL.: "Biological characteristics of stem cells from foetal, cord blood and extraembryonic tissues", JOURNAL OF THE ROYAL SOCIETY, INTERFACE, vol. 7, 2010, pages 689 - 706
HE YUAN ET AL: "The Implementation of TNFRSF Co-Stimulatory Domains in CAR-T Cells for Optimal Functional Activity", CANCERS, vol. 14, no. 2, 8 January 2022 (2022-01-08), CH, pages 299, XP093118067, ISSN: 2072-6694, DOI: 10.3390/cancers14020299 *
HENIKOFF SHENIKOFF J G, PROC NATL ACAD SCI, vol. 89, 1992, pages 10915 - 9
HOLLIGER ET AL., PROC. NATL. ACAD SCI. USA, vol. 90, 1993, pages 6444 - 6448
HOLLIGER, PHUDSON, J: "Engineered antibody fragments and the rise of single domains", NATURE BIOTECHNOLOGY, vol. 23, no. 9, 2005, pages 1126 - 1136, XP008076746, DOI: 10.1038/nbt1142
HONEGGERPLUCKTHUN, JOURNAL OF MOLECULAR BIOLOGY, vol. 309, 2001, pages 657 - 670
HSU ET AL., JOURNAL OF BIOLOGICAL CHEMISTR, vol. 272, no. 21, 1997, pages 13471 - 13474
HSU ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 21, 1997, pages 13471 - 13474
HSU: "ATAR, A Novel Tumor Necrosis Factor Receptor Family Member, Signals through TRAF2 and TRAF5", THE JOURNAL OF BIOLOGICAL, no. 21, 1997, pages 13471 - 13474
HUSTON ET AL., PROC. NATL. ACAD SCI. USA, vol. 85, 1988, pages 5879 - 5883
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
JOUNGSANDER, NAT REV MOL CELL BIOL, vol. 14, no. 1, 2013, pages 49 - 55
KABAT ET AL., U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, 1991
KAIVALEKAR ET AL., IMMUNITY, vol. 44, no. 2, 2016, pages 380
KERSHAW ET AL., CLIN. CANCER RES, vol. 12, 2006, pages 6106
KIM ET AL., GENOME RES, vol. 22, no. 7, 2012, pages 1327 - 33
KIM ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 93, 1996, pages 1156 - 1160
LEI ANHUA ET AL: "A Chimera Antigen Receptor Containing TLR4 Signaling Domain Enhances CAR-iMACs Polarization and Potency against Solid Tumors", RESEARCH SQUARE, 31 January 2022 (2022-01-31), XP093083090, Retrieved from the Internet <URL:https://assets.researchsquare.com/files/rs-1279112/v1/921a5da4-1419-48df-8df0-3586852613b5.pdf> [retrieved on 20230918], DOI: 10.21203/rs.3.rs-1279112/v1 *
LONG ET AL., NAT. MED., vol. 21, no. 6, 2015, pages 581
LUTZ, JOURNAL OF IMMUNOLOGICAL METHODS, vol. 223, no. 1, 1999, pages 77 - 92
MADDEN ET AL., METH. ETIN,MOL, vol. 266, 1996, pages 131 - 141
MATLUNG ET AL., CELL REPORTS, vol. 23, no. 13, 2018, pages 3946 - 3959
MAUS ET AL., BLOOD, vol. 123, no. 17, 2014, pages 2625
MILLER ET AL., NATURE BIOTECHNOLOGY, vol. 25, 2007, pages 778 - 785
MILLER ET AL., NATURE BIOTECHNOLOGY, vol. 29, 2011, pages 143 - 148
MILLER ET AL., THE EMBO JOURNAL, vol. 4, 1985, pages 1609 - 1614
MILONE ET AL., MOLECULAR THERAPY, vol. 17, no. 8, 2009, pages 1453 - 1464
MORRISSEY ET AL., ELIFE, 2018, pages 7
MOSCOUBOGDANOVE, SCIENCE, vol. 326, 2009, pages 1501 - 1512
MOSSEREDWARDS, NATURE REVIEWS IMMUNOLOGY, vol. 8, no. 12, 2008, pages 958 - 969
NACHEVABERZAL-HERRANZ, EUR. J. BIOCHEM., vol. 270, 2003, pages 1485 - 65
NING ET AL., FRONTIERS IN IMMUNOLOGY, vol. 12, 2021, pages 654960
NISHIKAWA ET AL., HUM GENE THER, vol. 12, no. 8, 2001, pages 861 - 70
PAN KEVIN ET AL: "CAR race to cancer immunotherapy: from CAR T, CAR NK to CAR macrophage therapy", JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH, vol. 41, no. 1, 31 March 2022 (2022-03-31), London UK, pages 119, XP093121023, ISSN: 1756-9966, Retrieved from the Internet <URL:https://link.springer.com/article/10.1186/s13046-022-02327-z/fulltext.html> DOI: 10.1186/s13046-022-02327-z *
PARK ET AL., CANCER IMMUNOL. IMMUNOTHER, vol. 61, no. 2, 2012, pages 203
PASEROOLIVE, IMMUNOLOGY LETTERS, vol. 151, no. 1-2, 2013, pages 71 - 75
PRESTA, CURR. OP. STRUCT. BIOL, vol. 2, 1992, pages 593 - 596
PROTEIN ENGINEERING, vol. 8, no. 7, 1995, pages 725 - 731
RANLIREZ ET AL., NUCL ACIDS RES, vol. 40, no. 12, 2012, pages 5560 - 8
REICHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
ROSENBERG ET AL., NEW ENG. J. OF MED, vol. 319, pages 1676 - 1988
SCHARENLΗERG ET AL., CURR GENE THER, vol. 13, no. 4, 2013, pages 291 - 303
SCHENBOMMIERENDORF, NUC ACIDS RES, vol. 13, 1985, pages 6223 - 36
SEDY ET AL., NATURE IMMUNOLOGY, vol. 6, no. 1, 2005, pages 90 - 98
SHUI ET AL., JOURNAL OF LEUKOCYTE BIOLOGY, vol. 89, no. 4, 2011, pages 517 - 523
SOROBETEABRODSKY, CELL HOST & MICROBE, vol. 24, no. 2, 2018, pages 187 - 188
STEINBERG ET AL., PLOS ONE, vol. 8, no. 10, 2013, pages 77992
STEPINSKI ET AL., RNA, vol. 7, 2001, pages 1468 - 95
UI-TEI ET AL., FEBS LETTERS, vol. 479, 2000, pages 79 - 82
URIBE-QUEROL EILEEN ET AL: "Phagocytosis: Our Current Understanding of a Universal Biological Process", FRONTIERS IN IMMUNOLOGY, vol. 11, 2 June 2020 (2020-06-02), Lausanne, CH, XP093197539, ISSN: 1664-3224, DOI: 10.3389/fimmu.2020.01066 *
URNOV ET AL., NATURE REVIEWS GENETICS, vol. 11, 2010, pages 636 - 646
VAN FURTH ET AL., BULLETIN OF THE WORLD HEALTH ORGANIZATION, vol. 46, no. 6, 1972, pages 845 - 852
VO, LINDA TGEORGE Q DALEY: "De novo generation of HSCs from somatic and pluripotent stem cell sources", BLOOD, vol. 125, no. 17, 2015, pages 2641 - 8, XP055323623, DOI: 10.1182/blood-2014-10-570234
WOLFE ET AL., ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE, vol. 29, 2000, pages 183 - 212
WUKABAT, J EXP MED., vol. 132, no. 2, 1970, pages 211 - 50
YE ET AL.: "The Structural Basis for the Recognition of Diverse Receptor Sequences by TRAF2", MOLECULAR CELL, vol. 4, pages 321 - 330, XP055835885, DOI: 10.1016/S1097-2765(00)80334-2
ZARNEGAR ET AL., NATURE IMMUNOLOGY, vol. 9, no. 12, 2008, pages 1371 - 1378
ZHANG ET AL., ONCOTARGET, vol. 6, no. 32, 2015, pages 33961
ZHANG, J. COMPUT. BIOL, vol. 7, no. 1-2, 2000, pages 203 - 14

Similar Documents

Publication Publication Date Title
US20240279665A1 (en) Altering Gene Expression in CART Cells and Uses Thereof
US11952408B2 (en) HPV-specific binding molecules
US11739297B2 (en) Method of increasing tumor killing activity of macrophages or monocytes comprising chimeric antigen receptor
US11535869B2 (en) CD8-specific antibody constructs and compositions thereof
KR20230069042A (ko) 원형 rna 조성물 및 방법
US20190175651A1 (en) Immortalized car-t cells genetically modified to elminate t-cell receptor and beta 2-microglobulin expression
CN108290956B (zh) 靶向psca的嵌合抗原受体
JP7085990B2 (ja) クロロトキシンドメインを含むキメラ抗原レセプター
CN110944652A (zh) T细胞抗原靶向的嵌合抗原受体(car)以及在细胞疗法中的用途
CA3204161A1 (fr) Utilisation de vecteurs viraux ciblant cd8
US20240316102A1 (en) Engineered natural killer (nk) cells and related methods
US20210246221A1 (en) Chimeric antigen receptor targeting sialyl lewis a and uses thereof
CN116113422A (zh) 免疫细胞的mRNA转染
WO2019097083A1 (fr) Cellule k562 modifiée
WO2023193003A2 (fr) Constructions d&#39;anticorps spécifiques de cd4 et compositions et utilisations associées
US20220411479A1 (en) Cd20 chimeric antigen receptors and methods of use for immunotherapy
US20250000975A1 (en) Preparation for chimeric antigen receptor immune cell constructed on basis of gas6 and use of chimeric antigen receptor immune cell
WO2024030583A2 (fr) Nouvelles constructions pour récepteurs antigéniques chimériques et leurs utilisations
WO2023150518A1 (fr) Vecteurs lentiviraux ciblant cd3 et leurs utilisations
WO2024236547A1 (fr) Cellules phagocytaires modifiées exprimant des récepteurs antigéniques chimériques comprenant un domaine co-stimulateur de médiateur d&#39;entrée du virus de l&#39;herpès (hvem) et utilisations associées
WO2025046513A1 (fr) Procédés de fabrication de cellules d&#39;origine myéloïde à partir de cellules souches hématopoïétiques et compositions et utilisations associées
US12258379B2 (en) Compositions and methods for switchable CAR T cells using surface-bound sortase transpeptidase
WO2024007020A1 (fr) Combinaison de cellules tueuses naturelles (nk) modifiées et d&#39;une thérapie par anticorps et méthodes associées
TW202430629A (zh) 利用慢病毒載體轉染之新穎大規模car-t免疫細胞製造方法

Legal Events

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

Ref document number: 24730782

Country of ref document: EP

Kind code of ref document: A1