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WO2025085846A1 - Interleukines modifiées et utilisations associées - Google Patents

Interleukines modifiées et utilisations associées Download PDF

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Publication number
WO2025085846A1
WO2025085846A1 PCT/US2024/052114 US2024052114W WO2025085846A1 WO 2025085846 A1 WO2025085846 A1 WO 2025085846A1 US 2024052114 W US2024052114 W US 2024052114W WO 2025085846 A1 WO2025085846 A1 WO 2025085846A1
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Prior art keywords
immune cell
seq
receptor
sequence
domain
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Jingli Zhang
Timothy P. RILEY
Carl Alexander Kamb
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A2 Biotherapeutics Inc
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A2 Biotherapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • 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
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4204—Epidermal growth factor receptors [EGFR]
    • 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
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—Mesothelin [MSLN]
    • 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
    • A61K40/4264—Cancer antigens from embryonic or fetal origin
    • A61K40/4266—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • 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
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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]
    • A61K2239/11—Antigen recognition 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]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • XML copy created on October 18, 2024 is named 061250-559001WO.xml and is 552 KB in size.
  • TECHNICAL FIELD [0003] The disclosure relates to the fields of adoptive cell therapy and cancer therapeutics.
  • These immune cell therapies such as CAR-T cell therapies have shown remarkable efficacy against some hematologic cancers (e.g., non-Hodgkin lymphoma and multiple myeloma). However, these cell therapies have shown little success targeting other hematologic cancer and solid tumors.
  • the low efficacy of these immune cell therapies in solid tumors is largely due to antigen selectivity profiles, systemic toxicity, short half-lives of immunostimulants, poor tissue penetration, and immune cell exhaustion.
  • the tumor microenvironment in solid cancers actively suppresses immune cell function of the natural and adoptive immune cells by decreasing proliferation and cytotoxicity, and increasing apoptosis.
  • engineered immune cells preferably achieve the challenging dual-objective of potency and selectivity to overcome the key obstacle in cancer therapy - selective
  • clinical studies have attempted to co-opt the major stimulatory receptors of the adaptive immune response: antigen receptors, co-stimulatory receptors, and cytokine receptors.
  • a promising new subtype of engineered immune cells involves synthetic logic gates; i.e., receptor systems designed to recognize and respond to complex molecular patterns on target cells.
  • examples of synthetic biology logic gates include OR, AND, and NOT gates that respond to different combinations of antigen pairs (see for review DiAndreth et al., 2022 PMID: 35561999; Labanieh and Mackall, 2023 PMID: 36813894).
  • a NOT gate is a type of cellular control system that activates when two conditions are satisfied by a target cell it encounters: (i) the target expresses an activating or A-antigen; and, (ii) the target cell lacks expression of a second blocker or B-antigen.
  • This molecular device exemplified by the Tmod dual-receptor system (see for review DiAndreth et al., Clin.
  • Immunol. (2022) Aug:241) can exploit situations where healthy normal cells express both the A- and B- antigens and cancer cells express the A- and not the B-antigen. Such situations occur, for example, when the cancer cells have lost expression of one allele via loss of heterozygosity (LOH; Hamburger et al., 2020 PMID: 33012527). Tmod can also be utilized in situations that do not involve genetic deletions such as LOH; for example, in blood cancer where malignant cells lack expression of a B-antigen for epigenetic reasons. [0008] .
  • immune cell therapies can be “armored” with a localized expression of pro-inflammatory cytokines or ligands.
  • “armored” CAR T been further engineered to secrete cytokines Docket No.: 061250-559001WO or express soluble or tethered ligands designed to further improve immune cell efficacy.
  • compositions and methods for engineered immune cells for logic- gated tumor cell killing are provided herein.
  • the present disclosure relates to an immune cell for treating a cancer, the immune cell comprising a membrane tethered cytokine and one or both of an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non- cancerous cell and an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by a cancer cell.
  • the cytokine is an interleukin, e.g., selected from an IL-12, an IL-2, an IL- 15, an IL-18, and an IL-21.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 1.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human IL-12, a human IL-2, a human IL- 15, a human IL-18, and a human IL-21.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one or both of SEQ ID NO: 23 or SEQ ID NO: 24.
  • the cytokine comprises a fusion protein comprising an IL-12A polypeptide and an IL-12B polypeptide.
  • the fusion protein comprises a linker separating the IL- 12A polypeptide and the IL-12B polypeptide.
  • the linker may comprise a polypeptide sequence of one or more of (GS)n (SEQ ID NO: 41), (GSGGS)n (SEQ ID NO: 42), (GGGS)n (SEQ ID NO: 43), (GGGGS)n (SEQ ID NO: 44), (GGGGGS)n (SEQ ID NO: 45), or (GGGGGGS)n (SEQ ID NO: 46), where n is an integer of at least one (and generally from 3 to 10), and/or GSGSSRGGSGSGGSGGGGSK (SEQ ID NO: 47), GGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 48), or (G4Q)4 (SEQ ID NO: 49) or the linker may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least at least 99%, or 100% identical any sequence disclosed in Table 7.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to each of SEQ ID NO: 23 and SEQ ID NO: 24.
  • the cytokine comprises a fusion protein comprising an IL-12A polypeptide and an IL-12B polypeptide.
  • the fusion protein comprises a linker separating the IL-12A polypeptide and the IL-12B polypeptide.
  • the linker may comprise a polypeptide sequence of one or more of (GS)n (SEQ ID NO: 41), (GSGGS)n (SEQ ID NO: 42), (GGGS)n (SEQ ID NO: 43), (GGGGS)n (SEQ ID NO: 44), (GGGGGS)n (SEQ ID NO: 45), or (GGGGGGS)n (SEQ ID NO: 46), where n is an integer of at least one (and generally from 3 to 10), and/or GSGSSRGGSGSGGSGGGGSK (SEQ ID NO: 47), GGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 48), or (G4Q)4 (SEQ ID NO: 49) or the linker may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 7.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 64.
  • the cytokine comprises a fusion protein comprising an IL-12A polypeptide and an IL-12B polypeptide.
  • the fusion protein comprises a linker separating the IL-12A polypeptide and the IL-12B polypeptide.
  • the linker may comprise a polypeptide sequence of one or more of (GS)n (SEQ ID NO: 41), (GSGGS)n (SEQ ID NO: 42), (GGGS)n (SEQ ID NO: 43), (GGGGS)n (SEQ ID NO: 44), (GGGGGS)n (SEQ ID NO: 45), or (GGGGGGS)n (SEQ ID NO: 46), where n is an integer of at least one (and generally from 3 to 10), and/or GSGSSRGGSGSGGSGGGGSK (SEQ ID NO: 47), GGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 48), or (G4Q)4 (SEQ ID NO: 49) or the linker may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 7.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one SEQ ID NO: 1 to SEQ ID NO: 22.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to either of SEQ ID NO: 27 or SEQ ID NO: 28.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 29, 30, 1046, 1047, and 1058.
  • the cytokine comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 31 or SEQ ID NO: 32.
  • the cytokine is tethered to the immune cell’s membrane via a transmembrane domain operably linked to the cytokine.
  • the transmembrane region comprises a PDGFRb transmembrane region, a B7 transmembrane region, a CD25 transmembrane region, a CD137 transmembrane region, a B7 transmembrane region, or a CD19 transmembrane region.
  • the cytokine is tethered to the immune cell’s membrane via a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 8.
  • the cytokine is tethered to the immune cell’s membrane via a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 55, 56, 57, 1048, 1076, 1077, or 1086.
  • the membrane tethered cytokine further comprises a hinge domain.
  • the hinge domain comprises a (G4Q) 2 , (G4Q) 5 , (G4Q) 10 , (G4Q) 10 -CD 2 5_full, CD25_hinge, IgG4, EGF3, or EGF7 hinge region.
  • the hinge domain comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 6.
  • the membrane tethered cytokine further comprises a linker domain.
  • the linker comprises a polypeptide sequence of one or more of (GS)n (SEQ ID NO: 41), (GSGGS)n (SEQ ID NO: 42), (GGGS)n (SEQ ID NO: 43), (GGGGS)n (SEQ ID NO: 44), (GGGGGS)n (SEQ ID NO: 45), or (GGGGGGS)n (SEQ ID NO: 46), where n is an integer of at least one (and generally from 3 to 10), and/or GSGSSRGGSGSGGSGGGGSK (SEQ ID NO: 47), GGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 48), or (G4Q) 4 (SEQ ID NO: 49) or the linker domain comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 7.
  • the membrane tethered cytokine further is expressed by a polynucleotide comprising one or both of a constitutive promoter and an inducible promoter. In some cases, the membrane tethered cytokine further is expressed by a polynucleotide comprising both of a constitutive promoter and an inducible promoter. In some cases, the inducible promoter is an activation induced promoter. In some cases, the activation induced promoter comprises one or more nuclear factor of activated cells response elements (NFAT RE). In some cases, the activation induced promoter comprises 1, 2, 3, 4, 5, 6, or more NFAT REs.
  • NFAT RE nuclear factor of activated cells response elements
  • the constitutive promotor comprises one or both of a eukaryotic translation elongation factor 1 alpha (EF1a) promoter or a TATA box promoter.
  • the membrane tethered cytokine further comprises an intracellular domain.
  • the intracellular domain is capable of providing a stimulatory signal.
  • the intracellular domain comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 12.
  • the intracellular domain comprises a PDGFRB, B7, CD137, CD27, OX40, Dap10, DAP12, MyD88, MyD88/CD40, CD3e Intracellular domain.
  • the membrane tethered cytokine comprises an PDGFRB transmembrane and intracellular domain.
  • the PDGFRB transmembrane and intracellular domain comprises a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1086.
  • the membrane tethered cytokine further comprises a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus
  • the immune cell further expresses a receptor for the cytokine.
  • the receptor is selected from an IL-12 receptor (e.g., one or both of IL-12R-beta-1 and IL- 12R-beta-2), an IL-2 receptor (e.g., one or both of L-2R-alpha and IL-2R-beta), an IL-18 receptor (e.g., one or both of IL-18R-alpha and IL-18R beta), or an IL-21 receptor.
  • the receptor for the cytokine binds to the membrane tethered cytokine and when bound transmits an intracellular signal consistent with natural cytokine binding.
  • the immune cell expresses the inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non-cancerous cell.
  • the inhibitor antigen is an HLA class I allele.
  • the HLA class I allele comprises HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.
  • the HLA- A allele comprises an HLA-A*02 allele, an HLA-A*03 allele, or HLA-A*11 allele
  • the HLA-B allele comprises an HLA-B*07 allele
  • the C allele comprises an HLA-C*07 allele
  • the HLA class I allele comprises an HLA-E allele. Docket No.: 061250-559001WO [0033]
  • the immune cell expresses the inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non-cancerous cell.
  • the inhibitor antigen is an HLA-A*02 allele and the extracellular ligand binding domain of the inhibitor receptor comprises a set of complementarity-determining region (CDR) sequences underlined in any one of SEQ ID NO: 153 to SEQ ID NO: 165.
  • CDR complementarity-determining region
  • the inhibitor antigen is an HLA-A*02 allele and the extracellular ligand binding domain of the inhibitor receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region as disclosed in any one of SEQ ID NO: 153 to SEQ ID NO: 165 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto, wherein the VH and VL are separated in SEQ ID NO: 153 to SEQ ID NO: 165 by the GGGGSGGGGSGGGGSGG (SEQ ID NO: 152) linker.
  • VH heavy chain variable
  • VL light chain variable
  • the inhibitor antigen is an HLA-A*02 allele and the extracellular ligand binding domain of the inhibitor receptor comprises an antibody fragment or a single chain Fv antibody fragment (scFv) as disclosed in any one of SEQ ID NO: 153 to SEQ ID NO: 165 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • scFv single chain Fv antibody fragment
  • the inhibitor antigen is an HLA-A*02 allele and the inhibitor antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of: SEQ ID NO: 121 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto or the inhibitor antigen is an HLA-A*02 allele and the inhibitor antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of SEQ ID NO: 123 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • the immune cell expresses the inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non-cancerous cell.
  • the inhibitor antigen is an HLA-A*03 allele and the extracellular ligand binding domain of the inhibitor receptor comprises a set of complementarity-determining region (CDR) sequences underlined in any one of the SEQ ID NOs in Table 13 or Table 14.
  • CDR complementarity-determining region
  • the inhibitor antigen is an HLA-A*03 allele and the extracellular ligand binding domain of the inhibitor receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region as disclosed in any one of SEQ ID NO: 1099 to SEQ ID NO: 1110 or SEQ ID NO: 1125- 1138, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto, wherein the VH and VL are separated in SEQ ID NO: 1099 to SEQ ID NO: 1110 or SEQ ID NO: 1125-1138 by the GGGGSGGGGSGGGGSGG (SEQ ID NO: 152) linker.
  • VH heavy chain variable
  • VL light chain variable
  • the inhibitor antigen is an HLA-A*03 allele and the extracellular ligand binding domain of the inhibitor receptor comprises an antibody or a single chain Fv antibody fragment (scFv) as disclosed in any one of SEQ ID NO: 1099 to SEQ ID NO: 1110 or SEQ ID NO: 1125-1138 or Docket No.: 061250-559001WO a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • scFv single chain Fv antibody fragment
  • the inhibitor antigen is an HLA-A*03 allele and the inhibitor antigen comprises HLA-A*03 and the inhibitory receptor comprises a sequence of: (SEQ ID NO: 1045) or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the immune cell expresses the inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non-cancerous cell.
  • the inhibitor antigen is an HLA-A*011 allele and the extracellular ligand binding domain of the inhibitor receptor comprises a set of complementarity-determining region (CDR) sequences underlined in any one of SEQ ID NO: 1139 to SEQ ID NO: 1147.
  • CDR complementarity-determining region
  • the inhibitor antigen is an HLA-A*011 allele and the extracellular ligand binding domain of the inhibitor receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region as disclosed in any one of SEQ ID NO: 1139 to SEQ ID NO: 1147 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto, wherein the VH and VL are separated in SEQ ID NO: 1139 to SEQ ID NO: 1147 by the GGGGSGGGGSGGGGSGG (SEQ ID NO: 152) linker.
  • VH heavy chain variable
  • VL light chain variable
  • the inhibitor antigen is an HLA-A*011 allele and the extracellular ligand binding domain of the inhibitor receptor comprises an antibody fragment or a single chain Fv antibody fragment (scFv) as disclosed in any one of SEQ ID NO: 1139 to SEQ ID NO: 1147 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor antigen is an HLA-A*011 allele and the inhibitor antigen comprises HLA-A*011 and the inhibitory receptor comprises a sequence of: (SEQ ID NO: 1045) or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the inhibitor receptor comprises a leukocyte immunoglobulin like receptor B1 (LILRB1) intracellular domain, comprises an LILRB1 hinge, and/or an LILRB1 transmembrane domains, or functional variants thereof.
  • LILRB1 leukocyte immunoglobulin like receptor B1
  • the LILRB1 intracellular domain, the LILRB1 hinge, and/or the LILRB1 transmembrane domains comprise a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 11.
  • the inhibitor receptor comprises a leukocyte immunoglobulin like receptor B1 (LILRB1) intracellular domain or a functional variant thereof.
  • the LILRB1 intracellular domain comprises the sequence of SEQ ID NO: 636, or a sequence having at least 85%, at least 90%, at least 95%, at 97%, at least 99% identity, or 100% identical thereto.
  • the inhibitor receptor comprises an LILRB1 hinge and an LILRB1 Docket No.: 061250-559001WO transmembrane domains, or functional variants thereof.
  • the LILRB1 transmembrane domain comprises the sequence of SEQ ID NO: 640, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 hinge domain comprises the sequence of SEQ ID NO: 639, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 hinge and LILRB1 transmembrane domains comprise the sequence of SEQ ID NO: 641, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 transmembrane and LILRB1 intracellular domains comprise the sequence of SEQ ID NO: 642, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 hinge, LILRB1 transmembrane, and LILRB1 intracellular domains comprise the sequence of SEQ ID NO: 643 or SEQ ID NO: 644, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the inhibitor receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • the immune cell expresses the activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by a cancer cell.
  • binding of the activator antigen by the activator receptor activates or promotes activation of the immune cell.
  • the activator antigen is selected from the antigens listed in Table 9.
  • the extracellular ligand binding domain specific to an activator antigen comprises an antibody listed in Table 9, comprises an antibody fragment of an antibody listed in Table 9, comprises a heavy chain variable (VH) region and/or a light chain variable (VL) region of an antibody listed in Table 9, or comprises a set of complementarity-determining region (CDR) sequences of an antibody listed in Table 9.
  • the activator antigen is selected from the group consisting of epidermal growth factor receptor (EGFR), mesothelin (MSLN), CEA cell adhesion molecule 5 (CEA), transferrin receptor (TFRC), HLA-E, erb-b2 receptor tyrosine kinase 2 (HER2), mesothelin (MSLN), PSMA, CD33, CD19 molecule (CD19), CLL-1, CD53, SPN, ITGA4, SELPLG, and CLEC12A or a peptide antigen thereof.
  • the activator receptor comprises extracellular ligand binding domain specific to an activator receptor listed in Table 10, comprises an scFv of an activator receptor listed in Table 10, comprises a heavy chain variable (VH) region and/or a light chain variable (VL) region of activator receptor listed in Table 10, or comprises a set of complementarity-determining region (CDR) sequences of activator receptor listed in Table 10.
  • VH heavy chain variable
  • VL light chain variable
  • CDR complementarity-determining region
  • the activator receptor comprises a CD28 co-stimulatory domain comprising an acid sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 65) or a Docket No.: 061250-559001WO sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor comprises a 4-1BB co-stimulatory domain comprising an amino acid sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 616) or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor comprises a CD3 ⁇ activation domain comprising an amino acid sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 610) or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor comprises a CD28 co-stimulatory domain, a 4-1BB costimulatory domain, and a CD3 ⁇ activation domain.
  • the activator receptor comprises an amino acid sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ ID NO: 618) or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor comprises a sequence of one of SEQ ID NO: 983 to SEQ ID NO: 1044, as set forth in Table 10, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor receptor and the activator receptor are expressed by a single polynucleotide.
  • the single polynucleotide comprises one or both of a constitutive promoter and an inducible promoter.
  • the constitutive promotor comprises a eukaryotic translation elongation factor 1 alpha (EF1a) promoter.
  • the coding sequence for the inhibitor receptor precedes the coding sequence for the activator receptor in the single polynucleotide.
  • the coding sequence for the activator receptor precedes the coding sequence for the inhibitor receptor in the single polynucleotide.
  • the single polynucleotide encodes a self-cleaving peptide between the coding sequence for the inhibitor receptor and the coding sequence activator receptor.
  • the self-cleaving peptide is selected from the T2A, P2A, E2A and F2A self-cleaving peptides.
  • the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 653)
  • the P2A self-cleaving peptide comprises a sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 654)
  • the E2A self-cleaving peptide comprises a sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 655)
  • the F2A self- peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 656)
  • the T2A self-cleaving peptide comprises Docket No.: 061250-559001WO a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 657), or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor receptor is expressed by a first polynucleotide and the activator receptor is expressed by a second polynucleotide.
  • the first polynucleotide and/or the second polynucleotide comprises one or both of a constitutive promoter and an inducible promoter.
  • the constitutive promotor comprises a eukaryotic translation elongation factor 1 alpha (EF1a) promoter.
  • the immune cell is a T cell, e.g., a CD8+ CD4- T cell or a CD8- CD4+ T cell. In some cases, the T cell is a cytotoxic T cell.
  • the immune cell is a natural killer (NK) cell.
  • the immune cell is modified to reduce or eliminate expression of the B2M gene product.
  • the shRNA comprises a first sequence, having from 5′ to 3′ end a sequence complementary to the B2M mRNA; and a second sequence, having from 5′ to 3′ end a sequence complementary to the first sequence, wherein the first sequence and second sequence form the shRNA.
  • the membrane tethered cytokine promotes T cell survival in vitro and in vivo, maintains selective killing of cancer cells (“logic-gated selectivity”) when expressed with the inhibitor antigen and the activator receptor in vitro and in vivo, and/or enhances antigen dependent expansion in vitro and in vivo.
  • the membrane tethered cytokine provides increased activation, proliferation, and killing capacity while maintaining the selectivity of a logic gated dual receptor system.
  • the immune cell has enhanced antigen-dependent expansion compared to a cell lacking the membrane tethered cytokine. In some cases, the immune cell does not exhaust after continued antigen-dependent expansion.
  • the immune cell maintains antigen-dependent expansion longer when compared to a cell lacking the membrane tethered cytokine. In some cases, the immune cell does not exhaust after continued antigen-dependent expansion.
  • the immune cell proliferates at least 10% more rapidly than a cell lacking the membrane tethered cytokine. Docket No.: 061250-559001WO [0047] In embodiments, the immune cell continues to proliferate in the absence of exogenous IL- 2. [0048] In embodiments, the immune cell overcomes TGF- ⁇ immune suppression better than a cell lacking the membrane tethered cytokine. [0049] In embodiments, the immune cell expresses and/or secretes greater amounts of IFN ⁇ compared to a cell lacking the membrane tethered cytokine.
  • the immune cell expresses greater amounts of the transcription factor T- bet compared to a cell lacking the membrane tethered cytokine.
  • the cancer is acute myelogenous leukemia, adult T-cell leukemia/lymphoma, B-cell lymphoma, breast cancer, chronic lymphocytic leukemia, colorectal cancer, diffuse large B-cell lymphoma, gastric cancer, glioblastoma multiforme, head & neck, urothelial cancer, a hematological cancer, hematological malignancy, Hodgkin's lymphoma, leukemia, lymphoma, malignant melanoma, melanoma, multiple myeloma, neuroblastoma, non- small cell lung cancer, non-small cell lung carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, psoriasis, renal cell carcinoma, small cell lung cancer, small cell lung carcinoma, ovarian cancer, pancreatic cancer, prostate cancer,
  • a further aspect of the present disclosure is a pharmaceutical composition, including a plurality any herein-disclosed immune cells and a pharmaceutically acceptable diluent, carrier, or excipient.
  • the pharmaceutical composition is comprised in a kit.
  • the pharmaceutical composition is used in a method for treating cancer.
  • An additional aspect of the present disclosure is a method of treating a cancer comprising administering to a subject in need therefore, an effective amount of any herein-disclosed immune cell.
  • An aspect of the present disclosure is a kit including any herein-disclosed immune cell.
  • the kit includes instructions for use, e.g., for killing a cancer cell or for treating a cancer.
  • a further aspect of the present disclosure is a kit including any herein-disclosed pharmaceutical composition.
  • the kit includes instructions for use, e.g., for killing a cancer cell or for treating a cancer.
  • Another aspect of the present disclosure is a polynucleotide or polynucleotide system, including one or more polynucleotides including one or more polynucleotide sequences encoding the membrane tethered cytokine, the inhibitor and/or the activator receptor.
  • the polynucleotide or polynucleotide system is comprised in a nanocarrier.
  • Docket No.: 061250-559001WO nanocarrier is capable of delivering the polynucleotide or polynucleotide system to an immune cell in vivo or ex vivo.
  • the nanocarrier is a lipid nanoparticle (LNP).
  • An additional aspect of the present disclosure is a method of making an immune cell therapy. The method comprising transforming immune cells with including any herein-disclosed polynucleotide or polynucleotide system. In some cases, the method comprises contacting immune cells with any herein disclosed nanocarrier.
  • a host cell comprises any herein- disclosed polynucleotide or polynucleotide system.
  • Any immune cell, polynucleotide, pharmaceutical composition, or method disclosed herein is applicable to any herein-disclosed immune cell, polynucleotide, pharmaceutical composition, or method.
  • any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
  • FIG. 1 is a series of plots showing the percent of specific killing of target cells by engineered T cells.
  • Target cells were engineered to express (A) CEA or (AB) CEA and HLA- A*02.
  • T-cells were engineered to express a CEA activating receptor or a CEA activating receptor and an HLA-A*02 inhibitory receptor. Both groups of T cells were treated with exogenous, soluble IL-2.
  • the bottom data curve that intersects the vertical line at hour 50 is the CEA receptor alone without exogenous IL-2.
  • the remaining data curves that have ⁇ 50% specific killing percentage were supplemented with exogenous IL-2 of various concentrations; the CEA activator receptor expressed alone supplemented with 3.3 IU/ml of IL-2 had the highest specific killing percentage.
  • the bottom data curve that intersects with the vertical line at hour 50 is the CEA receptor alone without exogenous IL-2.
  • FIG. 2 is a series of plots showing percent of specific killing of target cells by engineered T cells. Target cells were engineered to express (A) CEA or (AB) CEA and HLA- Docket No.: 061250-559001WO A*02.
  • T-cells were engineered to express a CEA activating receptor and an HLA-A*02 inhibitory receptor. Both groups of T cells were treated with exogenous, soluble IL-12, IL-15, or IL-21.
  • engineered immune cells were co-cultured with A target cells and the bottom data curve that intersects with the vertical line at hour 50 was not treated with exogenous IL-12.
  • the group supplemented with 0.4IU/ml had the highest percentage of specific killing at hour 50.
  • those same cells grown with AB target cells top right graph
  • none of the groups show above 10% specific killing percentage at hour 50.
  • FIG. 3A is a schematic illustrating a membrane tethered (mt) interleukin in the dual receptor system.
  • mt membrane tethered interleukin
  • Each engineered T-cell will express an antigen dependent activator receptor, an antigen dependent inhibitor receptor and an antigen independent membrane tethered interleukin that stimulates the endogenous interleukin receptor.
  • FIG.3B is a graphic representation of a dual receptor system.
  • the dual receptor system which consists of an activator and a blocker receptor, selectively kills tumor cells that express only the activator antigens, while protecting normal cells expressing both activator and blocker antigens.
  • FIG.4 is a schematic illustrating the structural rationale for each mt-interleukin design.
  • Mt-IL-2 (left) includes a flexible hinge to engage the IL-2 receptor components, whereas mt-IL- 12 (right) includes a longer, more rigid hinge to access the more distal binding region of the IL-12 receptor in addition to a linker connecting the IL-12A and IL-12B subunits.
  • FIG.5 shows quantification of IFN ⁇ secretion from T cells expressing mt-IL-2 or mt-IL- 12.
  • FIG. 6 shows quantification of the amount of the transcription factor T-bet in T cells expressing a mt-IL-2 or mt-IL-12.
  • FIG. 5 shows quantification of IFN ⁇ secretion from T cells expressing mt-IL-2 or mt-IL- 12.
  • FIG. 6 shows quantification of the amount of the transcription factor T-bet in T cells expressing a mt-IL-2 or mt-IL-12.
  • FIG. 7 plots the results of an IL-2 starvation assay with T cells transfected with a membrane-tethered IL-2 construct.
  • FIG. 8 shows the expression human PBMCs transfected with a construct encoding a HLA-A*02 inhibitory receptor and a CEA activator receptor and a construct encoding Docket No.: 061250-559001WO membrane tethered IL-12 (mt-IL-12).
  • HLA-A*02 tetramer was used to detect expression of the HLA-A*02 inhibitor receptor and an IL-12p70 specific antibody used to detect mt-IL-12 expression.
  • FIG. 8 shows the expression human PBMCs transfected with a construct encoding a HLA-A*02 inhibitory receptor and a CEA activator receptor and a construct encoding Docket No.: 061250-559001WO membrane tethered IL-12 (mt-IL-12).
  • HLA-A*02 tetramer was used
  • FIG. 10 is a series of plots showing the percent of specific killing of target cells by T cells at a 1:1 Effector: Target ratio. Target cells were engineered to express (A) CEA only, (AB) CEA and HLA-A*02, or (B) HLA-A*02 only.
  • FIG.11A is a schematic illustrating a two-part vector system used to generate engineered immune cells described herein. The two-part vector system co-expresses, from two separate constructs, a dual receptor and a membrane tethered interleukin.
  • the first construct encodes the blocker receptor and the activator receptor separated by the coding sequence for a self-cleaving linker (T2A).
  • the second construct encodes the membrane tethered interleukin.
  • both vectors constitutively express their respective polypeptides under the control of the EF1a promoter (pEF1a).
  • FIG. 11B shows the expression profile of human PBMCs transfected with a construct encoding an HLA-A*02 inhibitor receptor and a CEA activator receptor and a construct encoding membrane tethered IL-12 (mt-IL-12).
  • FIG.11C is a plot showing the percent of specific killing of target cells by engineered T cells expressing 1) a CEA activator receptor, an HLA-A*02 inhibitor receptor, and a B2Msh 2) T cells expressing a CEA activator receptor, an HLA-A*02 inhibitor receptor, a B2Msh and a mt- IL-12 or 3) a CEA activator receptor, an HLA-A*02 inhibitor receptor, a B2Msh and a secreted IL-12 at 48 hours with an Effector cell: Target cell (E:T) ratio ranging from 1:9 to 27:1.
  • Target cells were engineered to express (A) CEA only or (AB) CEA and HLA-A*02.
  • A CEA only or
  • AB CEA and HLA-A*02.
  • the selectivity window is the ratio of ET50 for AB divided by ET 50 for A and demonstrates changes in specificity in cell killing and cell for various engineered T cells. Docket No.: 061250-559001WO [0075]
  • FIG. 12A is a drawing illustrating the antigen induced proliferation experiment.
  • FIG. 12B are series of flow cytometry plots showing the histogram for the presence of CellTrace Violet.
  • CellTrace Violet is a reagent used to track proliferation in cells.
  • FIG. 13 is a series of plots showing the percent of specific killing of target cells by T cells at an effective 1:8 Effector: Target ratio.
  • Target cells were engineered to express (A) CEA only or (AB) CEA and HLA-A*02. T cells were transduced with a construct encoding a CEA activator receptor and an HLA-A*02 inhibitor receptor.
  • mt-IL-2 (C125A) was encoded on a separate construct and co-transduced.
  • FIG.14 represents the computational model used to select IL-2 attenuation mutations to de-emphasize Treg(CD25High) interactions in trans and promote cis-binding in Teff.
  • Each IL-2 mutation was modeled in Rosetta and scored for self, common ⁇ chain, IL2R ⁇ , and CD25 interactions. Mutations were selected that stabilized self-interactions while weakening interactions with CD25 and IL2R ⁇ .
  • FIG.15 shows the binding profiles for mt-IL-2 mutations expressed in Jurkat cells.
  • FIG.16A characterizes the expression and CD25 interaction of lead mt-IL-2 muteins in a Jurkat expression system.
  • FIG.16B plots the proliferation of IL-2+ cells from an IL-2 starvation assay. Jurkat cells expressing mt-IL-2 muteins with reduced CD25 affinity were measured for their percentage of IL- 2+ cells at 16, 19, 21, and 23 days post removal of IL-2.
  • FIG.17A is a schematic illustrating a two-part vector system used to generate engineered immune cells described herein.
  • the two-part vector system co-expresses, from two separate constructs, a dual receptor and a membrane interleukin.
  • the first construct encodes the blocker receptor and the activator receptor separated by the coding sequence for a self-cleaving Docket No.: 061250-559001WO linker (T2A).
  • the second construct encodes the membrane tethered IL-12.
  • both vectors constitutively express their respective polypeptides under the control of the EF1a promoter (pEF1a).
  • 17B is a schematic illustrating another version of two-part vector system used to generate engineered immune cells described herein.
  • the two-part vector system co-expresses, from two separate constructs dual receptor and membrane tethered interleukin are.
  • the first construct encodes the blocker receptor and the activator receptor separated by the coding sequence for a self- cleaving linker (T2A).
  • the second construct encodes the membrane tethered interleukin.
  • the activator and blocker are constitutively expressed under the control of the control of the EF1a promoter (pEF1a).
  • the membrane tethered IL-12 is expressed when the T cell is activated under the control of NFAT response elements (NFAT REs).
  • NFAT REs NFAT response elements
  • FIG.17C shows the expression profile of human PBMCs transfected with the two-part vector system shown in FIG.17A. HLA-A*02 tetramer was used to detect expression of the HLA- A*02 inhibitor receptor and an IL-12 P70 specific antibody was used to detect cell surface IL-12.
  • FIG.17D shows the expression profile of human PBMCs transfected with the two-part vector system described in FIG. 17B. HLA-A*02 tetramer was used to detect expression of the HLA-A*02 inhibitor receptor and an IL-12 P70 specific antibody was used to detect cell surface IL-12 ⁇ heterodimer expression.
  • FIG. 18 shows cell surface expression level of mt-IL-12.
  • T cells were transduced with mt-IL-12 constructs that were constitutively expressed or under the control of NFAT response elements (RE).6x NFAT Response Elements (RE) or 4X NFAT RE were tested by treating T cells with TransActTM (Miltenyi Biotec) to activate T cells.
  • TransActTM Miltenyi Biotec
  • FIG.19 shows the level of IL-12p70 present in the media of T cells engineered to express secreted IL-12, constitutively expressed mt-IL-12 or mt-IL-12 under the control of NFAT RE.
  • Media was collected on Day 1 and Day 4 and subjected to a cytometric bead array assay (CBA) to determine the level of IL-12p70.
  • LOD limit of detection.
  • FIG. 20 includes plots showing specific killing of target cells expressing A antigen (MSLN+) or AB antigen (MSLN+ and HLA-A*02+) by T cells from two donors.
  • T cells expressing a MSLN activator, an HLA-A*02 blocker and B2M shRNA grown with A target cells are represented by the solid black circle line and those cells grown with AB target cells are represented by the empty black circle dashed line.
  • T cells expressing a MSLN activator, an HLA- A*02 blocker, and B2M shRNA and a constitutively expressed mt-IL-12 (expressed by an eF1a promoter) grown with A target cells are by the solid gray circle line and those cells grown with AB target cells are represented by the empty gray circle dash line.
  • FIG.21 is a table showing the ET50 on A, ET50 on AB and the ET 50 AB/A from both donor T cells populations used in FIG.20.
  • FIG.22 is a diagram of the antigen dependent proliferation experiment quantified in FIG. 23A and FIG.23B.
  • MS751 cells were plated at 2e4 (2 x 10 4 ) cells per well of 96-well plates.24 hrs later, 1e4 (1 x 10 4 ) transduced T cells per well were plated with the MS751 target cells in 200 ⁇ L XVIVO-15 media.
  • 20% of T cells were removed from the original well and replated with freshly plated MS751 cells.
  • 10% of the T cells were removed from the previous well and replated with freshly plated MS751 cells. The number of transduced cells for each round were evaluated using FLOW CYTOMETRY.
  • FIG. 23A shows the cumulative fold expansion of T cells expressing MSLN activator CAR, HLA-A*02 blocker, B2M shRNA, and mt-IL-12 after co-culture with MS751 cells (MSLN+ and HLA-A*02 negative). A similar fold expansion was seen when the mt-IL-12 was expressed constitutively or under the control of the NFAT RE. [0092] FIG.
  • FIG. 23B shows the cumulative fold expansion of T cells expressing MSLN activator CAR (square dashed line), MLSN activator CAR, HLA-A*02 blocker, B2M shRNA, and constitutively expressed mt-IL-12 (gray circle dashed line), MLSN activator CAR, HLA-A*02 blocker, B2M shRNA, and mt-IL-12 under the control of NFAT RE (triangle dashed line), or MLSN activator CAR, HLA-A*02 blocker, and B2M shRNA (black circle dashed line) in the presence of 10ng/mL of TGF ⁇ 1 that was added each time T cells were plated or replated with targets.
  • MSLN activator CAR square dashed line
  • MLSN activator CAR HLA-A*02 blocker
  • B2M shRNA constitutively expressed mt-IL-12
  • NFAT RE triangle dashed line
  • FIG.24 is a plot showing the specific killing percentage of A and AB target cells from the engineered immune cells from two different donors used in FIG.23. Engineered immune cells from FIG. 23 were cocultured with A and AB target cells after undergoing repeated antigen exposure.
  • FIG.25 is a series of flow cytometry plots showing the surface expression of HLA-A*02 blocker receptor and mt-IL-12 from engineered immune cells expressing 1) MLSN activator receptor, HLA-A*02 blocker receptor, and B2M shRNA, 2) MSLN activator receptor, HLA-A*02 blocker receptor, B2M shRNA, and expressed mt-IL12 (EF1a) or 3) MSLN activator receptor, HLA-A*02 blocker receptor, B2M shRNA, and activation induced mt-IL-12.
  • FIG. 26A is a schematic showing the in vivo study design for testing mt-IL-12.
  • Tumor cell growth was established by engraftment of 5e4 (5 x 10 4 ) MSLN(+) HLA-A*02(-) MS751 cells (tumor; left flank of mouse). Normal cell growth was established by engraftment of 5e4 MSLN(+) HLA-A*02(+) MS751 cells (“normal”; right flank of mouse). T cells were infused eleven days after engraftment.
  • FIG. 26B quantifies tumor cells growth over time before and after infusion with the indicated T cells.
  • Tumors were established by engraftment of 5e4 MSLN(+) HLA-A*02(-) MS751 cells.
  • Two different concentrations of T cells were infused eleven days after tumor cell engraftment (7.5e4 vs.7.5e5 T cells infused, i.e., 7.5 x 10 4 or 7.5 x 10 5 T cells).
  • In vivo bioluminescent imaging (BLI) was performed twice a week to measure tumor volume. BLI was measured in photons per second (p/sec).
  • T cells were either untreated (solid circle), engineered to express MLSN activator CAR, HLA-A*02 blocker CAR, B2M shRNA, and constitutively expressed mt-IL-12 (solid square), MLSN activator CAR, HLA-A*02 blocker CAR, B2M shRNA, and mt-IL-12 under the control of NFAT RE (solid triangle), or MLSN activator CAR and HLA-A*02 blocker CAR and B2M shRNA (inverted empty triangle).
  • FIG.27A quantifies the number of HLA-A*02 blocker positive T cells at the indicated time points post T cell infusion from the in vivo experiment described in FIG.26.
  • T cells Two different concentrations of T cells were infused eleven days after tumor cell engraftment (7.5e4 vs.7.5e5 T cells infused).
  • T cells were either untreated (solid circle), engineered to express MLSN activator CAR, HLA-A*02 blocker CAR, B2M shRNA, and constitutively expressed mt-IL-12 (solid triangle), MLSN activator CAR, HLA-A*02 CAR, B2M shRNA, and mt-IL-12 under the Docket No.: 061250-559001WO control of NFAT RE (inverted empty triangle), or MLSN activator CAR and HLA-A*02 blocker CAR and B2M shRNA (solid square).
  • FIG.27B quantifies the number of hCD3+ positive T cells at the indicated time points post T cell infusion from the in vivo experiment described in FIG.26. Two different concentrations of T cells were infused eleven days after tumor cell engraftment (7.5e4 vs.7.5e5 T cells infused).
  • T cells were either untreated (solid circle), engineered to express MLSN activator CAR, HLA- A*02 blocker CAR, B2M shRNA, and constitutively expressed mt-IL-12 (solid triangle), MLSN activator CAR, HLA-A*02 blocker CAR, B2M shRNA, and mt-IL-12 under the control of NFAT RE (inverted empty triangle), or MLSN activator CAR and HLA-A*02 blocker CAR and B2M shRNA (solid square).
  • FIG.27C is a table comparing the average peak blocker+ T cells (per ul) in the peripheral blood between 7.5e4 infusion group of T cells engineered to express MLSN activator CAR, HLA- A*02 blocker CAR, B2M shRNA, and mt-IL-12 and 7.5e5 infusion group of T cells engineered to express MLSN activator CAR and HLA-A*02 blocker CAR and B2M shRNA, from the in vivo experiment described in FIG.26.
  • FIG.27D quantifies the amount of circulating IFN- ⁇ at the indicated time points post T cell infusion from the in vivo experiment shown in FIG. 26.
  • T cells Two different concentrations of T cells were infused eleven days after tumor cell engraftment (7.5e4 vs. 7.5e5 T cells infused).
  • T cells were either untreated (solid circle), engineered to express MLSN activator CAR, HLA-A*02 blocker CAR, B2M shRNA, and constitutively expressed mt-IL-12 (solid triangle), MLSN activator CAR, HLA-A*02 blocker CAR, B2M shRNA, and mt-IL-12 under the control of NFAT RE (inverted empty triangle), or MLSN activator CAR and HLA-A*02 blocker CAR and B2M shRNA (solid square).
  • FIG.27E is a series of plots showing the serum level of IL-12p70 from samples taken from the in vivo experiment described in FIG.26. Blood samples were harvested on Day 3, Day 10, Day 17 and Day 25. Any value below the dotted line was below the level of detection for the assay.
  • FIG.27F is a series of plots showing the body weight of mice over the course of the in vivo experiment described in FIG.26.
  • FIG.28 is a series of flow cytometry plots showing the surface expression of either the mEGFR activator receptor or the HLA-A*02 blocker receptor and mt-IL-12 from engineered immune cells expressing 1) mEGFR activator receptor (i.e.
  • FIG.29 is a plot showing specific killing of target cells expressing A antigen (mEGFR+) or AB target cells (mEGFR+ H-2Db+) by T cells.
  • FIG.30B are Kaplan-Meyer survival plots for the in vivo experiment described in FIG. 30A. Mice were infused with 1.2e7, 3.79e6 or 1.2e6 T cells per mouse 11 days after tumor cell engraftment. In the 1.2e7 infusion group, all mice infused with T cells expressing only EGFR activator receptor died within 5 days of T cell infusion.
  • FIG.30C are a series of graphs showing the Bioluminescent imaging (BLI) results from the in vivo experiment described in FIG.30A. On day 15 of the 1.2e7 infusion group (shown as a vertical line), all data curves that are below 10 ⁇ 9 total photon flux per second were infused with the 4x and 6x NFAT mt-IL-12 expressing T cells.
  • the remaining lines above the 10 ⁇ 9 total photon flux per second on day 15 are from the UTD group and the EGFR activator receptor and H-2Db blocker receptor group.
  • On day 15 of the 3.79e6 group (shown as a vertical line) all but one mouse from each of the 4x NFAT and 6xNFAT mt-IL-12 groups were below 10 ⁇ 9 total photon flux per second. The remaining mice from each group showed no reduction in total photon flux per second at the conclusion of the experiment.
  • On day 15 of the 1.2e6 group (shown by the vertical line) only one mouse from the 6xNFAT mt-IL12 group had a total photon flux per second below 10 ⁇ 9. The remaining mice from each group showed no reduction in total photon flux per second at the conclusion of the experiment.
  • FIG.30D is a series of plots showing the body weight of mice over the course of the in vivo experiment described in FIG.30.
  • FIG.31 is a series of plots showing the serum level of IL-12p70 from samples taken from the in vivo experiment described in FIG.30. Blood samples were harvested on Day 2, Day 9, Day 16, Day 23 and Day 29 or Day 30.
  • FIG.32A quantifies the number of dual receptor positive (EGFR activator receptor and H-2Db blocker receptor) T cells at the indicated time points post T cell infusion from the in vivo experiment described in FIG.31.
  • FIG.32B quantifies the number of hCD3+ T cells at the indicated time points post T cell infusion from the in vivo experiment described in FIG.31. Blood samples were harvested on Day 2, Day 9, Day 16, Day 23 and Day 29 or Day 30.
  • FIG.32C quantifies the amount of circulating IFN- ⁇ at the indicated time points post T cell infusion from the in vivo experiment described in FIG.31. Blood samples were harvested on Day 2, Day 9, Day 16, Day 23 and Day 29 or Day 30.
  • FIG.33 is a series of plots showing the serum level of IL-12p70 from samples taken from the in vivo experiment described in FIG.31.
  • FIG.34B shows the expression profile of human PBMCs transfected with the two-part vector system described in FIG. 34A.
  • HLA-A*02 tetramer was used to detect expression of the HLA-A*02 inhibitor receptor and an IL-18 specific antibody was used to detect cell surface IL-18 heterodimer expression.
  • FIG.35 shows the fold expansion of primary T cells from two donors. The expansion of untreated primary T cells (dashed line), T cells expressing MSLN activator and HLA-A*02 blocker and B2M shRNA (dark color line), or T cells expressing MSLN activator, HLA-A*02 blocker, B2M shRNA, and mt-IL-18 (light color line) was quantified over 21 days.
  • FIG. 36 includes plots showing specific killing of target cells expressing A antigen (MSLN+) or AB antigen (MSLN+ and HLA-A*02+) by T cells from two donors.
  • T cells expressing MSLN activator and HLA-A*02 blocker and B2M shRNA grown with A target cells are represented by the solid circle dashed line.
  • T cells expressing MSLN activator and HLA-A*02 blocker and B2M shRNA grown with AB target cells are represented by the empty circle dashed line.
  • T cells expressing MSLN activator, HLA-A*02 blocker, B2M shRNA, and mt-IL-18 grown with A target cells are represented by the solid circle solid line.
  • T cells expressing MSLN activator, HLA-A*02 blocker, B2M shRNA, and mt-IL-18 grown with AB target cells are represented by Docket No.: 061250-559001WO the empty circle solid line. The percentage of specific killing was quantified after 48hrs of co- culture. Effector: Target (E:T) ratios ranging from 1:81 to 3:1 were used in this experiment. [0119] FIG.
  • FIG. 37A shows the cumulative fold expansion of T cells from two donors expressing MSLN activator CAR (solid triangle dashed line), MSLN activator and HLA-A*02 blocker and B2M shRNA (solid circle solid line), or MSLN activator, HLA-A*02 blocker, B2M shRNA, and mt-IL-18 (solid circle dashed line) after co-culture with MS751 cells (MSLN+ and HLA-A*02 negative).
  • MS751 cells were plated at 2e4 cells per well of 96-well plates. 24 hr later, 1e4 transduced T cells per well were plated with the MS751 target cells in 200 ⁇ L XVIVO-15 media.
  • FIG.37B shows the cumulative fold expansion of T cells from two donors expressing MSLN activator CAR (solid triangle dashed line) or MSLN activator and HLA-A*02 blocker and B2M shRNA (solid circle solid line) or MSLN activator, HLA-A*02 blocker, B2M shRNA, and mt-IL-18 (solid circle dashed line) after co-culture with MS751 cells (MSLN+ and HLA-A*02 negative).
  • MS751 cells were plated at 2e4 cells per well of 96-well plates. 24 hr later, 1e4 transduced T cells per well were plated with the MS751 target cells in 200uL XVIVO-15 media.
  • FIG. 38A is a schematic showing the in vivo study design for testing mt-IL-18. Tumor cell growth was established by engraftment of 5e4 MSLN(+) HLA-A*02(-) MS751 cells (tumor; left flank of mouse).
  • FIG. 38B quantifies tumor cell growth over time before and after infusion with the indicated T cells. Tumors were established by engraftment of 5e4 MSLN(+) HLA-A*02(-) MS751 cells.7.5e5 T cells were infused eleven days after tumor cell engraftment. In vivo bioluminescent imaging (BLI) was performed twice a week to measure tumor volume. BLI was measured in photons per second (p/sec).
  • T cells were either untreated (solid circle), engineered to express MSLN activator and HLA-A*02 blocker and B2M shRNA (solid square), or MSLN activator, HLA-A*02 blocker, B2M shRNA, and expressed mt-IL-18 (solid triangle). Docket No.: 061250-559001WO [0123]
  • FIG. 38C quantifies “normal” cell growth over time before and after infusion with the indicated T cells. “Normal” cell growth was established by engraftment of MSLN(+) HLA- A*02(+) MS751 cells.5e4 MSLN(+) HLA-A*02(+) MS751 cells were engrafted into the left flank of mice.
  • FIG.39A is a schematic illustrating a vector used to generate engineered immune cells described herein. The construct encodes the membrane tethered interleukin.
  • FIG.39B is a schematic illustrating a vector used to generate engineered immune cells described herein.
  • the construct encodes the membrane tethered interleukin-12.
  • the membrane tethered interleukin-12 is expressed when the T cell is activated under the control of NFAT response elements (NFAT REs).
  • NFAT REs NFAT response elements
  • This version of the vector also includes a TATA box promoter.
  • FIG.39C is a schematic illustrating a vector used to generate engineered immune cells described herein.
  • the construct encodes the membrane tethered interleukin-12.
  • the vector constitutively expresses the membrane tethered interleukin-12 under the control of the TATA box.
  • This version of the vector also includes a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE).
  • WSV Woodchuck Hepatitis Virus
  • WPRE Woodchuck Hepatitis Virus
  • FIG.39D is a schematic illustrating a vector used to generate engineered immune cells described herein.
  • the construct encodes the membrane tethered interleukin-12.
  • the vector constitutively expresses the membrane tethered interleukin-12 under the control of the TATA box.
  • This version of the vector also includes a TATA Box promoter and a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus
  • FIG.40A is a schematic illustrating a two-part vector system used to generate engineered immune cells described herein. The two-part vector system co-expresses, from two separate constructs, a dual receptor and a membrane tethered interleukin.
  • the first construct encodes the blocker receptor and the activator receptor by the coding sequence for a self-cleaving linker (T2A).
  • the second construct encodes the membrane tethered interleukin with a Docket No.: 061250-559001WO transmembrane and intracellular domain.
  • the activator receptor and blocker receptor are constitutively expressed under the control of the EF1a promoter (pEF1a).
  • the membrane tethered interleukin is expressed upon activation through the 6X NFAT promoter.
  • FIG.40B is a schematic illustrating a single vector system used to generate engineered immune cells described herein.
  • the construct encodes the blocker receptor, the activator receptor, and the membrane tethered IL-18 separated by the coding sequence for a self-cleaving linker (P2A).
  • P2A self-cleaving linker
  • the blocker receptor, the activator receptor and the membrane tethered IL-18 constitutively expressed the control of the EF1a promoter (pEF1a).
  • FIG. 40C is a series of graphs showing antigen dependent expansion of engineered immune cells.
  • FIG.41A is a schematic illustrating a two-part vector system used to generate engineered immune cells described herein.
  • the two-part vector system co-expresses, from two separate constructs, a dual receptor and a modified constitutively active cytokine receptor.
  • the first construct encodes the blocker receptor and the activator receptor separated by the coding sequence for a self-cleaving linker (T2A).
  • the second encodes the modified constitutively active cytokine receptor with a non-native extracellular domain fused to the IL7R ⁇ transmembrane and Docket No.: 061250-559001WO intracellular domains.
  • the activator receptor, the blocker receptor and the modified constitutively active cytokine receptor are constitutively expressed the control of the EF1a promoter (pEF1a).
  • FIG. 41B is a scatter plot showing the expression profile of JNL cells transfected with the two-part vector system described in FIG. 41A. JNL cells were engineered to express a CEA activator receptor and an HLA-A*02 blocker receptor with or without the constitutively active IL7R ⁇ construct.
  • FIG.41C is a graph representing the total cell count of engineered JNL cells 1) that were untransduced (UTD), 2) that expressed an HLA-A*02 blocker receptor and a CEA activator receptor, or 3) that expressed a CEA activator receptor, an HLA-A*02 blocker receptor and the constitutively active IL7R ⁇ construct.
  • Each group of JNL cells was grown without the addition of exogenous IL2 and proliferation was measured on day two, day five, day seven, day ten and day thirteen.
  • FIG.41D is a plot showing specific killing of target cells that were CEA(+)HLA-A*02(- ), target cells that were CEA(+)HLA-A*02(+), or targets that were CEA(-)HLA-A*02(-).
  • Engineered JNL cells and target cells were cultured at the indicated effector: target (E:T) cell ratios. JNL cells were engineered to express a CEA activator receptor, an HLA-A*02 blocker receptor with or without the constitutively active IL7R ⁇ construct.
  • FIG. 42A is a scatter plot showing the expression profile of JNL cells transfected with the two part vector system described in FIG.41A.
  • FIG.42B is a plot showing specific killing of target cells that were either MSLN(+)HLA- A*02(-) or MSLN(+)HLA-A*02(+). JNL cells were engineered to express a MSLN activator receptor, a HLA-A*02 blocker receptor with or without the constitutively active IL7R ⁇ construct.
  • DETAILED DESCRIPTION Provided herein are, inter alia, compositions and methods for engineered interleukins and engineered immune cells expressing the engineered interleukins for the treatment of cancers.
  • the engineered interleukins provide a localized immunostimulatory effect that can advantageously enhance immune cell survival and/or anti- activity in a patient recipient.
  • the compositions and methods disclosed herein provide effective cancer therapies. Docket No.: 061250-559001WO [0138] Also provided herein are, inter alia, compositions and methods for engineered immune cells for logic gated tumor cell killing expressing engineered interleukins for the treatment of cancers. Definitions [0139] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. [0140] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the “administration” of an agent e.g., an anti-HLA-A*03 antibody or CAR-expressing cell, to a subject or subject includes any route of introducing or delivering to a subject a compound to perform its intended function.
  • Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and target cell or tissue.
  • Non-limiting examples of route of administration include parenteral, enteral, and topical routes of administration.
  • Administration includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some or medically relevant result is achieved.
  • the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • the term “mammal” includes both human and non-human mammals.
  • the term “subject” or “patient” includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, sheep, mice, horses, and cows.
  • antibody collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins.
  • antibody includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103 M ⁇ 1 greater, at least 104 M ⁇ 1 greater or at least 105 M ⁇ 1 greater than a binding constant for other molecules in a biological sample).
  • antibody also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies).
  • antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
  • Antibody fragments or “antigen binding fragments” include proteolytic antibody fragments (such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragments and Fab fragments as are known in the art), recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies, and triabodies (as are known in the art), and camelid antibodies (see, for example, U.S. Pat. Nos.
  • proteolytic antibody fragments such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragments and Fab fragments as are known in the art
  • recombinant antibody fragments such as sFv fragments, dsFv fragments, bispecific sFv fragment
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins.
  • Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
  • amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence which is determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence.
  • a variant has a deletion relative to an aligned reference sequence
  • that insertion will not correspond to a numbered amino acid position in the reference sequence.
  • truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.
  • a binding affinity can be measured as a dissociation constant, which for a specific binding pair (such as an antibody/antigen pair) can be lower than 1 ⁇ 10 ⁇ 5 M, lower than 1 ⁇ 10 ⁇ 6 M, lower than 1 ⁇ 10 ⁇ 7 M, lower than 1 ⁇ 10 ⁇ 8 M, lower than 1 ⁇ 10 ⁇ 9 M, lower than 1 ⁇ 10 ⁇ 10 M, lower than 1 ⁇ 10 ⁇ 11 M or lower than 1 ⁇ 10 ⁇ 12 M.
  • binding affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979.
  • binding affinity is measured by a binding constant.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay.
  • the term “human antibody” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis or by somatic mutation in vivo).
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a rabbit, have been grafted onto human framework sequences.
  • human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L , C H domains (e.g., C H1 , C H2 , C H3 ), hinge, V L , V H ) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
  • antibodies designated primate monkey, baboon, chimpanzee, etc.
  • rodent mouse, rat, rabbit, guinea pig, hamster, and the like
  • other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies.
  • chimeric antibodies include any combination of the above.
  • a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • linker peptides are considered to be of human origin.
  • HLA-A*02 refers to HLA-A*02 polypeptide.
  • HLA-A*02 antibodies of the disclosure may bind to proteins falling within the HLA- A*02 allele group (for example HLA-A*02:01, HLA-A*02:02 and the like), and may not bind, or bind with lower affinity, to proteins falling within other HLA-A allele groups (for example, HLA- A*01, HLA-A*11, and the like).
  • HLA-A*02 antibodies of the Docket No.: 061250-559001WO disclosure will still be considered to be specific to HLA-A*02.
  • specificity is considered in the context of the subject to be treated with an HLA-A*02 antibody or receptor of the disclosure.
  • the HLA-A*02 antibody is specific to the HLA-A*02 allele of the subject.
  • HLA-A*03 antibody and “anti-HLA-A*03 antibody” are used interchangeably, and refer to an antibody that specifically binds to an HLA-A*03 polypeptide.
  • HLA-A*03 refers to HLA-A*03 polypeptide.
  • HLA-A*03 antibodies of the disclosure may bind to proteins falling within the HLA- A*03 allele group (for example HLA-A*03:01, HLA-A*03:02 and the like), and may not bind, or bind with lower affinity, to proteins falling within other HLA-A allele groups (for example, HLA- A*02, HLA-A*11, and the like).
  • HLA-A*03 antibodies of the disclosure will still be considered to be specific to HLA-A*03. In some cases, specificity is considered in the context of the subject to be treated with an HLA-A*03 antibody or receptor of the disclosure.
  • the HLA-A*03 antibody is specific to the HLA-A*03 allele of the subject.
  • the terms “inhibitor receptor”, “inhibitory receptor”, and “blocker receptor” and the like are synonyms.
  • the terms “engineered cell”, “engineered immune cell”, “immune cell”, and the like are synonyms.
  • the term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
  • isolated means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.
  • subject refers to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny Docket No.: 061250-559001WO of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • Non-human primates and, preferably, human patients, are included.
  • treatment includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • prevent and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease.
  • prevention and similar words also includes reducing the intensity, effect, symptoms and/or burden of disease prior to onset or recurrence.
  • the term “amount” refers to “an amount effective” or “an effective amount” of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
  • a “therapeutically effective amount” of a virus or cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects.
  • terapéuticaally effective amount includes an amount that is effective to “treat” a subject (e.g., a patient).
  • An “increased” or “enhanced” amount of a physiological response is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) the level of activity in an untreated cell.
  • a “decreased” or “reduced” amount of a physiological response is typically a “statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) the level of activity in an cell.
  • techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health.
  • the BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol.215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res.25:3389- 3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences.
  • the program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program.
  • the program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween.
  • the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
  • a “polynucleotide system” refers to one or more polynucleotides.
  • the one or more polynucleotides may be designed to work in concert for a particular application, or to produce a desired transformed cell.
  • exogenous is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular and the like that originate from outside the Docket No.: 061250-559001WO organism.
  • the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).
  • a target cell can be cancer cell, which can be killed by the transplanted T cells of the adoptive cell therapy.
  • Target cells of the disclosure express a target antigen, as described herein, and do not express a non-target antigen.
  • a “non-target cell” refers to cell that is not targeted by an adoptive cell therapy.
  • normal, healthy, non-cancerous cells are non-target cells.
  • Some, or all, non-target cells in a subject may express both the target antigen and the non-target antigen.
  • Non-target cells in a subject may express the non-target antigen irrespective of whether or not these cells also express the target antigen.
  • a “non-target allelic variant” refers to an allele of a gene whose product is expressed by non-target cells, but is not expressed by target cells.
  • a non-target allelic variant is an allele of a gene that is expressed by normal, non-cancer cells of subject, but not expressed by cancer cells of the subject.
  • the expression of the non-target allelic variant can be lost in the cancer cells by any mechanism, including, but not limited to, loss of heterozygosity, mutation, or epigenetic modification of the gene encoding the non-target allelic variant.
  • a ligand binding domain refers to a ligand binding domain that has a high specificity for a named target.
  • Antibody specificity can be viewed as a measure of the goodness of fit between the ligand binding domain and the corresponding ligand, or the ability of the ligand binding domain to discriminate between similar or even dissimilar ligands.
  • affinity is a measure of the strength of the binding between the ligand binding domain and ligand, such that a low-affinity ligand binding domain binds weakly and high- affinity ligand binding domain binds firmly.
  • a ligand binding domain that is specific to a target allele is one that can discriminate between different alleles of a gene.
  • a interleukin 2 IL-2
  • IL-12 receptor interleukin-2 receptor
  • the person of skill in the art will appreciate that a ligand Docket No.: 061250-559001WO binding domain can be said to be specific to a particular target, and yet still have low levels of binding to one or more additional targets that do not affect its function in the receptor systems described herein.
  • a “target antigen,” whether referred to using the term antigen or the name of a specific antigen, refers to an antigen expressed by a target cell, such as a cancer cell. Expression of target antigen is not limited to target cells. Target antigens may be expressed by both cancer cells and normal, non-cancer cells in a subject.
  • a “non-target antigen” (or “blocker antigen”) whether referred to using the term antigen or the name of a specific antigen, refers to an antigen that is expressed by normal, non-cancer cells and is not expressed in cancer cells.
  • Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population.
  • a polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion.
  • a polymorphism includes e.g., a simple sequence repeat (SSR) and a single nucleotide polymorphism (SNP), which is a variation, occurring when a single nucleotide of adenine (A), thymine (T), cytosine (C) or guanine (G) is altered.
  • SSR simple sequence repeat
  • SNP single nucleotide polymorphism
  • affinity refers to strength of binding of a ligand to a single ligand binding site on a receptor, for example an antigen for the antigen binding domain of any of the receptors described herein.
  • Ligand binding domains can have a weaker interaction (low affinity) with their ligand, or a stronger interaction (high affinity).
  • Kd or dissociation constant, is a type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, such as, for example, when a macromolecular complex comprising receptor and its cognate ligand separates into the ligand and the receptor.
  • a receptor that is “responsive” or “responsive to” refers to a receptor comprising an intracellular domain, that when bound by a ligand (i.e. antigen) generates a signal corresponding to the known function of the intracellular domain.
  • a ligand i.e. antigen
  • An activator receptor bound to a target antigen can generate a signal that causes activation of an immune cell expressing the activator receptor.
  • an inhibitory receptor bound to a non-target antigen can generate an inhibitory signal that prevents or reduces an activation of an immune cell expressing the activator receptor. Responsiveness of receptors, and their ability activate or inhibit immune cells expressing the Docket No.: 061250-559001WO receptors, can be assayed by any means known in the art and described herein, including, but not limited to, reporter assays and cytotoxicity assays. [0181] As used herein, “activation” of an immune cell or an immune cell that is “activated” refers to an immune cell that can carry out one or more functions characteristic of an immune response. These functions include proliferation, release of cytokines, and cytotoxicity, i.e. killing of a target cell.
  • activated immune cells express markers that will be apparent to persons of skill in the art.
  • activated T cells can express one or more of CD69, CD71, CD25 and HLA-DR.
  • An immune cell expressing an activator receptor e.g., a CEA CAR
  • a target antigen e.g., CEACAM
  • a “target antigen” can also be referred to an “activator antigen” and may be isolated or expressed by a target cell.
  • Activation of an immune cell expressing an inhibitory receptor can be prevented when the inhibitory receptor becomes responsive to a non-target antigen (e.g., HLA-A*02 or HLA-A*03), even when the activator receptor is bound to the target activator ligand.
  • a “non-target antigen” can also be referred to as an “inhibitory ligand” or a “blocker”, and may be isolated or expressed by a target cell.
  • Receptor expression on an immune cell can be verified by assays that report the presence of the activator receptors and inhibitory receptors described herein.
  • expression of a interleukin on an immune cell can be verified by assays that report the presence of the interleukin described herein.
  • a population of immune cells can be stained with a labeled molecule (e.g., a fluorophore labeled receptor-specific antibody or a fluorophore-labeled receptor-specific ligand), and quantified using fluorescence activated cell sorting (FLOW CYTOMETRY) flow cytometry.
  • FLOW CYTOMETRY fluorescence activated cell sorting
  • This method allows a percentage of immune cells in a population of immune cells to be characterized as expressing an activator receptor, an inhibitory receptor, or both receptors, and a mt-interleukin.
  • the ratio of activator receptor, inhibitory receptor, and interleukin expressed by the immune cells described herein can be determined by, for example, digital droplet PCR.
  • a suitable percentage of immune cells expressing an activator receptor, an inhibitory receptor, and an interleukin of the disclosure is determined specifically for the methods described herein.
  • a suitable percentage of immune cells expressing both an activator receptor, an inhibitory receptor, and an interleukin of the disclosure can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • immune cells can express both the activator receptor, the inhibitory receptor, and an interleukin of the disclosure.
  • a suitable ratio of activator receptor and inhibitory receptor in an immune cell can be about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5.
  • a responsive receptor expressed by the immune cells described herein can be verified by assays that measure the generation of a signal expected to be generated by the intracellular domain of the receptor.
  • Reporter cell lines such as Jurkat-Luciferase NFAT cells (Jurkat cells) can be used to characterize a responsive receptor.
  • Jurkat cells are derived from T cells and comprise a stably integrated nuclear factor of activated T-cells (NFAT)-inducible luciferase reporter system.
  • NFAT is a family of transcription factors required for immune cell activation, whose activation can be used as a signaling marker for T cell activation.
  • Jurkat cells can be transduced or transfected with the activator receptors and/or inhibitory receptors described herein.
  • the activator receptor is responsive to the binding of a ligand if the Jurkat cell expresses a luciferase reporter gene, and the level of responsiveness can be determined by the level of reporter gene expression.
  • the presence of luciferase can be determined using any known luciferase detection reagent, such as luciferin.
  • an inhibitory receptor is responsive to the binding of a ligand if, when co-expressed with an activator receptor in Jurkat cells, it prevents a normally responsive immune cell from expressing luciferase in response to the activator receptor.
  • the responsiveness of an inhibitory receptor can be determined and quantified in a Jurkat cell expressing both an activator and an inhibitor by observing the following: 1) the Jurkat cell expresses luciferase in the presence of activator receptor ligand and absence of inhibitory receptor ligand; and 2) luciferase expression in the Jurkat cell is reduced or eliminated in the presence of both an activator receptor ligand and an inhibitory receptor ligand.
  • This approach can be used to determine the sensitivity, potency, and selectivity of activator receptors and specific pairs of activator receptors and inhibitory receptors.
  • the sensitivity, potency, and selectivity can be quantified by EC50 or IC50 values using dose- response experiments, where an activator receptor ligand and/or inhibitory receptor ligand is titrated into a culture of Jurkat cells expressing an activator receptor or a specific pair of activator and inhibitory receptors.
  • the EC50 and IC50 values can be determined in a co-culture of immune cells (e.g., Jurkat cells or primary immune cells) expressing an activator receptor or a specific pair of activator and inhibitory receptors and target cells expressing an increasing amount of an activator ligand or inhibitor ligand.
  • An amount of activator ligand or inhibitor ligand can be accomplished in the target cell by, for example, titration of activator ligand or Docket No.: 061250-559001WO inhibitor ligand encoding mRNA into target cells, or use of target cells that naturally express different levels of the target ligands.
  • Illustrative suitable EC 50 and IC 50 values for the activator and inhibitory receptors as determined used target cells expressing varying amounts of the target and non-target ligands include an EC50 of 10 transcripts per million (TPM) or less for the activator receptor, for example an EC50 of between 2-10 TPM, and an IC50 of 25 TPM or less for the inhibitory receptor, for example an IC 50 of 5-21 TPM.
  • TPM transcripts per million
  • IC50 10 transcripts per million
  • the immune cells are co-incubated with target cells that express an activator receptor ligand, an inhibitory receptor ligand, or both an activator and inhibitory receptor ligand.
  • viability of the target cell is measured using any method to measure viability in a cell culture. For example, viability can be determined using a mitochondrial function assay that uses a tetrazolium salt substrate to measure active mitochondrial enzymes. Viability can also be determined using imaging-based methods.
  • Target cells can express a fluorescent protein, such as green fluorescent protein or red fluorescent protein. Reduction in total cell fluorescence indicates a reduction in viability of the target cell.
  • a reduction in viability of the target cell following incubation with immune cells expressing an activator receptor or a specific pair of activator and inhibitory receptors is interpreted as target cell-mediated activation of the immune cell.
  • a measure of the selectivity of the immune cells can also be determined using this approach.
  • the immune cell expressing a pair of activator and inhibitory receptors is selective if the following is observed: 1) viability is reduced in target cells expressing the activator receptor ligand but not the inhibitory receptor ligand; 2) viability is not reduced in target cells expressing both an activator receptor ligand and an inhibitory receptor ligand.
  • a “specific killing” value can be derived that quantifies the percentage of immune cell activation based on the reduction in viability of target cell as a percentage of a negative control (immune cells that do not express an activator receptor).
  • a “selectivity ratio” value can be derived that represents the ratio of the specific killing observed in target cells expressing an activator receptor ligand in the absence of inhibitory receptor ligand to the specific killing observed in target cells expressing both an activator receptor ligand and an inhibitory receptor ligand. This approach can be used to characterize the population of cells for the production and manufacturing of the immune cells, pharmaceutical compositions, and kits described herein.
  • a suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be, for example, the following criteria: 1) at least 40%, at least least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% specific killing following a 48 hour co-incubation Docket No.: 061250-559001WO of immune cells and target cells expressing activator receptor ligand in the absence of inhibitory receptor ligand; and 2) less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 5%, less than or equal to 3% or less than or equal to 1% specific killing of target cell expressing both an activator receptor ligand and an inhibitory receptor ligand.
  • a suitable specific killing value for the immune cells, pharmaceutical compositions and kits can be the following criteria: 1) between 30% and 99%, between 40% and 99%, between 50% and 99%, between 55% and 95%, between 60% and 95%, between 60% and 90%, between 50% and 80%, between 50% and 70% or between 50% and 60% of target cells expressing the activator ligand but not the inhibitor ligand are killed; and 2), between 1% and 40%, between 3% and 40%, between 5% and 40%, between 5% and 30%, between 10% and 30%, between 15% and 30% or between 5% and 20% of target cells expressing the activator ligand and the inhibitor ligand are killed.
  • a suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be, for example, the following criteria: 1) at least 50% specific killing following a 48 hour co-incubation of immune cells and target cells expressing activator receptor ligand in the absence of inhibitory receptor ligand; and 2) less than or equal to 20% specific killing of target cell expressing both an activator receptor ligand and an inhibitory receptor ligand.
  • the immune cells are capable of killing at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% of target cells expressing the activator ligand and not the inhibitor ligand over a period of 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, or 60 hours, while killing less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3% or less than 1% of target cells expressing the activator and inhibitor ligands over the same time period.
  • a suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%.
  • a suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.
  • a suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at most about 50%, at most about 55%, at most about at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, or at most about 95%.
  • a Docket No.: 061250-559001WO suitable specific killing value of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand for the immune cells, pharmaceutical compositions, and kits can be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%.
  • the suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be determined following about 6 hours, about 12 hours, about 18 hours, about 24, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours of co-incubation of immune cells with target cells.
  • a suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%.
  • a suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.
  • a suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, or at most about 95%.
  • a suitable specific killing value of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand for the immune cells, pharmaceutical compositions, and kits can be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%.
  • the suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be determined following about 6 hours, about 12 hours, about 18 hours, about 24, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours of co-incubation of immune cells with target cells.
  • immune cell refers to a cell involved in the innate or adaptive (acquired) immune systems.
  • Illustrative innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymorphonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages and mast cells.
  • NK Natural Killer
  • Immune cells with roles in acquired immunity include lymphocytes such as T-cells and B-cells.
  • T-cell refers to of lymphocyte that originates from a bone marrow precursor that develops in the thymus gland.
  • T-cells Docket No.: 061250-559001WO which develop upon migration to the thymus, which include, helper CD4+ T-cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memory T-cells.
  • helper CD4+ T-cells cytotoxic CD8+ T cells
  • memory T cells memory T cells
  • regulatory CD4+ T-cells stem memory T-cells.
  • Different types of T- cells can be distinguished by the ordinarily skilled artisan based on their expression of markers. Methods of distinguishing between T-cell types will be readily apparent to the ordinarily skilled artisan.
  • T cell presents a unique T cell receptor (TCR) on its surface that recognizes MHC molecules presenting a peptide.
  • TCR T cell receptor
  • a “NK-cell” or “Natural killer cell” are a type of cytotoxic lymphocyte. NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3 ⁇ ).
  • the term “functional variant” refers to a protein that has one or more amino-acid substitutions, insertions, or deletions as compared to a parental protein, and which retains one or more desired activities of the parental protein.
  • a functional variant may be a fragment of the protein (i.e.
  • an “engineered” object generally indicates that the object has been modified by human intervention.
  • a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid may synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire a new function or property.
  • An “engineered” system comprises at least one engineered component.
  • variants of any of the interleukins described herein with one or more conservative amino acid substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide.
  • Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally, or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues) without altering the basic functions of the encoded proteins.
  • Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about Docket No.: 061250-559001WO 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the interleukin protein sequences described herein.
  • such conservatively substituted variants are functional variants.
  • interleukin 2 As used herein, “interleukin 2,” “IL-2”, “IL2,” and “TCGF” (e.g., Genbank Accession numbers: NM_000586 and NP_000577 (human) all refer to a member of a cytokine that binds to the IL-2 receptor.
  • the IL-2 receptor has three forms, generated by different combinations of three different proteins, often referred to as "chains": ⁇ (alpha) (also called IL-2R ⁇ , CD25, or Tac antigen), ⁇ (beta) (also called IL-2R ⁇ , or CD122), and ⁇ (gamma) (also called IL-2R ⁇ , ⁇ c, common gamma chain, or CD132); these subunits are also parts of receptors for other cytokines.
  • IL-2 enhances activation-induced cell death (AICD).
  • IL-2 also promotes the differentiation of T cells into effector T cells and into memory T cells when the initial T cell is also stimulated by an antigen.
  • IL-2 stimulates naive CD4+ T cell differentiation into Th1 and Th2 lymphocytes and impedes differentiation into Th17 and follicular Th lymphocytes.
  • IL-2 also increases the cell killing activity of both natural killer cells and cytotoxic T cells.
  • Enhanced Il-2 stimulation of T cells can result in increased T cell proliferation, enhanced effector functions, Treg overabundance, and immunosuppression and T cell exhaustion.
  • interleukin 12 refers to an interleukin that is a heterodimeric cytokine encoded by the IL-12A and IL-12B genes (Genbank Accession numbers: NM_000882 (IL-12A) and NM_002187 (IL-12B)).
  • IL-12 is composed of a bundle of four alpha helices and is involved in the differentiation of native T cells into TH1 cells. It is encoded by two separate genes, IL-12A (p35) and IL-12B (p40).
  • the active heterodimer referred to as 'p70'
  • a homodimer of p40 are formed following protein synthesis.
  • IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12R- ⁇ 1 and IL-12R- ⁇ 2.
  • IL-12 is known as a T cell-stimulating factor that can stimulate the growth and function of T cells.
  • IL-12 can stimulate the production of interferon gamma (IFN- ⁇ ), and tumor necrosis factor-alpha (TNF- ⁇ ) from T cells and natural killer (NK) cells and reduce IL-4 mediated suppression of IFN- ⁇ .
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumor necrosis factor-alpha
  • NK natural killer cells
  • IL- 12 can further mediate enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes.
  • IL-12 can also have anti-angiogenic activity by increasing production of interferon gamma, which in turn increases the production of the chemokine inducible protein-10 (IP-10 or CXCL10). IP-10 then mediates this anti-angiogenic effect.
  • interleukin 15 “IL-15,” and “IL15” all refer to an interleukin that binds to and signals through a complex composed of an IL-15 specific receptor alpha chain (IL-15R ⁇ ), an IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132) (e.g., Genbank Accession numbers: NP_000576 and NP_751915 (human); and NM_001254747 and NP_001241676 (mouse)).
  • IL-15 has been shown to stimulate T cell Docket No.: 061250-559001WO proliferation inside tumors. IL-15 also is able to extend the survivability of effector memory CD8+ T cells and is critical for the development of NK cells. IL-15 has a short half-life of less than 40 minutes in vivo. Modifications to IL-15 monomer can improve its in vivo pharmacokinetics in the treatment of cancers. These modifications have generally centered on improving the trans- presentation of IL-15 with the alpha subunit of IL-15 receptor, IL-15R ⁇ .
  • Such modifications include: 1) pre-association of IL-15 and its soluble receptor a-subunit-Fc fusion to form IL-15: IL- 15R ⁇ -Fc complex (see, e.g., Rubinstein et al., Proc Natl Acad Sci U.S.A.103:9166–71 (2006)); 2) expression of the super agonist IL-15-sIL-15R ⁇ -sushi protein (see, e.g., Bessard et al., Molecular cancer therapeutics 8: 2736-45 (2009)); and 3) pre-association of human IL-15 mutant IL-15N72D with IL-15R ⁇ -Fc sushi-Fc fusion complex (see, e.g., Zhu et al., Journal of Immunology 183: 3598- 6007 (2009)).
  • interleukin 18 As used herein, “interleukin 18,” “IL-18,” “IL18,” “IGIF,” “IL-1g,” “interferon-gamma inducing factor,” and “IL1F4,” all refer to an interleukin that is a heterodimeric cytokine encoded by the IL-18 gene (e.g., Genbank Accession numbers: NM_001243211, NM_001562 and NM_001386420).
  • IL-18 structurally similar to IL-1 ⁇ , is a member of IL-1 superfamily of cytokines. This cytokine, which is expressed by many human lymphoid and nonlymphoid cells, has an important role in inflammatory processes.
  • IL-18 in combination with IL-12 can activate cytotoxic T cells (CTLs), as well as natural killer (NK) cells, to produce IFN- ⁇ and, therefore, contributes to tumor immunity.
  • CTLs cytotoxic T cells
  • NK natural killer
  • IL-21 IL-21
  • IL21 e.g., Genbank Accession numbers: NM_001207006 and NP_001193935 (human); and NM_0001291041 and NP_001277970 (mouse)
  • NK natural killer
  • microenvironment may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment.
  • the tumor microenvironment refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al., Cancer Res., 2012, 72, 2473.
  • a regulatory element which may comprise promoter or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.
  • a “vector” as used herein generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles.
  • the vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.
  • genetic elements e.g., regulatory elements
  • an expression cassette and “a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression.
  • an expression cassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.
  • the present disclosure provides an engineered interleukin (e.g., IL-2 and IL-18).
  • an “engineered interleukin” indicates that the interleukin has been modified by human intervention.
  • an interleukin can be engineered by changing the nucleic acid sequence to a sequence that does not occur in nature; a interleukin may be modified by addition of an N-terminal or C-terminal polypeptide that it does not associate with in nature, such that the combined product possesses a function not present in the original interleukin; an engineered interleukin may be modified by changing its amino acid sequence to a sequence that does not exist in nature; or an engineered interleukin may acquire a new function or property.
  • the engineered interleukin, the engineered interleukin is an IL-2, an IL-12, an IL-15, an IL-18, or an IL-21.
  • the engineered interleukin is selected from Tables 1-5. In some embodiments, the engineered interleukin comprises a mutation, a deletion, or an insertion compared to the wild type sequence. In some embodiments, the engineered interleukin comprises a fusion, for example with a stabilizing protein or a transmembrane anchor (e.g., a transmembrane anchor). In some embodiments, the engineered interleukin is a soluble polypeptide that is secreted by an immune cell. In some embodiments, the engineered interleukin is a tethered polypeptide that is presented on the surface of an immune cell. In some embodiments, the engineered interleukin is a stabilized interleukin.
  • mt-IL membrane-tethered interleukin
  • the mt-IL refers to an interleukin that is linked to the cellular membrane by a transmembrane domain. Due to its linkage to the membrane, unlike natural interleukins, mt-IL are not secreted from the cell.
  • the mt-IL has a signal sequence that when expressed directs the interleukin to the extracellular surface of the cell membrane. Docket No.: 061250-559001WO [0209]
  • the immune cell expressing engineered interleukin proliferates at least 10% more rapidly than immune cells without the engineered interleukin.
  • the immune cell expressing engineered interleukin proliferates by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50% more rapidly than immune cells without the engineered interleukin.
  • the rate of cellular proliferation may be measured by any reproducible means of measurement.
  • the rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.
  • Stabilized interleukins may have advantages compared with wild type interleukins: - reduced stress on cell production machinery; - improved longevity of interleukins; and/or - higher and more consistent expression levels of interleukins.
  • IL-2 [0211] In one aspect, the present disclosure provides an engineered interleukin 2 (IL-2).
  • the engineered interleukin 2 is selected from the group disclosed in Table 1.
  • Illustrative IL-2 amino acid sequences are provided in 1 below.
  • Table 1 Illustrative IL-2 protein sequences Pro Na WT RM IL- GSE Sig Seq (Un P60 Hu CL 2 NR (C1 Hu CL 2 ( NR G2 F78 Hu CL 2 ( NR E6 Hu CL 2 ( NR P65 Human IL- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL 2 (C125A) EEMLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR E62M WITFAQSIISTLT (SEQ ID NO: 5) Docket No.: 061250-559001WO Human IL- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL 5 (C125
  • the engineered interleukin 2 (IL-2) comprises a C125A mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a G27C and F78C mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a E62Q mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a P65R mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a E62M mutation with regard to SEQ ID NO: 1.
  • the engineered interleukin 2 comprises a F42V mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a R38D mutation with regard to SEQ ID NO: 1. [0214] In some embodiments, the engineered interleukin 2 (IL-2) comprises a C125A, G27C and F78C mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a C125A and E62Q mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a C125A and P65R mutation with regard to SEQ ID NO: 1.
  • the engineered interleukin 2 (IL-2) comprises a C125A and E62M mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a C125A and F42V mutation with regard to SEQ ID NO: 1. In some embodiments, the engineered interleukin 2 (IL-2) comprises a C125A and R38D mutation with regard to SEQ ID NO: 1. [0215] In some embodiments, the engineered interleukin 2 (IL-2) comprises one or more engineered disulfide bonds to improve stability. In some embodiments, the engineered interleukin 2 (IL-2) comprises one or more mutations that improve stability and decrease CD25 interactions.
  • the C125A mutation in IL-2 may reduce intrachain disulfide bond formation and aggregation.
  • the C125A mutations may reduce interchain disulfide bond formation and aggregation.
  • the engineered interleukin 2 (IL-2) comprises one or more engineered cysteine mutations to resolve a free cysteine.
  • the engineered interleukin 2 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 1) (human IL-2 C125A).
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 1.
  • the engineered interleukin 2 comprises a polypeptide sequence at least 80%, at least 90%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: Docket No.: 061250-559001WO APTSSSTKKTQLQLEHLLLDLQMILNCINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNCHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 2).
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 2.
  • the engineered interleukin 2 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 3).
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 3.
  • the engineered interleukin 2 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 4).
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 4.
  • the engineered interleukin 2 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEMLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 5).
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 5.
  • the engineered interleukin 2 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 6).
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 6.
  • the engineered interleukin 2 comprises a polypeptide sequence at least 80%, at least 90%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: Docket No.: 061250-559001WO APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 7).
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 7.
  • the engineered interleukin 2 is a fusion protein comprising an IL-2 (optionally a soluble IL-2 or a tethered IL-2), and a CD25 or a fragment thereof.
  • the engineered interleukin 2 comprises a hinge region.
  • the hinge region is a hinge region selected from Table 6.
  • the hinge region is a (G4Q)2, (G4Q)5, (G4Q)10, (G4Q)10-CD25_full, CD25_hinge, IgG4, EGF3, or EGF7 hinge region.
  • the engineered interleukin 2 is a soluble IL-2 or a tethered IL-2 (e.g., a soluble IL-2).
  • the fusion protein comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8-14.
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to: SEQ ID NO: 8-14.
  • the engineered interleukin 2 (IL-2) is a fusion protein comprising an IL-2 (optionally a soluble IL-2 or a tethered IL-2), and a hinge region.
  • the hinge region is a hinge region selected from Table 6. In some embodiments, the hinge region is a (G4Q)2, (G4Q)5, (G4Q)10, IgG4, EGF3, or EGF7 hinge region.
  • the engineered interleukin 2 comprises a linker between the IL-2 and the CD25. In some embodiments, the linker is selected from Table 7. [0228] In some embodiments, the engineered interleukin 2 (IL-2) is a fusion protein comprising a tethered IL-2 and a CD25 or a fragment thereof. In some embodiments, the engineered interleukin 2 is a CD25-independent IL-2.
  • the engineered interleukin 2 has reduced CD25 binding compared to wild type IL-2. In some embodiments, the engineered interleukin 2 has reduced CD25 recognition compared to wild type IL-2. CD25 recognition and binding can be measured by methods known in the art, for example by FLOW CYTOMETRY. In some embodiments, the engineered interleukin 2 (IL-2) engages CD25 in cis on the same cell. [0229] In some embodiments, the tethered interleukin comprises a transmembrane domain selected from Table 8.
  • tethered interleukin comprises a transmembrane domain of PDGFRb, B7, CD25, CD137, or CD19. Docket No.: 061250-559001WO [0230]
  • the fusion protein comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15-21.
  • the engineered IL-2 comprises a polypeptide sequence 100% identical to SEQ ID NO: 15-21.
  • IL-12 includes two subunits called IL-12 alpha (p35) and IL-12 beta (p40). These two subunits can be linked by a peptide linker to form a single chain IL-12 (see, e.g., U.S. Patent No. 7,226,998, the contents of which is incorporated herein by reference in its entirety).
  • the single chain form of IL-12 (SEQ ID NO 25) comprises IL-12 alpha and IL-12 beta linked by a peptide linker.
  • IL-12 may be secreted by phagocytic cells.
  • IL-12R IL-12 receptor
  • NK natural killer cells
  • STAT4 signal transducer and activator of transcription 4
  • IFN- ⁇ interferon gamma
  • Signaling downstream of IFN- ⁇ includes activation of T-box transcription factor TBX21 (Tbet) which, in part, induces pro-inflammatory functions of T helper 1 (TH1) cells, thereby linking innate and adaptive immune responses.
  • IL- 12A is linked, via the peptide linker, to the N-terminus of IL-12B.
  • IL-12B is linked, via the peptide linker, to the N-terminus of IL-12A.
  • Peptide linkers having proper length and flexibility can be selected such that the single-chain IL-12 has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the activity of a corresponding wild-type IL-12.
  • the peptide linker comprises an amino acid sequence from Table 7 below.
  • the peptide linker comprises the amino acid sequence of SEQ ID NO: 47. [0233]
  • Illustrative IL-12 amino acid sequences are provided in Table 2 below.
  • the engineered IL-12 comprises a polypeptide sequence 100% identical to SEQ ID NO: 23.
  • the engineered interleukin 12 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24.
  • the engineered IL-12 comprises a polypeptide sequence 100% identical to SEQ ID NO: 24.
  • the engineered interleukin 12 is a single chain IL-12 and comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25.
  • the engineered IL-12 comprises a polypeptide sequence 100% identical to SEQ ID NO: 25.
  • the engineered interleukin 12 comprises a polypeptide sequence at 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or Docket No.: 061250-559001WO 100% identical to SEQ ID NO: 26.
  • the engineered IL-12 comprises a polypeptide sequence 100% identical to SEQ ID NO: 26.
  • the fusion protein comprises an IL-12 protein that has one or more mutations relative to the corresponding wild-type IL-12 (e.g., human IL-12).
  • the IL-12 protein comprises a mutation that reduces affinity for an IL-12 receptor.
  • the IL-12 protein comprises an IL-12B (p40) subunit comprising one more mutations that reduce affinity for IL-12RP1 (see, e.g., Glassman et al. (2021) Cell 184:983-99).
  • IL-12B p40 subunit comprising one more mutations that reduce affinity for IL-12RP1 (see, e.g., Glassman et al. (2021) Cell 184:983-99).
  • CD8+ T cells after stimulation (e.g., via TCR), upregulates IL-12RP1 expression to increase sensitivity to IL-12.
  • the engineered interleukin 12 is a fusion protein comprising an IL-12 (optionally a soluble IL-12 or a tethered IL-12), and a hinge region.
  • the hinge region is a hinge region selected from Table 6.
  • the hinge region is a (G4Q)2, (G4Q)5, (G4Q)10, IgG4, EGF3, or EGF7 hinge region.
  • the engineered interleukin 12 comprises a linker between the IL- 12 and the hinge region. In some embodiments, the linker is selected from Table 7. [0239] In some embodiments, the tethered interleukin 12 comprises a transmembrane domain selected from Table 8. In some embodiments, the tethered interleukin 12 comprises a transmembrane domain of PDGFRb, B7, CD25, CD137, or CD19.
  • the present disclosure provides an engineered interleukin 15 (IL-15).
  • IL-15 Illustrative IL-15 amino acid sequences are provided in Table 3 below. Tabl Pro Na Hu IED 15 LSS Human IL- NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH 15 no SS DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 28)
  • the engineered interleukin 15 (IL-15) comprises a polypeptide sequence at least 80%, at least 90%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 27.
  • the engineered interleukin 15 comprises a polypeptide sequence 100% identical to SEQ ID NO: 27.
  • the engineered interleukin 15 (IL-15) comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 28.
  • the engineered interleukin 15 (IL-15) comprises a polypeptide sequence 100% identical to SEQ ID NO: 28.
  • the engineered interleukin 15 is a fusion protein comprising an IL-15 (optionally a soluble IL-15 or a tethered IL-15), and a hinge region.
  • the hinge region is a hinge region selected from Table 6.
  • the hinge region is a (G4Q)2, (G4Q)5, (G4Q)10, IgG4, EGF3, or EGF7 hinge region.
  • the engineered interleukin 15 is a fusion protein of IL-15 and IL- 15R ⁇ (IL-15/IL-15R ⁇ ).
  • the engineered interleukin 15 comprises a linker between the IL- 15 and the hinge region.
  • the linker is selected from Table 7.
  • the tethered interleukin 15 comprises a transmembrane domain selected from Table 8.
  • the tethered interleukin 15 comprises a transmembrane domain of PDGFRb, B7, CD25, CD137, or CD19.
  • IL-18 [0247] In one aspect, the present disclosure provides an engineered interleukin 18 (IL-18). IL- 18 is released primarily from macrophages and DCs as an inactive pro-form.
  • IL-18 amino acid sequences are provided in Table 4 below.
  • the engineered interleukin 18 (IL-18) comprises a polypeptide sequence 100% identical to SEQ ID NO: 29. In some embodiments, the engineered interleukin 18 (IL-18) comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the engineered interleukin 18 (IL-18) comprises a polypeptide sequence 100% identical to SEQ ID NO: 30.
  • the engineered interleukin 18 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1046. In some embodiments, the engineered interleukin 18 (IL-18) comprises a polypeptide sequence 100% identical to SEQ ID NO: 1046.
  • the engineered interleukin 18 comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1047.
  • the engineered interleukin 18 (IL-18) comprises a polypeptide sequence 100% identical to SEQ ID NO: 1047.
  • the engineered interleukin 18 (IL-18) is a fusion protein comprising an IL-18 (optionally a soluble IL-18 or a tethered IL-18), and a hinge region.
  • the hinge region is a hinge region selected from Table 6. In some embodiments, the hinge region is a (G4Q)2, (G4Q)5, (G4Q)10, IgG4, EGF3, or EGF7 hinge region.
  • the engineered interleukin 18 comprises a linker between the IL- 18 and the hinge region. In some embodiments, the linker is selected from Table 7.
  • the tethered interleukin 18 comprises a transmembrane domain selected from Table 8. In some the tethered interleukin 18 comprises a transmembrane domain of PDGFRb, B7, CD25, CD137, or CD19.
  • the present disclosure provides an engineered Interleukin 21 (IL-21).
  • IL-21 Illustrative IL-21 amino acid sequences are provided in Table 5 below. Table 5 Illustrative IL-21 Proteins Protein Amino Acid Sequence Na Hu PE 21 HR Hu AQ 21 SLL [0255]
  • the engineered interleukin 21 (IL-21) comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 31.
  • the engineered interleukin 21 comprises a polypeptide sequence 100% identical to SEQ ID NO: 31. In some embodiments, the engineered interleukin 21 (IL-21) comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, the engineered interleukin 21 (IL-21) comprises a polypeptide sequence 100% identical to SEQ ID NO: 32.
  • the engineered interleukin 21 is a fusion protein comprising an IL-21 (optionally a soluble IL-21 or a tethered IL-21), and a hinge region.
  • the hinge region is a hinge region selected from Table 6.
  • the hinge region is a (G4Q)2, (G4Q)5, (G4Q)10, IgG4, EGF3, or EGF7 hinge region.
  • the engineered interleukin 21 comprises a linker between the IL- 21 and the hinge region. In some embodiments, the linker is selected from Table 7.
  • the tethered interleukin 21 comprises a transmembrane domain selected from Table 8. In some embodiments, the tethered interleukin 21 comprises a transmembrane domain of PDGFRb, B7, CD25, CD137, or CD19.
  • Hinges [0259] In one aspect, the present disclosure provides an engineered interleukin comprising a hinge or fusion protein that allows for the correct spacing of the components of the engineered interleukin (e.g., the interleukin, the hinge, and membrane anchor). [0260] Illustrative hinge amino acid sequences are provided in Table 6 below.
  • the engineered interleukin is fused with a hinge region, for example a (G 4 Q) 2 , (G 4 Q) 5 , (G 4 Q) 10 , (G 4 Q) 10 -CD25_full, CD25_hinge, IgG4, EGF3, or EGF7 hinge region.
  • a hinge region for example a (G 4 Q) 2 , (G 4 Q) 5 , (G 4 Q) 10 , (G 4 Q) 10 -CD25_full, CD25_hinge, IgG4, EGF3, or EGF7 hinge region.
  • the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 33. In some embodiments, the hinge region comprises a polypeptide sequence 100% identical to SEQ ID NO: 33.
  • the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 34. In some embodiments, the hinge region comprises a polypeptide sequence 100% identical to SEQ ID NO: 34. In some embodiments, the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35.
  • the hinge region comprises a polypeptide sequence 100% identical to SEQ 35. In some embodiments, the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least Docket No.: 061250-559001WO 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 36. In some embodiments, the hinge region comprises a polypeptide sequence 100% identical to SEQ ID NO: 36.
  • the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 37. In some embodiments, the hinge region comprises a polypeptide sequence 100% identical to SEQ ID NO: 37. In some embodiments, the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 38.
  • the hinge region comprises a polypeptide sequence 100% identical to SEQ ID NO: 38. In some embodiments, the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 39. In some embodiments, the hinge region comprises a polypeptide sequence 100% identical to SEQ ID NO: 39.
  • the hinge region comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 40. In some embodiments, the hinge region comprises a polypeptide sequence 100% identical to SEQ ID NO: 40.
  • Linkers [0263] In one aspect, the present disclosure provides an engineered interleukin comprising a linker that allows for the linkage of components of the engineered interleukin (e.g., the interleukin and a membrane anchor).
  • a peptide linker between the interleukin and the transmembrane domain is a peptide linker, which can be same linker or different linkers of different length.
  • the linker can play a role in the structure and function of the membrane tethered interleukin. If the linker is too short, it may not allow for the interleukin to bind it to its receptor. If the linker is the appropriate length, it may allow the interleukin to bind to its receptor on the cell surface and stimulate the immune cell. Additionally, in order to achieve proper expression and conformation of the membrane tethered interleukin of the present disclosure, in certain embodiments specific linkers are used between the interleukin and the transmembrane domain.
  • a “linker” as used herein, is a peptide that links two polypeptides.
  • a linker can link a interleukin to a transmembrane domain to produce a membrane tethered interleukin.
  • Suitable linkers include linkers that are at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29 or 30 amino acid residues in length.
  • the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, 50-60 Docket No.: 061250-559001WO amino acids in length.
  • Suitable linkers include, but are not limited to: a cleavable linker, a non- cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.
  • the linker is a peptide linker that optionally comprises Gly and Ser.
  • the peptide linker utilize a glycine-serine polymer, including for example (GS)n (SEQ ID NO:41), (GSGGS)n (SEQ ID NO:42), (GGGS)n (SEQ ID NO:43), (GGGGS)n (SEQ ID NO:44), (GGGGGS)n (SEQ ID NO:45), and (GGGGGGS)n (SEQ ID NO:46), where n is an integer of at least one (and generally from 3 to 10).
  • the linker is a peptide linker that optionally comprises glycine (G) and glutamine (Q) residues.
  • the linker consists essentially of glycine (G) and glutamine (Q) residues. In some embodiments, the linker consists of glycine (G) and glutamine (Q) residues. In certain embodiments, the peptide linker utilize a glycine-glutamine polymer, including for example (GQ)n (SEQ ID NO: 1051), (GQGGQ)n (SEQ ID NO: 1052), (GGGQ)n (SEQ ID NO:1053), (GGGGQ)n (SEQ ID NO:1054), (GGGGGQ)n (SEQ ID NO:1055), and (GGGGGGQ)n (SEQ ID NO:46), where n is an integer of at least one (and generally from 3 to 10).
  • GQ glycine-glutamine polymer
  • the peptide linker is GSGSSRGGSGSGGSGGGGSK (SEQ ID NO:47).
  • (GxQ)y linkers the X indicates to the number of glycine residues present in the linker and the Y indicates the number of GxQ repeats in the linker, for example, (G4Q)2 would represent GGGGQGGGGQ (SEQ ID NO: 33).
  • the linker is a cleavable linker.
  • the cleavable linker allows for the release of the engineered interleukin into the tumor microenvironment.
  • the linker is a self-cleaving 2A peptide. See, e.g., Liu et al., Sci.
  • 2A peptides are viral oligopeptides that mediate cleavage of polypeptides during translation in eukaryotic cells.
  • the 2A peptide includes a C-terminus having the amino acid sequence GDVEXiNPGP (SEQ NO:50), wherein Xi is any naturally occurring amino acid residue.
  • the 2A peptide is a porcine teschovirus-12A peptide Docket No.: 061250-559001WO (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO:51).
  • the 2A peptide is an equine rhinitis A virus 2A peptide (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO:52).
  • the 2A peptide is a foot-and-mouth disease virus 2A peptide: (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO:53).
  • the cleavable linker includes a furin-cleavable sequence. Illustrative furin-cleavable sequences are described for example, Duckert et al., Protein Engineering, Design & Selection 17(1):107-112 (2004), and US Patent No.
  • the linker includes a 2A peptide and a furin-cleavable sequence.
  • the furin-cleavable 2A peptide includes the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:54).
  • the engineered interleukin is attached in the cell membrane by a degradable linker (e.g., a disulfide linker) such that under physiological conditions, the linker degrades, thereby releasing the engineered interleukin.
  • the engineered interleukin is reversibly linked to functional groups through a degradable linker such that under physiological conditions, the linker degrades and releases the engineered interleukin.
  • Suitable degradable linkers include, but are not limited to: a protease sensitive linker that is sensitive to one or more enzymes present in biological media such as proteases in a tumor microenvironment such a matrix metalloproteases present in a tumor microenvironment or in inflamed tissue (e.g., matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9)).
  • MMP2 matrix metalloproteinase 2
  • MMP9 matrix metalloproteinase 9
  • the degradable linker comprises PLGLAG (SEQ ID NO: 1057).
  • the components of the engineered interleukin are linked by an enzyme-sensitive linker.
  • Illustrative cleavable linker include those that are recognized by one of the following enzymes: metalloprotease MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.
  • Transmembrane Domains [0268]
  • the present disclosure provides an engineered tethered interleukin comprising a transmembrane domain that tethers the interleukin to the cell membrane.
  • Illustrative transmembrane domain amino acid sequences are provided in Table 8 below.
  • the transmembrane domain comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 55. In some embodiments, the transmembrane domain comprises a polypeptide sequence 100% identical to SEQ ID NO: 55.
  • the transmembrane domain comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 56. In some embodiments, the transmembrane domain comprises a polypeptide sequence 100% identical to SEQ ID NO: 56.
  • the transmembrane domain comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 57. In some embodiments, the transmembrane domain comprises a polypeptide sequence 100% identical to SEQ ID NO: 57.
  • the transmembrane domain comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1048. In some embodiments, the transmembrane domain comprises a polypeptide sequence 100% identical to SEQ ID NO: 1048.
  • the transmembrane domain comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1076. In some embodiments, the transmembrane domain comprises a polypeptide sequence 100% identical to SEQ ID NO: 1076.
  • the transmembrane domain comprises a polypeptide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1077. In some embodiments, the transmembrane domain comprises a polypeptide sequence 100% identical to SEQ ID NO: 1077. Docket No.: 061250-559001WO Intracellular Signaling Domains [0271] In some embodiments, the engineered interleukin includes an intracellular signaling domain. In some embodiments the engineered interleukin includes a B7 intracellular signaling domain.
  • the B7 family consists of structurally related, cell-surface protein ligands, which bind to receptors on lymphocytes that regulate immune responses. Activation of T and B lymphocytes can be initiated by engagement of cell-surface, antigen-specific T-cell receptors or B-cell receptors, but additional signals delivered simultaneously by B7 ligands can determine the ultimate immune response. These 'costimulatory' or 'coinhibitory' signals can be delivered by B7 ligands through the CD28 family of receptors on lymphocytes. Interaction of B7-family members with costimulatory receptors augments immune responses, and interaction with coinhibitory receptors attenuates immune responses.
  • the intracellular domain of B7 may contain serine and threonine residues that could potentially serve as phosphorylation sites in signaling pathways.
  • the B7 intracellular signaling domain is SEQ ID NO: 1078.
  • the B7 intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1078.
  • the engineered interleukin includes a CD137 intracellular signaling domain.
  • the CD137 (4-1BB, TNFRSF9) cytoplasmic signaling domain can be a key component of approved chimeric antigen receptors (CARs).
  • CD137 is a surface receptor that can be activated and expressed by T cells.
  • CD137 signaling can increase the survival, proliferation, and effector function of antigen-primed T cells. CD137 signaling can give metabolic advantages to antigen primed cells. CD137 signaling can activate NK cells and dendritic cells. CD137 signaling can help form memory T cells. CD137 signaling can activate cytotoxic effects T cells.
  • the CD137 intracellular signaling domain is SEQ ID NO: 616. In some embodiments, the CD137 intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 616. [0273] In some embodiments the engineered interleukin includes a CD27 intracellular signaling domain.
  • CD27 which belongs to the TNFR superfamily, which can be constitutively expressed on both CD8 and CD4 T cells, natural killer T cells, and other immune cells.
  • CD27 co-stimulatory signaling can promote T cell activation, clonal expansion, and effector T cell differentiation, survival and memory.
  • CD27 signaling can also enhance CAR T cell expansion, effector functions, and survival in vitro, as well as augmenting CAR T cell persistence and antitumor activity in vivo.
  • the CD27 intracellular signaling domain is SEQ ID NO: 1079.
  • the CD27 intracellular signaling comprises an amino acid sequence having Docket No.: 061250-559001WO at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1079.
  • the engineered interleukin includes a OX40 intracellular signaling domain.
  • OX40 can be a costimulatory receptor that activates T cells by binding to its ligand, OX40L.
  • OX40L is a 32 kDa protein that has a cytoplasmic domain, a transmembrane segment, and an extracellular region.
  • the OX40 intracellular signaling domain is SEQ ID NO: 1080.
  • the OX40 intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1080.
  • the engineered interleukin includes a DAP10 intracellular signaling domain.
  • DNAX-activating protein 10 is a transmembrane adapter protein that can associate with an activation receptor (NKG2D).
  • DAP10 has a minimal extracellular region, but it also has a conserved cysteine to create a disulfide-bonded homodimer.
  • the tyrosine in the YINM sequence in DAP10 can be phosphorylated to allow binding of phosphatidylinositol-3 kinase (PI3K) and a Grb2 - Vav1 – son of sevenless 1 (SOS1) complex.
  • PI3K phosphatidylinositol-3 kinase
  • SOS1 sevenless 1
  • the DAP10 intracellular signaling domain is SEQ ID NO: 1080.
  • the DAP10 intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1081.
  • the engineered interleukin includes a DAP12 intracellular signaling domain.
  • DAP12 is an encoding gene of a transmembrane signaling polypeptide and possesses an immunoreceptor tyrosine-based activation motif in its cytoplasmic domain.
  • DAP12 can also be referred to as TYRO protein tyrosine kinase-binding protein (TYROBP).
  • TYROBP TYRO protein tyrosine kinase-binding protein
  • the DAP12 intracellular signaling domain is SEQ ID NO: 1082.
  • the DAP12 intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1082.
  • the engineered interleukin includes a MYD88 intracellular signaling domain. MyD88 can act as an adaptor for inflammatory signaling pathways downstream of members of the Toll-like receptor (TLR) and interleukin-1 (IL-1) receptor families.
  • TLR Toll-like receptor
  • IL-1 interleukin-1
  • the MYD88 intracellular signaling domain is SEQ ID NO: 1083.
  • the MYD88 intracellular domain comprises an amino acid sequence Docket No.: 061250-559001WO having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1083.
  • the engineered interleukin includes a MYD88/CD40 intracellular signaling domain.
  • the MYD88/CD40 intracellular signaling domain is SEQ ID NO: 1084.
  • the MYD88/CD40 intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1084.
  • the engineered interleukin includes a CD3E intracellular signaling domain.
  • the CD3e intracellular signaling domain is SEQ ID NO: 1085.
  • the CD3e intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1085.
  • the engineered interleukin includes a PDGFRB transmembrane intracellular signaling domain.
  • the PDGFRB transmembrane intracellular signaling domain is SEQ ID NO: 1086.
  • the PDGFRB transmembrane intracellular signaling domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1086.
  • Table 12 Polypeptide sequences for illustrative intracellular signaling domains Pro Na B7 CD 6 ID CD OX 80 Dap PY DA AL LL My AL LL K My SRI 40 SG CD PD Tra ane and Intracellular domain Docket No.: 061250-559001WO Promoter Sequences [0281]
  • the engineered interleukin is expressed under the control of a constitutive promoter.
  • the constitutive promotor is an eukaryotic translation elongation factor 1 alpha (EF1a) promoter.
  • the engineered interleukin is expressed under the control of an inducible promoter.
  • the inducible promoter is an activation induced promoter.
  • the activation induced promoter is a nuclear factor of activated cells response element (NFAT RE) promoter.
  • the NFAT RE promoter is a 1x NFAT RE.
  • the NFAT RE promoter is a 2x NFAT RE.
  • the NFAT RE promoter is a 3x NFAT RE.
  • the NFAT RE promoter is a 4x NFAT RE. In some embodiments, the NFAT RE promoter is a 5x NFAT RE. In some embodiments, the NFAT RE promoter is a 6x NFAT RE.
  • the engineered interleukin includes a signal sequence polypeptide that facilitates the translocation of the protein to the immune cell membrane. Any suitable signal peptide that facilities the localization of the fusion protein to the immune cell membrane can be used. In some embodiments, the signal peptide does not interfere with the bioactivity of the engineered interleukin.
  • Illustrative signal peptide sequences include, but are not limited to: human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence, human prolactin signal sequence, and human IgE signal sequence.
  • the fusion protein includes a human IgE signal sequence.
  • the human IgE signal sequence has the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO:58).
  • the human IgE signal sequence includes the amino acid sequence NIKGSPWKGSLLLLLVSNLLLCQSVAP (SEQ ID NO:59).
  • the signal peptide sequence is an IL-2 signal sequence having the amino acid sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:60).
  • the present disclosure further provides nucleic acids comprising a nucleotide sequence encoding a polypeptide, e.g., a fusion protein, comprising a signal peptide, an interleukin and, a receptor for the interleukin. It is contemplated that any interleukin described herein can be concurrently expressed with its cognate receptor, e.g., as separate constructs or as a fusion protein. In some embodiments, interleukin is soluble.
  • the interleukin is membrane tethered.
  • mt-IL-2 is concurrently expressed with IL-2R.
  • mt-IL-2 is concurrently expressed with IL-2R alpha.
  • mt-IL- 2 is concurrently expressed with IL-2R beta.
  • mt-IL-2 is concurrently expressed with IL-2R-alpha and IL-2R-beta.
  • mt-IL-12 is concurrently expressed with IL-12R-beta-1.
  • mt-IL-12 is concurrently expressed with IL- 12R-beta-2.
  • mt-IL-12 is concurrently expressed with IL-12R-beta-1 and IL-12R-beta-2.
  • mt-IL-15 is concurrently expressed with IL-15R-alpha.
  • mt-IL-18 is concurrently expressed with IL-18R-alpha.
  • mt-IL-18 is concurrently expressed with IL-18R-beta.
  • mt-IL-18 is concurrently expressed with IL-18R-alpha and IL-18R beta.
  • mt-IL-21 is concurrently expressed with IL-21R.
  • the polypeptide comprises an amino acid linker connecting the interleukin and its receptor.
  • the peptide linker utilize a glycine-serine polymer, including for example (GS)n (SEQ ID NO:41), (GSGGS)n (SEQ ID NO:42), (GGGS)n (SEQ ID NO:43), (GGGGS)n (SEQ ID NO:44), (GGGGGS)n (SEQ ID NO:45), and (GGGGGGS)n (SEQ ID NO:46), where n is an integer of at least one (and generally from 3 to 10). Additional linkers that can be used with the present compositions and methods are described in U.S. Patent Publication Nos.
  • the peptide linker is GSGSSRGGSGSGGSGGGGSK (SEQ ID NO:47).
  • the present disclosure further provides nucleic acids comprising a nucleotide sequence encoding a polypeptide, e.g. a fusion protein, comprising a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain derived from an interleukin receptor.
  • any receptor for any interleukin described here in can incorporated into a constitutively active interleukin receptor.
  • the constitutively active interleukin constructs comprise at least one extracellular domain, a transmembrane domain, and at least one intracellular domain, wherein the intracellular domain is derived from a cytokine receptor and wherein the extracellular domain is derived from the same or a different native protein than the transmembrane and intracellular domain components.
  • the transmembrane domain and the extracellular domain are from the different native interleukin receptors.
  • the transmembrane domain and the extracellular domain are from the same native interleukin receptor and the intracellular domain is from a different protein, but the receptor is still constitutively active because it includes a mutation that places the domain and/or intracellular domain in an active confirmation. Docket No.: 061250-559001WO [0286]
  • the constitutively active interleukin constructs are constitutively active because their transmembrane domain and/or intracellular domain components are configured to transmit an activating signal in the absence of a corresponding signal from the extracellular domain to which they are operably linked. In some embodiments, there is no ligand requirement for the cytokine receptor.
  • the transmembrane domain and/or intracellular domain of the constitutively active interleukin constructs of the disclosure may be configured such that they are able to homodimerize in a non-natural manner or situation or environment, thereby allowing the extracellular domain to remain in a state that transmits an activating signal to a corresponding entity downstream in a signaling pathway.
  • the transmembrane/intracellular domains of the constitutively active interleukin constructs independently give a positive cytokine signal in the absence of binding of a cytokine by the extracellular domain to which they are operably linked.
  • the constitutively active interleukin receptor comprises one, two, or more extracellular domains.
  • the extracellular domain is capable of binding a ligand but the signal itself is not transmitted, for example because of structural or other reasons.
  • the extracellular domain may or may not be from the same native protein as its corresponding transmembrane and/or intracellular domain.
  • the extracellular domain may or may not be from the same native protein as the intracellular domain to which it is operably linked.
  • the extracellular domain of the constitutively active interleukin receptor is derived from EGFR.
  • the extracellular domain of the constitutively active interleukin receptor comprises SEQ ID NO: 1170 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • the extracellular domain of the constitutively active interleukin receptor is SEQ ID NO: 1170 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • the extracellular domain of the constitutively active interleukin receptor is derived from CD33.
  • the extracellular domain of the constitutively active interleukin receptor comprises SEQ ID NO: 1171 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • the extracellular domain of the constitutively active interleukin receptor is SEQ ID NO: 1171 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • the constitutively active interleukin receptor comprises a transmembrane domain that is operably linked to the extracellular domain and to the intracellular domain.
  • the transmembrane domain may or not be from the same native protein as the intracellular domain to which it is operably linked.
  • the transmembrane is Docket No.: 061250-559001WO not from the same native protein as the extracellular domain to which it is operably linked.
  • the transmembrane domain is not natural, because it includes a mutation that causes dimerization.
  • the transmembrane domain may comprise one or more mutations compared to its corresponding wild type, native protein that allows the transmembrane domain to be self-active.
  • self-active refers to a transmembrane domain that in conjunction with the intracellular domain to which it is operably linked transmits a signal to the cell in the absence of a corresponding activating signal from an extracellular domain to which it is operably linked.
  • the transmembrane domain comprises one or more mutations that cause or facilitate homodimerization.
  • the transmembrane domain imparts a functional configuration to the receptor to allow self-activation, such as allowing the downstream intracellular domains to be oriented relative to each other in a manner that is conducive to signaling, such as permitting kinases that have associated with the intracellular domains to interact with each other and cross activate each other.
  • the transmembrane domain comprises one or more mutations that allow the transmembrane domain and intracellular domain to act in conjunction in a non- transient manner when they do not naturally do so.
  • the mutation renders the transmembrane and intracellular domain able to homodimerize artificially.
  • the transmembrane domain may comprise one or more mutations that allow homodimerization of two separate receptors each of which comprises the transmembrane domain and at least one intracellular domain, wherein the homodimerization of such receptors does not occur in nature.
  • the one or more mutations in the transmembrane domain induce a receptor homodimer to form a self- activating helical structure, for example.
  • the ability to determine whether or not a particular mutation will induce a structural configuration such that the transmembrane and intracellular domains self-activate may be determined with routine methods, such as assaying for STAT3 or STAT5 phosphorylation following growth of the cells harboring the particular receptor being tested in the absence of growth factors.
  • transmembrane domain components with such one or more gain-of- function mutations may be employed in methods and compositions of the disclosure.
  • the mutation(s) may be a substitution, insertion, deletion, or combination thereof, for example.
  • the one or more mutations comprises inclusion of at least one cysteine and/or at least one proline.
  • the transmembrane domain utilizes a mutation identified in a tumor patient as a gain-of-function mutation in the transmembrane domain.
  • the mutant versions include cysteine insertion(s) that induce disulfide bond formation in the transmembrane domain.
  • the one or more mutations lacks insertion of a cysteine.
  • transmembrane derivatives may be utilized that do not have a cysteine Docket No.: 061250-559001WO insertion(s) (and, therefore, no non-native disulfide bond) that still signals and is constitutively active, for example because the mutation rendered the transmembrane domain conformationally changed compared to a natural version of the transmembrane domain, thereby allowing induction of signaling.
  • the constitutively active interleukin receptor comprises one or more intracellular domains.
  • the intracellular domain is from the same native protein as the transmembrane domain.
  • the intracellular domain is from a different native protein as the transmembrane domain.
  • the intracellular domain is from a cytokine receptor, including an immunostimulatory cytokine receptor.
  • the cytokine receptor acts in a signaling pathway that includes STAT5, STAT3, and so forth.
  • Immunostimulatory cytokine intracellular domains useful for constitutively active interleukin constructs of the disclosure include IL-7Ra receptor alpha, CD 122 (the common receptor beta of IL-2 and IL-15), IL-21 receptor alpha, IL- 23 receptor alpha, and IL-12 receptor alpha, and IL-6 receptor, for example.
  • Other immunostimulatory cytokine intracellular domains known in the art are contemplated.
  • the immunostimulatory cytokine intracellular domains may or may not comprise one or more mutations.
  • the mutation causes or facilitates homodimerization.
  • the intracellular domain is selected based on the desired downstream pathway.
  • the intracellular domain may be selected based on the desire for the signal to be transmitted via JAK1, STAT5, STAT4, JAK3, STAT3, and so on. Or the desire to not signal from one or more of JAK1, STAT5, STAT4, JAK3, STAT3, and so on.
  • the signaling pathway includes STAT5.
  • STAT5 is a major downstream signaling node of the immunostimulatory cytokines IL-15 and IL-7, both of which are known to be useful in activating T-cells in the context of immunotherapy.
  • IL-15 and IL-7 both of which are known to be useful in activating T-cells in the context of immunotherapy.
  • Several publications have already shown that bypassing the cytokine-cytokine receptor interaction, and activating STAT5 directly (using constitutively active STAT5 mutants), CD8 T-cell function is enhanced via increased persistence in vivo and enhanced anti-tumor efficacy in vivo using preclinical models.
  • the same concept can be extended to activating STAT proteins downstream of other known immunostimulatory cytokines, such as STAT4, which is downstream of IL-12.
  • the intracellular domain comprises the intracellular domain from the IL7 receptor alpha chain (IL7R ⁇ ), which may or may not comprise one or more mutations. In some embodiments, it is operably linked to a transmembrane domain that comprises a mutation that causes or facilitates homodimerization.
  • the IL7R ⁇ transmembrane and intracellular domain comprises SEQ ID NO: or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • the constitutively active interleukin intracellular domain further comprises a first switch a first switch domain on a first polypeptide chain and a second switch domain on the second polypeptide chain, where the two switch domains are selected to homo- or heterodimerize in response to a cognate switch molecule.
  • Illustrative switch domain pairs and cognate switch molecules are provided in Table 15. Where the switch domains form heterodimers, it should be understood that the first and second switch domains may be interchanged between the two polypeptide chains. While representative sequences are provided, it is possible to identify functional variants of the provided sequences by varying the amino acid sequence and testing for selective dimerization in the presence of the switch molecule.
  • switch domain refers to a polypeptide molecule that, in the presence of a switch molecule, associates with another switch domain. The association of the polypeptides and small molecule results in a functional coupling of a first polypeptide molecule that comprises a switch domain to a second polypeptide molecule that comprises a switch domain.
  • the first and second switch domain are the same as one another, i.e., they are polypeptides having the same (or similar) amino acid sequence,and referred to collectively as a homodimerization switch.
  • the first and second switch domains are different from one another, i.e., they include polypeptide sequences having different amino acid sequence, and are referred to collectively as a heterodimerization switch.
  • the switch domain is intracellular. In embodiments, the switch domain is extracellular.
  • the switch is made up of a switch domain comprising FKBP and a second switch domain comprising FRB and the switch molecule is a small molecule, e.g., a rapalog.
  • the first switch domain comprises FKBP and the second switch domain comprises calcineurin and the switch molecule is a small e.g., a rapalog.
  • the first switch domain comprises FKBP and the second switch domain comprises cyclophilin and the Docket No.: 061250-559001WO switch molecule is a small molecule, e.g., a rapalog.
  • the first switch domain comprises FKBP and the second switch domain comprises bacterial dihydrofolate reductase (DHFR) and the switch molecule is a small molecule, e.g., a rapalog or methotrexate.
  • the first switch domain comprises calcineurin and the second switch domain comprises cyclophilin and the switch molecule is a small molecule, e.g., cyclosporin A or rapalog.
  • the first switch domain comprises PYR1-like 1 (PYL1) and the second switch domain comprises of abscisic acid insensitive 1 (ABI1) and the switch molecule is a small molecule, e.g., abscisic acid.
  • the first switch domain comprises GIB1 and the second switch domain comprises GAI and the switch molecule is a small molecule, e.g. gibberellin.
  • the first switch domain comprises an estrogen receptor and the second switch domain comprises an estrogen receptor and the switch molecule is a small molecule, e.g. tamoxifen or 4-hydroxytamoxifen.
  • FKBP12 FKBP, or FK506 binding protein
  • Rapamycin binds to FKBP and to the large P13K homolog FRAP (RAFT, mTOR), thereby acting to dimerize molecules.
  • the FKBP switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1177. In embodiments, the FKBP switch domain comprises or consists essentially of SEQ ID NO: 1177. In embodiments, the FKBP switch domain comprises a F36V mutation.
  • an FKBP/FRAP based switch also referred to herein as an FKBP/FRB, based switch can use a heterodimerization molecule, e.g., rapamycin or a rapamycin analog.
  • FRB is a 93 amino acid portion of FRAP, that is sufficient for binding the FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L. (1995). Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue has been reported in Proc Natl Acad Sci USA 92: 4947-51).
  • the FRB and FKBP protein can be mutated at certain residues to increase or decrease their affinity for rapamycin or rapalogs. In embodiments, the FKBP is mutated at F36V.
  • FKBP with F36V mutations has been reported to have 1000x higher affinity for Rim than the native FKBP ( Clackson, et al. "Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity.” PNAS 95.18 (1998): 10437-10442)
  • the F36V mutations may also allow the FKBP interaction with Rim to be a reversible reaction (Rollins, et al. "A ligand-reversible dimerization system for controlling protein–protein interactions.” PNAS 97.13 (2000): 7096- 7101).
  • the F36V mutation is underlined table Mutations that can increase or decrease binding affinity are described in U.S. Patent App. No. which is incorporated here in its entirety.
  • the switch domain comprises an FRB binding fragment or analog of Docket No.: 061250-559001WO FKBP and an FKBP binding fragment or analog of FRB
  • the FKBP binding fragment or analog of FRB comprises one or more mutations which enhances the formation of a complex between FKBP or FRB and the switch molecule.
  • the FKBP binding fragment or analog of FRB may comprise: an E2032 mutation, e.g., an E2032I mutation or E2032L mutation; a T2098 mutation, e.g., a T2098L mutation; or an E2032 and a T2098 mutation, e.g., an E2032I and a T2098L or an E2032L and a T2098L mutation.
  • the FRB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1178. In embodiments, the FRB switch domain comprises or consists essentially of SEQ ID NO: 1178.
  • calcineurin catalytic subunit A also known as PPP3CA; CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit; calcineurin A alpha; calmodulin- dependent calcineurin A subunit alpha isoform; protein phosphatase 2B, catalytic subunit, alpha isoform; etc.
  • Calcineurin protein phosphatase 2B
  • Calcineurin protein phosphatase 2B
  • RyR ryanodine receptor
  • the association between calcineurin and RyR involves FKB12, an accessory unit of RyR.
  • the calcineurin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1179.
  • the calcineurin switch domain comprises or consists essentially of SEQ ID NO: 1179.
  • the switch domain is a domain of a cyclophilin (also known as cyclophilin A, PPIA, CYPA, CYPH, PPIase A, etc.) capable of dimerizing.
  • Cyclophilins are part of a group of proteins known as immunophilins that have peptidyl-prolyl cis-trans isomerase activity. Mammalian cyclophilin A is the major cellular target for, and thus mediates the actions of, the immunosuppressive drug cyclosporin A. The cyclophilins and FKBP naturally bind to cyclosporin and rapamycin to mediate their immunosuppressive and toxic effects.
  • the cyclophilin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1180. In embodiments, the cyclophilin switch domain comprises or consists essentially of SEQ ID NO: 1180.
  • the switch domain is a domain of a DHFR (also known as dihydrofolate reductase, DHFRP1, and DYR) capable of dimerizing in response to a small molecule.
  • the DHFR switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1181.
  • the cyclophilin switch domain or consists essentially of SEQ ID NO: 1181.
  • the switch domain is a domain of a PYL protein (also known as abscisic acid receptor and as RCAR) capable of dimerizing in response to a small molecule.
  • a switch domain can be derived from proteins such as those of Arabidopsis thaliana: PYR1, RCAR1(PYL9), PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8 (RCAR3), PYL10, PYL11, PYL12, PYL13.
  • the PYL1 switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1182. In embodiments, the PYL1 switch domain comprises or consists essentially of SEQ ID NO: 1182. [0306] In embodiments, the switch domain is a domain of a ABI protein (also known as Abscisic Acid-Insensitive) capable of dimerizing in response to a small molecule.
  • ABI protein also known as Abscisic Acid-Insensitive
  • a switch domain can be derived from proteins such as those of I: ABI1 (Also known as ABSCISIC ACID- INSENSITIVE 1, Protein phosphatase 2C56, AtPP2C56, P2C56, and PP2C ABI1) and/or ABI2 (also known as P2C77, Protein phosphatase 2C77, AtPP2C77, ABSCISIC ACID-INSENSITIVE 2, Protein phosphatase 2C ABI2, and PP2C ABI2).
  • the ABI switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1183.
  • the ABI switch domain comprises or consists essentially of SEQ ID NO: 1183.
  • the switch domain can comprise a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of any of the amino acid in SEQ ID NO: 1183.
  • the switch domain is a domain of a GID1 Arabidopsis thaliana protein (also known as Gibberellin receptor GID1) capable of dimerizing in response to a small molecule.
  • the GID1 switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1184.
  • the GID1 switch domain comprises or consists essentially of SEQ ID NO: 1184.
  • the switch domain is a domain of a GAI I protein (also known as Gibberellic Acid Insensitive, and DELLA protein GAI) capable of dimerizing in response to a small molecule.
  • the GAI switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1185. In embodiments, the GAI switch domain comprises or consists essentially of SEQ ID NO: 1185. Docket No.: 061250-559001WO [0309] In embodiments, the switch domain is a domain of GyrB (also known as DNA gyrase subunit B) capable of dimerizing in response to a small molecule. In embodiments, the GyrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1186.
  • GyrB also known as DNA gyrase subunit B
  • the GyrB switch domain comprises or consists essentially of SEQ ID NO: 1186.
  • the switch domain is a domain of DmrB (i.e. DmrB homodimerization domain) capable of dimerizing in response to a small molecule.
  • the DmrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1187.
  • the DmrB switch domain comprises or consists essentially of SEQ ID NO: 1187.
  • the switch domain is a domain of the estrogen receptor capable of dimerizing in response to a small molecule, e.g., tamoxifen.
  • the estrogen switch domain comprises the estrogen receptor.
  • the estrogen switch domain comprises a portion of the estrogen receptor.
  • the estrogen switch domain comprises estrogen receptor alpha.
  • the estrogen switch domain comprises estrogen receptor beta.
  • the estrogen switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 1188, or a fragment thereof and which is capable of binding the small molecule, e.g., tamoxifen.
  • the estrogen switch domain comprises or consists essentially of SEQ ID NO: 1188.
  • the switch domains dimerize upon treatment with a small molecule or “switch molecule” selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, tamoxifen or a derivative thereof (e.g.4-hydroxytamoxifen) or any combination thereof.
  • a small molecule or “switch molecule” selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, met
  • 'Rapalog may refer to an analog or derivative of rapamycin, examples include everolimus (RAD001), temsirolimus (CCL-779), ridaforolimus, dactolisib (NVP-BEZ235), GSK2126458, XL 765, AZD8055, INK128/MLN0128, OS1027, AP1903 (Rimiducid) and rapalinks. Additional examples of rapalogs are known in the art and are described in Abdel-Magid et al., 2019 PMID: 31223435.
  • Tamoxifen may refer to an analog or derivative of tamoxifen and is capable of acting as a selective estrogen receptor modulator.
  • the switch domains listed in the present specification are meant to be examples not limiting. The switch domains can be combined combination and can be truncated or mutated to increase or decrease affinity for a specific small molecule. Docket No.: 061250-559001WO [0316]
  • the constitutively active cytokine construct comprises a switch domain.
  • the switch domain forms a homodimer upon binding of the small molecule.
  • the constitutively active cytokine construct comprises a single switch domain.
  • the constitutively active cytokine construct comprises more than one switch domain. In some embodiments, the constitutively active cytokine construct comprises two switch domains. In some embodiments, the constitutively active cytokine construct comprises three switch domains. [0317] In some embodiments, the switch domain of the constitutively active cytokine construct is a FKBP switch domain. In embodiments, the FKBP switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1177. In embodiments, the FKBP switch domain comprises or consists essentially of SEQ ID NO 177.
  • the switch domain of the constitutively active cytokine construct is a FRB switch domain.
  • the FRB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1178.
  • the FRB switch domain comprises or consists essentially of SEQ ID NO 1178.
  • the switch domain of the constitutively active cytokine construct is a calcineurin switch domain.
  • the calcineurin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1179.
  • the calcineurin switch domain comprises or consists essentially of SEQ ID NO 1179.
  • the switch domain of the constitutively active cytokine construct is a cyclophilin switch domain.
  • the cyclophilin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1180.
  • the cyclophilin switch domain comprises or consists essentially of SEQ ID NO 1180.
  • the switch domain of the constitutively active cytokine construct is a bacterial DHFR switch domain.
  • the bacterial DHFR switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1181. In embodiments, the bacterial DHFR switch domain comprises or consists essentially of SEQ ID NO 1181. [0322] In some embodiments, the switch domain of the constitutively active cytokine construct is a PYLI switch domain. In embodiments, the switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% Docket No.: 061250-559001WO identity or is identical to SEQ ID NO: 1182.
  • the PYLI switch domain comprises or consists essentially of SEQ ID NO 1182.
  • the switch domain of the constitutively active cytokine construct is a ABI1 switch domain.
  • the ABI1 switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1183.
  • the bacterial ABI1 switch domain comprises or consists essentially of SEQ ID NO 1183.
  • the switch domain of the constitutively active cytokine construct is a GIB1B switch domain.
  • the GIB1B switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1184. In embodiments, the GIB1B switch domain comprises or consists essentially of SEQ ID NO 1184. [0325] In some embodiments, the switch domain of the constitutively active cytokine construct is a GAI switch domain. In embodiments, the GAI switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1185. In embodiments, the GAI switch domain comprises or consists essentially of SEQ ID NO: 1185.
  • the switch domain of the constitutively active cytokine construct is a GyrB switch domain.
  • the GyrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1186.
  • the GyrB switch domain comprises or consists essentially of SEQ ID NO 1186.
  • the switch domain of the constitutively active cytokine construct is a DmrB switch domain.
  • the DmrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1187.
  • the DmrB switch domain comprises or consists essentially of SEQ ID NO 1187.
  • the switch domain of the constitutively active cytokine construct is an estrogen receptor switch domain.
  • the estrogen switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 1188, or a fragment thereof and which is capable of binding the small molecule, e.g., tamoxifen.
  • the estrogen switch domain comprises or consists essentially of SEQ ID NO: 1188.
  • the intracellular is of a certain length.
  • the length of the intracellular domain is between 70-250 amino acids, 70-225 amino acids, 70-200 Docket No.: 061250-559001WO amino acids, 70-175 amino acids, 70-150 amino acids, 70-125 amino acids, 70-100 amino acids, 80-250 amino acids, 80-225 amino acids, 80-200 amino acids, 80-175 amino acids, 80-150 amino acids, 80-100 amino acids, 90-250 amino acids, 90-225 amino acids, 90-200 amino acids, 90-175 amino acids, 90-150 amino acids, 90-125 amino acids,, 90-100 amino acids, 100-250 amino acids, 100-225 amino acids, 100-200 amino acids, 100-175 amino acids, 100-150 amino acids, 100-125 amino acids, 125-250 amino acids, 125-225 amino acids, 125-200 amino acids, 125-175 amino acids, 125-150 amino acids, 150-250 amino acids, 150-225 amino acids, 150-200 amino acids, 150-175 amino acids, 175-250 amino acids, 175-225 amino acids, 17
  • engineered cells of the present disclosure may comprise a constitutively active interleukin receptor with an activator receptor and a blocker receptor.
  • the engineered immune cell may be produced by introducing a vector(s) encoding the constitutively active interleukin receptor and/or encoding the activator receptor and the blocker receptor.
  • a nucleic acid molecule(s) or vector(s) encoding the constitutively active interleukin receptor and/or encoding the activator encoding and the blocker receptor introduced into the host cell may either integrate into the genome of the host or it may be maintained extra chromosomally.
  • the engineered immune cell is a T cell that expresses the constitutively active interleukin receptor with or without an activator receptor and with or without a blocker receptor.
  • the constitutively active interleukin receptor is expressed under the control of a constitutive promoter.
  • the constitutive promotor is a eukaryotic translation elongation factor 1 alpha (EF1a) promoter.
  • the constitutively active interleukin receptor is expressed under the control of an inducible promoter.
  • the inducible promoter is an activation induced promoter.
  • the activation induced promoter is a nuclear factor of activated cells response element (NFAT RE) promoter.
  • NFAT RE nuclear factor of activated cells response element
  • the NFAT RE promoter is a 1x NFAT RE.
  • the NFAT RE promoter is a 2x NFAT RE.
  • the NFAT RE promoter is a 3x NFAT RE.
  • the NFAT RE promoter is a 4x NFAT RE.
  • the NFAT RE promoter is a 5x NFAT RE.
  • the NFAT RE promoter is a 6x NFAT RE.
  • the constitutively active interleukin receptors of the present invention are functional and are capable of driving immune cell proliferation in the absence of exogenous IL2.
  • the constitutively active receptors of the present invention e.g., the IL7R ⁇ construct, when incorporated into the dual receptor system of the present disclosure, robust Docket No.: 061250-559001WO on-target activity and high normal cell selectivity of the dual receptor system, e.g., sparing of off- target cells, is maintained.
  • Such constitutively active interleukin constructs that activate cells independent of cytokine binding are beneficial in developing better versions of engineered immune cells.
  • the disclosure provides an activator receptor, comprising a first extracellular ligand binding domain specific to a target antigen comprising a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).
  • MHC-I major histocompatibility complex class I
  • the activator receptor mediates activation of an immune cell expressing the activator receptor upon binding of the target antigen by the extracellular ligand binding domain of the activator receptor.
  • the activator receptor is responsive to a target antigen (i.e. activator ligand).
  • the activator receptor when a target antigen binds to or contacts the activator receptor, the activator receptor is responsive and activates an immune cell expressing the first receptor upon binding of the target antigen by the extracellular ligand binding domain of the activator receptor.
  • the activator receptor is a chimeric antigen receptor (CAR).
  • the activator receptor is a T cell receptor (TCR).
  • activator ligand binding domains for the activator receptor may be isolated or derived from any source known in the art, including, but not limited to, art recognized T cell receptors, chimeric antigen receptors and antibody binding domains.
  • the first ligand binding domain may be derived from any of the antibodies disclosed in Table 9, and bind to a first ligand selected from the antigens described in Table 9. Accordingly, the immune cells comprising the two receptor system described can be used to treat any of the diseases or disorders described in Table 9. Selection of an appropriate first, activator receptor ligand binding domain to treat any the cancers described herein will be apparent to those of skill in the art.
  • the activator antigen is selected from the group consisting of CD33, CD19 molecule (CD19), CLL-1, CD53, SPN, ITGA4, SELPLG, and CLEC12A.
  • Chimeric Antigen Receptors [0338] The disclosure provides an activator receptor and immune cells comprising same. In some embodiments, the activator receptor is a chimeric antigen receptor. [0339] The term “chimeric antigen receptors (CARs)” as used herein, may refer to artificial receptors derived from T-cell receptors and encompasses engineered receptors that graft an artificial specificity onto a particular immune effector cell.
  • CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy.
  • CARs direct specificity of the cell to a tumor associated antigen, for example.
  • Illustrative CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region.
  • CARs further comprise a hinge domain.
  • CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to a CD3 transmembrane domain and endodomain.
  • CARs may be derived from ligands of receptors (e.g., peptides).
  • CARs comprise domains for additional co-stimulatory signaling, such as CD3, 4-1BB, FcR, CD27, CD28, CD137, DAP10, and/or OX40.
  • molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors.
  • the extracellular ligand binding domain of the activator receptor is fused to the extracellular domain of a CAR.
  • the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo.
  • Illustrative extracellular hinges can be isolated or derived from IgD and CD8 domains, for example IgG1.
  • the hinge is isolated or derived from CD8 ⁇ or CD28.
  • the hinge is or derived from CD8 ⁇ or CD28.
  • the CD8 ⁇ hinge comprises an amino acid sequence having at least 80% identity, at Docket No.: 061250-559001WO least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 61).
  • the CD8 ⁇ hinge comprises SEQ ID NO: 61.
  • the CD8 ⁇ hinge consists essentially of SEQ ID NO: 61.
  • the CD8 ⁇ hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGA GGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 62).
  • the CD8 ⁇ hinge is encoded by SEQ ID NO: 62.
  • the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 63). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO:63.
  • the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGA ACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCT TCTAAGCCC (SEQ ID NO: 605).
  • the CD28 hinge is encoded by SEQ ID NO: 605.
  • the CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • 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 may be isolated or derived from (i.e.
  • the Docket No.: 061250-559001WO transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the CARs comprise a CD28 transmembrane domain.
  • the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 606).
  • the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 606.
  • the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of [0347] TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGG TGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 607).
  • the CD28 transmembrane domain is encoded by SEQ ID NO: 607.
  • the CARs comprise an IL-2Rbeta transmembrane domain.
  • the IL-2Rbeta transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 608).
  • the IL-2Rbeta transmembrane domain comprises or consists essentially of SEQ ID NO: 608.
  • the IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of [0349] ATTCCGTGGC TCGGCCACCT CCTCGTGGGC CTCAGCGGGG CTTTTGGCTT CATCATCTTA GTGTACTTGC TGATC (SEQ ID NO: 609).
  • the IL- 2Rbeta transmembrane domain is encoded by SEQ ID NO: 609.
  • the cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the instant disclosure is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed.
  • effector function refers to a specialized function of a cell.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain can be employed, in many cases it is not necessary to use the entire domain.
  • intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.
  • the intracellular domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain.
  • the intracellular activation domain ensures that there is T-cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell.
  • the at least one cytoplasmic activation is a CD247 molecule (CD3 ⁇ ) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX-activating protein of 12 kDa (DAP12) activation domain.
  • CD3 ⁇ CD247 molecule
  • KIR stimulatory killer immunoglobulin-like receptor
  • DAP12 DNAX-activating protein of 12 kDa
  • the CD3 ⁇ activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 610).
  • the CD3 ⁇ activation domain comprises or consists essentially of SEQ ID NO: 610.
  • the CD3 ⁇ activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of [0355] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCA GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGG ACAAGCGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC TCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTG AGATTGGGATGAAAGGCGAGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAG GGACTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCC CCTCGC (SEQ ID NO: 611).
  • the CD3 ⁇ activation domain is encoded by SEQ ID NO: 611).
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • the ITAM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids (YxxL/I (SEQ ID NO: 612)).
  • the cytoplasmic domain contains 1, 2, 3, 4 or 5 ITAMs.
  • An illustrative ITAM containing cytoplasmic domain is the CD3 ⁇ activation domain.
  • ITAM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the CD3 ⁇ activation domain comprising a single ITAM comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 613).
  • the CD3 ⁇ activation domain comprises SEQ ID NO: 613.
  • the CD3 ⁇ activation domain comprising a single ITAM consists essentially of an amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 613).
  • the CD3 ⁇ activation domain comprising a single ITAM is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of AGAGTGAAGT TCAGCAGGAG CGCAGACGCC CCCGCGTACC AGCAGGGCCA GAACCAGCTC TATAACGAGC TCAATCTAGG ACGAAGAGAG GAGTACGATG TTTTGCACAT GCAGGCCCTG CCCCCTCGC (SEQ ID NO: 614).
  • the CD3 ⁇ activation domain is encoded by SEQ ID NO: 614.
  • the cytoplasmic domain of the CAR can be designed to comprise the CD3 ⁇ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the instant disclosure.
  • the cytoplasmic domain of the CAR can comprise a CD3 ⁇ chain portion and a co-stimulatory domain.
  • the co-stimulatory domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • Examples of such Docket No.: 061250-559001WO molecules include the co-stimulatory domain is selected from the group consisting of IL-2R ⁇ , Fc Receptor gamma (FcR ⁇ ), Fc Receptor beta (FcR ⁇ ), CD3g molecule gamma (CD3 ⁇ ), CD3 ⁇ , CD3 ⁇ , CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), T
  • the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain.
  • the co-stimulatory domain is isolated or derived from CD28.
  • the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain.
  • the co-stimulatory domain is isolated or derived from CD28.
  • the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of [0361] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 65).
  • the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 65.
  • the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of [0362] AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCC CCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTT CGCCGCCTACCGGAGC (SEQ ID NO: 615).
  • the CD28 co-stimulatory domain is encoded by SEQ ID NO: 615.
  • the co-stimulatory domain is isolated or derived from 4-1BB.
  • the 4-1BB co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 616).
  • the 4-1BB co-stimulatory domain comprises or consists essentially of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 616).
  • the 4-1BB co-stimulatory domain s encoded by a nucleotide sequence having at least 80% identity, at least 90% at least 95% identity, at least 99% identity or is identical to a sequence of Docket No.: 061250-559001WO AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCAGT ACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAG GAGGATGTGAACTG (SEQ ID NO: 617).
  • the intracellular domain of the CAR comprises a CD28 co- stimulatory domain, a 4-1BB costimulatory domain, and a CD3 ⁇ activation domain.
  • the intracellular domain of the CAR comprises a sequence of [0365] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 618), or a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity thereto.
  • the cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example between 2 and 10 amino acids in length may form the linkage.
  • a glycine-serine doublet provides an example of a suitable linker.
  • An illustrative linker comprises a sequence of GGGGSGGGGSGGGGSGG (SEQ ID NO: 619).
  • a short oligo- or polypeptide linker for example between 2 and 10 amino acids in length may form the linkage.
  • a glycine-serine doublet provides an example of a suitable linker.
  • Illustrative full length activator receptors of the disclosure are described in Table 10 below.
  • the activator receptor comprises a sequence of SEQ ID NOS: 983-1044, as set forth in Table 10. In some embodiments, the activator receptor comprises a sequence of SEQ ID NO: 985, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the activator receptor comprises a sequence of SEQ ID NO: 994, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the activator receptor comprises a sequence of SEQ ID NO: 998, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the activator receptor comprises a sequence of SEQ ID NO: 999, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the activator receptor comprises a sequence of SEQ ID NO: 1000, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the activator receptor comprises a sequence of SEQ ID NO: 1011, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the activator receptor comprises a sequence of SEQ ID NO: 1032, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the activator receptor sequence of SEQ ID NO: 1037, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some Docket No.: 061250-559001WO embodiments, the activator receptor comprises a sequence of SEQ ID NO: 1041 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example between 2 and 10 amino acids in length may form the linkage.
  • a glycine-serine doublet provides an example of a suitable linker.
  • T Cell Receptors TCRs
  • the disclosure provides an activator receptor and immune cells comprising same.
  • the activator receptor is a T cell receptor (TCR).
  • a “TCR”, sometimes also called a “TCR complex” or “TCR/CD3 complex” refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits.
  • the TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes.
  • TCR alpha/beta heterodimer engages peptide-MHC
  • conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal.
  • the TCR alpha and TCR beta polypeptides form a heterodimer
  • CD3 epsilon and CD3 delta form a heterodimer
  • two CD3 zeta form a homodimer.
  • any suitable ligand binding domain may be fused to an extracellular domain, hinge domain or transmembrane of the TCRs described herein.
  • the ligand binding domain can be an antigen binding domain of an antibody or TCR, or comprise an antibody fragment, a V ⁇ only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb).
  • the ligand binding domain is fused to one or more extracellular domains or transmembrane domains of one or more TCR subunits.
  • the TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma or CD3 zeta.
  • the ligand binding domain can be fused to TCR alpha, or TCR beta, or portions of the ligand binding can be fused to two subunits, for example portions of the ligand binding domain can be fused to both TCR alpha and TCR beta.
  • TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivative be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity.
  • TCR transmembrane domains isolated or derived from any source are envisaged as within the scope of the disclosure.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target.
  • a transmembrane domain of particular use in this disclosure may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can be attached to the extracellular region of a polypeptide of the TCR, e.g., the antigen binding domain of the TCR alpha or beta chain, via a hinge, e.g., a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • the hinge is isolated or derived from CD8 ⁇ or CD28.
  • the extracellular ligand binding domain is attached to one or more transmembrane domains of the TCR.
  • the transmembrane domain comprises a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or both. In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region).
  • one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region
  • additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain.
  • the transmembrane domain may be a membrane anchor domain.
  • a natural or artificial transmembrane domain may comprise a hydrophobic a-helix of about 20 amino acids, often with positive flanking the transmembrane segment.
  • the transmembrane domain may have one transmembrane segment or more than one transmembrane Docket No.: 061250-559001WO segment. Prediction of transmembrane domains/segments may be made using publicly available prediction tools (e.g., TMHMM, Krogh et al. Journal of Molecular Biology 2001; 305(3):567-580; or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993; 347: 166).
  • TMHMM Krogh et al. Journal of Molecular Biology 2001; 305(3):567-580
  • TMpred Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993; 347: 166.
  • Non-limiting examples of membrane anchor systems include platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (added post- translationally to a signal sequence) and the like.
  • the transmembrane domain comprises a TCR alpha transmembrane domain.
  • the TCR alpha transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 620).
  • the TCR alpha transmembrane domain comprises, or consists essentially of, SEQ ID NO: 621. In some embodiments, the TCR alpha transmembrane domain is encoded by a sequence of GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACG CTGCGGCTGTGG (SEQ ID NO: 621). [0383] In some embodiments, the transmembrane domain comprises a TCR beta transmembrane domain.
  • the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 622).
  • the TCR beta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 622.
  • the TCR beta transmembrane domain is encoded by a sequence of ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGT GCCCTCGTGCTG (SEQ ID NO: 623).
  • TCRs of the disclosure can comprise one or more intracellular domains.
  • Illustrative TCRs comprising intracellular domains for use in the instant disclosure are described in PCT/US2020/045250 filed on September 6, 2020, the contents of which are incorporated herein by reference.
  • the intracellular domain comprises one or more domains capable of providing a stimulatory signal to a transmembrane domain.
  • the intracellular domain comprises a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal.
  • the intracellular domain comprises a first, second and third intracellular domain capable of providing a stimulatory signal.
  • the intracellular domains capable of providing a stimulatory signal are selected from the group of a CD28 molecule (CD28) domain, a LCK proto-oncogene, Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily Docket No.: 061250-559001WO member 9 (4-1BB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.
  • an intracellular domain comprises at least one intracellular signaling domain.
  • An intracellular signaling domain generates a signal that promotes a function a cell, for example an immune effector function of a TCR containing cell, e.g., a TCR-expressing T- cell.
  • the intracellular domain of the activator receptor of the disclosure includes at least one intracellular signaling domain.
  • the intracellular domains of CD3 gamma, delta or epsilon comprise signaling domains.
  • the extracellular domain, transmembrane domain and intracellular domain are isolated or derived from the same protein, for example T-cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta.
  • TCR T-cell receptor
  • Examples of intracellular domains for use in activator receptors of the disclosure include the cytoplasmic sequences of the TCR alpha, TCR beta, CD3 zeta, and 4-1BB, and the intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • the intracellular signaling domain comprises a primary intracellular signaling domain.
  • Illustrative primary intracellular signaling domains include those derived from the proteins responsible for primary stimulation, or antigen dependent stimulation.
  • the intracellular domain comprises a CD3 delta intracellular domain, a CD3 epsilon intracellular domain, a CD3 gamma intracellular domain, a CD3 zeta intracellular domain, a TCR alpha intracellular domain or a TCR beta intracellular domain.
  • the intracellular domain comprises a TCR alpha intracellular domain.
  • a TCR alpha intracellular domain comprises Ser-Ser.
  • a TCR alpha intracellular domain is encoded by a sequence of TCCAGC.
  • the intracellular domain comprises a TCR beta intracellular domain.
  • the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or is identical to a sequence of: MAMVKRKDSR (SEQ ID NO: 624).
  • the TCR beta intracellular domain comprises, or consists essentially of SEQ ID NO: 624.
  • the TCR beta intracellular domain is encoded by a sequence ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 625).
  • the intracellular signaling domain comprises at least one stimulatory intracellular domain.
  • the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, for example a co-stimulatory domain.
  • the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.
  • co-stimulatory intracellular signaling domains include those derived from proteins responsible for co-stimulatory signals, or antigen independent stimulation.
  • Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA, a Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18) 4-1BB (CD137, TNF receptor superfamily member 9), and CD28 molecule (CD28).
  • a co-stimulatory protein can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • TNF receptor proteins TNF receptor proteins
  • Immunoglobulin-like proteins TNF receptor proteins
  • cytokine receptors cytokine receptors
  • integrins signaling lymphocytic activation molecules
  • activating NK cell receptors examples include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically binds with CD83, CD4, and the like.
  • the co-stimulatory domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional variant thereof.
  • the stimulatory domain comprises a co-stimulatory domain.
  • the co-stimulatory domain comprises a CD28 or 4-1BB co-stimulatory domain.
  • CD28 and 4-1BB are well characterized co-stimulatory molecules required for full T cell activation and known to enhance T cell effector function.
  • CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to boost cytokine release, cytolytic function, and persistence over the first-generation CAR containing only the CD3 zeta signaling domain.
  • CARs chimeric antigen receptors
  • co-stimulatory domains for example CD28 and 4-1BB domains
  • the stimulatory domain comprises a CD28 intracellular domain or a 4-1BB intracellular domain.
  • Docket No.: 061250-559001WO Inhibitory Receptors The disclosure provides an inhibitory receptor, comprising an extracellular ligand binding domain specific to an inhibitor antigen, for example a HLA class I allele (MHC-I) and an inhibitory domain (for example an inhibitory intracellular signaling domain).
  • MHC-I HLA class I allele
  • inhibitory domain for example an inhibitory intracellular signaling domain
  • the inhibitor antigen is an HLA class I allele comprising HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.
  • the MHC-I comprises a human leukocyte antigen HLA-A*01, HLA-A*02, HLA-A*03, HLA-A* 11, HLA-B*07, or HLA-C*07.
  • the MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).
  • the MHC-I comprises a human leukocyte antigen A*03 allele (HLA-A*03).
  • the disclosure provides an inhibitory receptor, comprising an extracellular ligand binding domain specific to an HLA that has been lost in a cancer cell.
  • the HLA can be lost in the cancer cell through any mechanism, such as, without limitation, epigenetic changes that effect non-target allelic variant expression, mutations to the gene encoding the HLA, disruption of cellular signaling that regulates expression of the HLA, chromosome loss, partial or complete deletion of the genomic locus, gene silencing through modification of nucleic acids or heterochromatin, or loss of expression through other mechanisms.
  • the cells or subject treated may exhibit a loss of expression of the HLA because of non- genetic changes.
  • the disclosure provides compositions and methods for killing cells and/or treating subject lacking expression of the non-target antigen from any cause, including but not limited to, loss of heterozygosity.
  • the non-target antigen can be an HLA protein, for example HLA-A*03 protein, or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC-I), where the non-target antigen comprises a polymorphism.
  • MHC-I major histocompatibility complex class I
  • the inhibitory receptor is humanized.
  • the disclosure provides an inhibitory receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can discriminate between single amino-acid variant alleles of a non-target antigen.
  • the disclosure provides an inhibitory receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can discriminate between different levels of expression of a non-target antigen.
  • LILRB1 Inhibitory Receptors [0403] The disclosure provides an inhibitory receptor comprising a leukocyte immunoglobulin like receptor B1 (LILRB1) inhibitory domain, and optionally, a LILRB1 transmembrane and/or hinge domain, or functional variants thereof.
  • LILRB1 leukocyte immunoglobulin like receptor B1
  • the inclusion of the LILRB1 transmembrane domain and/or the LILRB1 hinge domain in the inhibitory receptor may increase the inhibitory signal generated by the inhibitory receptor compared to a reference inhibitory receptor having another transmembrane domain or another hinge domains.
  • the inhibitory receptor comprising the LILRB1 inhibitory domain may be a CAR or TCR, as described herein. Any suitable ligand binding domain, as described herein, may be fused to the LILRB1-based inhibitory receptors.
  • LILRB1 Leukocyte immunoglobulin-like receptor subfamily B member 1
  • Leukocyte immunoglobulin-like receptor B1 also known as Leukocyte immunoglobulin-like receptor B1, as well as ILT2, LIR1, MIR7, PIRB, CD85J, ILT- 2 LIR-1, MIR-7 and PIR-B
  • LIR leukocyte immunoglobulin-like receptor
  • the LILRB1 protein belongs to the subfamily B class of LIR receptors. These receptors contain two to four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs).
  • ITIMs cytoplasmic immunoreceptor tyrosine-based inhibitory motifs
  • the LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. LILRB1 is thought to regulate inflammatory responses, as well as cytotoxicity, and to play a role in limiting auto-reactivity. Multiple transcript variants encoding different isoforms of LILRB1 exist, all of which are contemplated as within the scope of the instant disclosure. [0405] In some embodiments of the inhibitory receptors described herein, the inhibitory receptor comprises one or more domains isolated or derived from LILRB1.
  • the one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least or is identical to a sequence or subsequence of SEQ ID NO:645.
  • the one or more domains of LILRB1 comprise an amino Docket No.: 061250-559001WO acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 645.
  • the one or more domains of LILRB1 consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 645. In some embodiments, the one or more domains of LILRB1 consist of an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 645.
  • an inhibitory receptor comprising a polypeptide, wherein the polypeptide comprises one or more of: an LILRB1 hinge domain or functional variant thereof; an LILRB1 transmembrane domain or a functional variant thereof; and an LILRB1 intracellular domain or an intracellular domain comprising at least one, or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • an “immunoreceptor tyrosine-based inhibitory motif” or “ITIM” refers to a conserved sequence of amino acids with a consensus sequence of S/I/V/LxYxxI/V/L (SEQ ID NO: 66), or the like, that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-possessing inhibitory receptors interact with their ligand, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or the inositol-phosphatase called SHIP.
  • the polypeptide comprises an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), at least two ITIMs, at least 3 ITIMs, at least 4 ITIMs, at least 5 ITIMs or at least 6 ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.
  • the polypeptide comprises an intracellular domain comprising at least one ITIM selected from the group of ITIMs consisting of NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • each ITIM is independently selected from NLYAAV (SEQ ID NO:626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises both ITIMs NLYAAV (SEQ ID NO: 626) and VTYAEV (SEQ ID NO: 627).
  • the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 630.
  • the intracellular domain comprises or consists of a sequence identical to SEQ ID NO: 630. Docket No.: 061250-559001WO [0412] In some embodiments, the intracellular domain comprises both ITIMs VTYAEV (SEQ ID NO: 627) and VTYAQL (SEQ ID NO: 628). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 631. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 631. [0413] In some embodiments, the intracellular domain comprises both ITIMs VTYAQL (SEQ ID NO: 628) and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 632. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO:632. [0414] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), and VTYAQL (SEQ ID NO: 628). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 633. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 633.
  • the intracellular domain comprises the ITIMs VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 634. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 634. [0416] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 635. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 635. [0417] In some embodiments, the intracellular domain comprises a sequence at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 636). In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to the LILRB1 intracellular domain (SEQ ID NO: 636). [0418] LILRB1 intracellular domains or functional variants thereof of the disclosure can have at least 1, at least 2, at least 4, at least 4, at least 5, at least 6, at least 7, or at least 8 ITIMs.
  • the LILRB1 intracellular domain or functional variant thereof has 2, 3, 4, 5, or 6 ITIMs.
  • the intracellular domain comprises two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • the intracellular domain comprises three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises five immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • the intracellular domain comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • the LILRB1 protein has four immunoglobulin (Ig) like domains termed D1, D2, D3 and D4.
  • the LILRB1 hinge domain comprises an LILRB1 D3D4 domain or a functional variant thereof.
  • the LILRB1 D3D4 domain comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to SEQ ID NO: 637. In some embodiments, the LILRB1 D3D4 domain comprises or consists essentially of SEQ ID NO: 637.
  • the polypeptide comprises the LILRB1 hinge domain or functional variant thereof. In embodiments, the LILRB1 hinge domain or functional variant thereof comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO: 638.
  • the Docket No.: 061250-559001WO LILRB1 hinge domain or functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO: 638.
  • the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO: 638.
  • the LILRB1 hinge domain consists essentially of a sequence identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO:638.
  • the transmembrane domain is a LILRB1 transmembrane domain or a functional variant thereof.
  • the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% to SEQ ID NO: 640.
  • the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 640.
  • the LILRB1 transmembrane domain comprises a sequence identical to SEQ ID NO:640.
  • the LILRB1 transmembrane domain consists essentially of a sequence identical to SEQ ID NO: 640.
  • the transmembrane domain can be attached to the extracellular region of the inhibitory receptor, e.g., the antigen binding domain or ligand binding domain, via a hinge, e.g., a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8a hinge or an LILRB1 hinge.
  • the inhibitory receptor comprises an inhibitory domain.
  • the inhibitory receptor comprises an inhibitory intracellular domain and/or an inhibitory transmembrane domain.
  • the inhibitory domain is isolated or derived from LILR1B.
  • Inhibitory Receptors Comprising Combinations of LILRB1 Domains [0433]
  • the LILRB1-based inhibitory receptors of the disclosure comprise more than one LILRB1 domain or functional equivalent thereof.
  • the inhibitory receptor comprises an LILRB1 transmembrane domain and intracellular domain, or an LILRB1 hinge domain, transmembrane domain and intracellular domain.
  • the inhibitory receptor comprises an LILRB1 hinge domain or functional variant thereof, and the LILRB1 transmembrane domain or a functional variant thereof.
  • the polypeptide comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to SEQ ID NO: 641.
  • the polypeptide comprises a sequence identical to SEQ ID NO: 641.
  • the inhibitory receptor comprises: the LILRB1 transmembrane domain or a functional variant thereof, and an LILRB1 intracellular domain and/or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • the polypeptide comprises the LILRB1 transmembrane domain or a functional variant thereof, and an LILRB1 intracellular domain and/or an intracellular domain comprising at least two ITIM, wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO:628), and SIYATL (SEQ ID NO: 629).
  • the inhibitory receptor comprises a LILRB1 transmembrane domain and intracellular domain.
  • the polypeptide comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to SEQ ID NO: 642. In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 642. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 642.
  • the inhibitory receptor comprises: an LILRB1 hinge domain or functional variant thereof; an LILRB1 transmembrane domain or a functional variant thereof; and an LILRB1 intracellular domain and/or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from LYAAV (SEQ ID NO: 626), VTYAE (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • the inhibitory receptor comprises a sequence at least 95% identical to SEQ ID NO: 643 or SEQ ID NO: 643, or at least 99% identical to SEQ ID NO: 643 or SEQ ID NO: 644, or identical to SEQ ID NO: 643 or SEQ ID NO: 644.
  • the polypeptide comprises a sequence at least 99% identical to SEQ ID NO: 641, or at least 99% identical to SEQ ID NO: 641, or identical to SEQ ID NO: 641.
  • the polypeptide comprises a sequence at least 99% identical to SEQ ID NO: 642 or at least 99% identical to SEQ ID NO: 642 or identical to SEQ ID NO: 642.
  • the LILRB1 hinge comprises a sequence of SEQ ID NO: 639
  • the LILRB1 transmembrane domain comprises a sequence of SEQ ID NO: 640
  • the LILRB1 intracellular domain comprises a sequence of SEQ ID NO: 636.
  • the non-target antigen comprises HLA-A*03
  • the inhibitory receptor comprises a sequence of: EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEIRLKSTN YATHYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTLITPDYWGQGTTVTVS SGGGGSGGGGSGGGGSGGDIQMTQSPSSVSASVGDRVTITCKASQDVSTTVAWYQQKP GKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPPTFGGG TKVEIKYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQS GLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPT DRGLQWRSSPAADA
  • the non-target antigen comprises HLA-A*03 and the inhibitory receptor comprises a sequence of SEQ ID NO: 1045.
  • the non-target antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of: MDMRVPAQLLGLLLLWLRGARCDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTY LEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPRTSGGGTKLEIKGGGGSGGGGSGGGGSGGQVQLQSGPELVKPGASVRISCKASGY TFTSYHIHWVKQRPGQGLEWIGWIYPGNVNTEYNEKFKGKATLTADKSSSTAYMHLSS LTSEDSAVYFCAREEITYAMDYWGQGTSVTVSSYGSQSSKPYLLTHPSDPLELVVSGPSG GPSSPTTGPTSTSGPEDQPL
  • the non-target antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of SEQ ID NO: 121.
  • the non-target antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTSGGGTKLEIKGGGGSGGG GSGGGGSGGQVQLQSGPELVKPGASVRISCKASGYTFTSYHIHWVKQRPGQGLEWIG WIYPGNVNTEYNEKFKGKATLTADKSSSTAYMHLSSLTSEDSAVYFCAREEITYAMDY WGQGTSVTVSSYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPT GSDPQSGL
  • the non-target antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of SEQ ID NO: 123. Docket No.: 061250-559001WO
  • the non-target antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIHWVRQAPGQGLEWIGWIYPGNVNT EYNEKFKGKATITADESTNTAYMELSSLRSEDTAVYYCAREEITYAMDYWGQGTLVTV SSGGGGSGGGGSGGGGSGGDIQMTQSPSTLSASVGDRVTITCRSSQSIVHSNGNTYLEW YQQKPGKAPKLLIYKVSNRFSGVPARFSGSGSGTEFTLTISSLQPDDFATYYCFQGSHVPR TFGQGTKVEVKYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTPT
  • the non-target antigen comprises HLA-A*02 and the inhibitory receptor comprises a sequence of SEQ ID NO: 1167.
  • Immune Checkpoint Inhibitory Receptors [0445] The disclosure provides an inhibitory receptor comprising an immune checkpoint inhibitory domain.
  • the inhibitory domain is homologous to a signal transduction element of an immune checkpoint protein, such as an immune checkpoint protein selected from the group consisting of PD1; CTLA4; BTLA; 2B4; CD 160; CEACAM, such as CEACAM1; KIRs, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LIR1, LIR2, LIR3, LIR5, LIR8 and CD94- KG2A; LAG3; TIM3; V-domain Ig suppressor of T cell activation (VISTA); STimulator of INterferon Genes (STING); immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing proteins, T cell immunoglobulin and ITIM domain (TIGIT), and adenosine receptor (e.g., A2aR).
  • an immune checkpoint protein selected from the group consisting of
  • the immune checkpoint protein is a negative immune regulator.
  • the negative immune regulator is selected from the group consisting of 2B4, LAG- 3 and BTLA-4.
  • immune checkpoint protein is a natural killer cell inhibitory receptor, e.g., KIRs, such as KIR2DL1, KTR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KTR3DL3; or a Leukocyte Ig-like receptor, such as LIR1, LIR2, LIR3, LIR5, LIR8; and CD94-NKG2A, a C-type lectin receptor which forms heterodimers with CD94 and contains 2 ITIMs.
  • KIRs such as KIR2DL1, KTR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KTR3DL3
  • Leukocyte Ig-like receptor such as LIR1, LIR
  • the disclosure provides an inhibitory receptor that is an inhibitory chimeric antigen receptor.
  • the inhibitory receptor may comprise an extracellular ligand binding domain that binds to and recognizes HLA-A*02 or HLA-A*03 or a peptide derivative thereof in an MHC-I complex.
  • the term “inhibitory receptor” as used herein refers to a ligand-binding domain that is fused to an intracellular signaling domain capable of transducing an inhibitory signal that inhibits or suppresses the immune activity of an immune cell.
  • Inhibitory receptors have immune cell inhibitory potential, and are distinct and distinguishable from CARs, which are receptors with immune cell activating potential.
  • CARs are activating receptors as they include intracellular stimulatory and/or co-stimulatory domains.
  • Inhibitory receptors are inhibiting receptors that contain intracellular inhibitory domains.
  • inhibitory signal refers to signal transduction or changes in protein expression in an immune cell resulting in suppression of an immune response (e.g., decrease in cytokine production or reduction of immune cell activation). Inhibition or suppression of an immune cell can selective and/or reversible, or not selective and/or reversible.
  • Inhibitory receptors of the disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure.
  • Inhibitory receptors are responsive to non-target antigens (e.g., HLA-A*02 or HLA-A*03). For example, when a non-target antigen (e.g., HLA-A*02 or HLA-A*03) binds to or contacts the inhibitory receptor, the inhibitory receptor is responsive and activates an inhibitory signal in the immune cell expressing the inhibitory receptor upon binding of the non-target antigen by the extracellular ligand binding domain of the inhibitory receptor.
  • non-target antigens e.g., HLA-A*02 or HLA-A*03
  • Inhibitory receptors of the disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure.
  • the ligand binding domain is an antigen binding domain.
  • Illustrative antigen binding domains include, inter alia, scFv, SdAb, V ⁇ -only domains, and TCR antigen binding domains derived from the TCR ⁇ and ⁇ chain variable domains.
  • Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.
  • the extracellular ligand binding domain of the inhibitory receptor is an scFv.
  • the extracellular ligand binding domain of the inhibitory receptor binds to and recognizes HLA-A*02 or HLA-A*03.
  • the extracellular ligand binding domain of the inhibitory receptor is an scFv.
  • the extracellular ligand binding domain of the inhibitory receptor is fused to the extracellular domain of an inhibitory receptor.
  • the inhibitory receptors of the present disclosure comprise an extracellular hinge region.
  • Illustrative hinges can be isolated or derived from IgD and CD8 domains, for example IgG1. In some embodiments, the hinge is isolated or derived from CD8 ⁇ or CD28.
  • the inhibitory receptors of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the inhibitory receptor.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • 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 may be isolated or derived from (i.e.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular domain of the inhibitory receptor.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the disclosure provides an inhibitory receptor comprising an intracellular domain.
  • the intracellular domain of the inhibitory receptors of the instant disclosure is responsible for inhibiting activation of the immune cells comprising the inhibitory receptor, which would otherwise be activated in response to activation signals by the activator receptor.
  • the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM).
  • the inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1.
  • CTLA-4 and PD-1 are immune inhibitory receptors the surface of T cells, and play a pivotal role in attenuating or terminating T cell responses. Docket No.: 061250-559001WO [0461]
  • an inhibitory intracellular domain is isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.
  • TRAIL receptor comprises TR10A, TR10B or TR10D.
  • an inhibitory intracellular domain is isolated from phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1). In some embodiments, an inhibitory intracellular domain is isolated from leukocyte immunoglobulin like receptor B1 (LILRB1). [0463] In some embodiments, the inhibitory domain is isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, PD-1, PAG1 or LILRB1 protein. [0464] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region or a combination thereof.
  • the inhibitory domain is isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor B1 (LIR1, also called LIR-1 and LILRB1), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor K1 (NKG2D), CTLA- 4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N and C terminal SH2 domains), or ZAP70 KI_K369A (kinase inactive ZAP70).
  • KIR3DL2 three Ig domains and long cytoplasmic tail 2
  • KIR3DL3DL3DL3 three Ig domains and long cytoplasmic tail 2
  • LIR1 leukocyte immuno
  • the inhibitory domain is isolated or derived from a human protein.
  • the inhibitory receptor comprises an inhibitory domain.
  • the inhibitory receptor comprises an inhibitory intracellular domain and/or an inhibitory transmembrane domain.
  • the inhibitory intracellular domain is fused to an intracellular domain of an inhibitory receptor.
  • the inhibitory intracellular domain is fused to the transmembrane domain of an inhibitory receptor.
  • the inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain or a portion thereof isolated or derived isolated or derived from the same protein, for example an ITIM containing protein.
  • the inhibitory receptor comprises a hinge region isolated or derived from isolated or derived from the same protein as the intracellular domain and/or transmembrane domain, for example an ITIM containing protein.
  • the inhibitory receptor is a TCR comprising an inhibitory domain (an inhibitory TCR).
  • the inhibitory TCR comprises an inhibitory intracellular domain and/or an inhibitory domain.
  • the inhibitory intracellular domain is fused to the intracellular domain of TCR alpha, TCR beta, CD3 Docket No.: 061250-559001WO delta, CD3 gamma or CD3 epsilon or a portion thereof a TCR.
  • the inhibitory intracellular domain is fused to the transmembrane domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.
  • the inhibitory receptor is a TCR comprising an inhibitory domain (an inhibitory TCR).
  • the inhibitory domain is isolated or derived from LILRB1.
  • Inhibitor Ligands [0471]
  • the HLA for example HLA-A*02 or HLA-A*03
  • the HLA is not expressed by the target cells, and is expressed by non-target cells.
  • the HLA for example HLA-A*02 or HLA-A*03
  • the HLA is expressed by healthy cells, i.e.
  • the target cells are a plurality of cancer cells that have lost expression of the non-target antigen through loss of heterozygosity (LOH).
  • the non-target cells are a plurality of healthy cells (i.e., non-cancer cells), that express both the target and the non- target antigen.
  • the HLA for example HLA-A*02 or HLA-A*03
  • Non-target major histocompatibility complex class I MHC-I (or pMHC-I) antigens comprising any of HLA-A are envisaged as within the scope of the disclosure.
  • the non-target antigen comprises a Major Histocompatibility Complex (MHC) protein.
  • MHC is MHC class I.
  • MHC class I protein comprises a human leukocyte antigen (HLA) protein.
  • HLA human leukocyte antigen
  • the non-target antigen comprises an allele of an HLA Class I protein selected from the group consisting of HLA- A.
  • the HLA-A allele comprises HLA-A*03.
  • the HLA-A allele comprises HLA-A*02.
  • the ligand binding domain of the inhibitory receptor comprises an scFv.
  • the scFv binds to HLA-A*03.
  • the scFv binds to HLA-A*02.
  • the non-target antigen comprises HLA-A*03
  • the extracellular ligand binding domain of the inhibitory receptor comprises an HLA-A*03 ligand binding domain.
  • the HLA-A*03 ligand binding domain comprises an scFv domain.
  • the non-target antigen comprises HLA-A*03
  • the ligand binding domain of the inhibitory receptor comprises an HLA-A*03 ligand binding domain.
  • the ligand binding HLA-A*03 independent of the peptide in a Docket No.: 061250-559001WO pMHC complex comprising HLA-A*03.
  • the HLA-A*03 ligand binding domain comprises an scFv domain.
  • the HLA-A*03 antigen binding domain comprises a heavy chain and a light chain.
  • the antigen binding domain comprises a variable light chain region (VL) comprising a sequence set forth in 4A or 4B.
  • VL variable light chain region
  • the antigen binding domain comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence set forth in 4A or 4B.
  • the VH may be paired with any of the VLs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity.
  • the VH and VL are separated by a linker, for example GGGGSGGGGSGGGGSGG (SEQ ID NO: 152).
  • the VH and VL are ordered, from N to C terminal, VH, linker and VL.
  • the VH and VL are ordered, from N to C terminal, VL, linker and VH.
  • the non-target antigen comprises HLA-A*02
  • the extracellular ligand binding domain of the inhibitory receptor comprises an HLA-A*02 ligand binding domain.
  • the HLA-A*02 ligand binding domain comprises an scFv domain.
  • Such scFv’s include, for example and without limitation the following mouse and humanized scFv antibodies that bind HLA-A*02 in a peptide-independent way (complementarity determining regions underlined): C-001765 MMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTSGGGTKLEIKGGGGS GGGGSGGGGSGGQVQLQQSGPELVKPGASVRISCKASGYTFTSYHIHWVKQRPGQGLE WIGWIYPGNVNTEYNEKFKGKATLTADKSSSTAYMHLSSLTSEDSAVYFCAREEITYAM DYWGQGTSVTVSSYG (SEQ ID NO: 153); or DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQ
  • the HLA-A*03 ligand binding domain comprises an scFv domain.
  • scFv include, for example and without limitation the following mouse and humanized scFv antibodies that bind HLA-A*03 in a peptide-independent way (complementarity determining regions underlined): Tabl N am e sc v sequence acid s equence A3-26 EVQLVESGGGLVQPGRS CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAATTATTGGAT LRLSCAASGFTFSNYWM Docket No.: 061250-559001WO GAACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTGTCTGAG NWVRQAPGKGLEWVSE ATTAGATTGAAATCTACTAATTATGCAACACATTATGCGGAGTCTGTGAAA IRLKSTNYATHYAESVKG QM LITP GG GDI DRV WY SYR DFT QQ IK
  • the HLA-A*11 ligand binding domain comprises an scFv domain.
  • scFv include, for example and without limitation the following mouse and humanized scFv antibodies that bind HLA-A*11 in a peptide-independent way (complementarity determining regions underlined): Tabl scFv NP GS 9 AA NO: 1139) 8 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWNDDKRYSPS L KSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHRHMRLSCFDYWGQGTLVTVSSGGGGS Docket No.: 061250-559001WO scFv Protein Sequence GGGGSGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA NO: TK VSS 7 PK SEQ PG GG 6 LIY Q
  • the HLA-B*07 ligand binding domain comprises an scFv domain.
  • scFv include, for example and without limitation the following mouse and humanized scFv antibodies that bind HLA-B*07 in a peptide-independent way (complementarity determining regions underlined): Tabl HL QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYSWHWIRQPPGK 1.10_scFv GLEWIGYIHFSGSTHYHPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCARGGVVSHYAMDCWGQGTTVTVSSGGGGSGGGGS Docket No.: 061250-559001WO CQ GGG (SEQ ID NO: 1156) Docket No.: 061250-559001WO CQ GGG (SEQ ID NO: 1157) Differentially Expressed Inhibitor Ligands [0485] The disclosure provides inhibitor ligands (non-target) (non-
  • Activation of the inhibitory receptor is mediated by the presence of HLA-A*03 on the surface of a cell.
  • a cell that expresses HLA-A*03 will activate the inhibitory receptor based on the level of expression of the HLA-A*03.
  • the HLA-A*03 is expressed by both target and non-target cells.
  • the HLA-A*03 is expressed by non- target cells at a higher level than the target cells. The higher levels of HLA-A*03 expressed by the non-target cells activate the inhibitory receptor, thereby preventing activation of the immune cell.
  • the lower levels of HLA-A*03 expressed by the target are not sufficient to activate the inhibitory receptor, leading to activation of the immune cell.
  • the HLA-A*03 is expressed by non-target cells but not by target cells. In the absence of expression of the HLA-A*03, the target cells activate the target receptor, thereby activating the immune cells.
  • Differential expression can be determined by any techniques known in the art used to measure expression. These include, inter alia, techniques for measuring mRNA and/or protein levels of a target gene in a cell. Methods of measuring protein levels in samples include immunohistochemistry, enzyme-linked immunosorbent assays (ELISA), and analytical methods such as liquid chromatography-mass spectrometry (LC-MS). Methods of measuring mRNA levels include real time quantitative reverse transcription PCR (qRT-PCR), as well as high throughput sequencing.
  • qRT-PCR real time quantitative reverse transcription PCR
  • HLA-A*03 Activation of the inhibitory receptor by HLA-A*03 can occur according to various modalities known in the art. Activation of the inhibitory receptor by HLA-A*03 can be determined by methods known in the art. For example, the level of downstream intracellular signaling in a cell expressing the inhibitory receptor can be measured through the use of a reporter gene. [0490] Without wishing to be bound by theory, whether or not expression of HLA-A*03 inhibits activation of an immune cell via activation of the inhibitory receptor can occur according to the ratio of the HLA-A*03 to the inhibitor receptor.
  • the expression levels of the HLA-A*03 and the Docket No.: 061250-559001WO inhibitory receptor, and the ratio thereof, can be determined by methods known in the art, including, inter alia, immunohistochemistry and fluorescence activated cell sorting (FLOW CYTOMETRY). Analysis of the expression levels of the HLA-A*03 on target and non-target cells can be used to predict selective targeting of the immune cells expressing the inhibitory receptor. Low or no expression of the HLA-A*03 on a target or non-target cell can indicate, for example, that the inhibitory receptor will not be activated in an immune cell of the disclosure.
  • inhibition of immune cell activation by HLA-A*03 via activation of the inhibitory receptor can depend on the affinity of the HLA-A*03 for the inhibitory receptor. Methods of measuring affinity are known in the art, and include, inter alia, enzyme-linked immunosorbent assay or radioimmunoassay methods.
  • inhibition of immune cell activation by HLA-A*03 via activation of the inhibitory receptor can occur according to cross talk between the inhibitory receptor and the activator receptor, leading to down-regulation of the activity of the activator receptor.
  • HLA-A*03 activation of the inhibitory receptor by HLA- A*03 can lead to reduced expression of the activator receptor on the surface of the immune cell.
  • HLA-A*03 is expressed at a lower level in a target cell than a normal cell.
  • the HLA-A*03 is expressed by healthy cells, i.e. cells that are not cancer cells.
  • HLA-A*03 expression level is at least about 10 times less, at least about 30 times less, at least about 50 times less, at least about 70 times less, at least about 90 times less, at least about 100 times less, at least about 110 times less, at least about 150 times less, at least about 200 times less, at least about 250 times less, at least about 300 times less, at least about 350 times less, at least about 400 times less, at least about 450 times less, at least about 500 times less, at least about 600 times less, at least about 700 times less, at least about 800 times less, at least about 900 times less or at least about 1000 times less in the target cell than in the non- target cell.
  • the HLA-A*03 expression level is about 10 times less, about 30 times less, about 50 times less, about 70 times less, about 90 times less, about 100 times less, or about 110 times less than the plurality of healthy cells. In some embodiments, the HLA-A*03 expression level is at least about 5 times less in the plurality of cancer cells than in the plurality of healthy cells. In some embodiments, the HLA-A*03 expression level is at least about 5 times less in a target cell than a non-target cell. In some embodiments, the target cells are a plurality of cancer cells that have low or no expression of HLA-A*03.
  • polynucleotide or polynucleotide system including one or more polynucleotides including one or more polynucleotide sequences encoding the membrane tethered cytokine, the inhibitor receptor, and the activator receptor.
  • the polynucleotide or polynucleotide system is comprised in a nanocarrier.
  • nanocarrier is capable of delivering the polynucleotide or polynucleotide system to an immune cell in vivo or ex vivo.
  • the nanocarrier is a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • An additional aspect of the present disclosure is a method of making an immune cell therapy, comprising transforming immune cells with including any herein-disclosed polynucleotide or polynucleotide system .
  • the method comprises contacting immune cells with any herein disclosed nanocarrier.
  • a host cell comprises any herein- disclosed polynucleotide or polynucleotide system.
  • the present disclosure provides polynucleotides encoding the engineered interleukins, activator receptor, and/or inhibitory receptor described herein.
  • the sequence of the engineered interleukin, the first and/or inhibitory receptor is operably linked to a promoter.
  • the sequence encoding the engineered interleukin is operably linked to a first promoter
  • the sequence encoding the activator receptor is operably linked to a second promoter
  • the sequence encoding the inhibitory receptor is operably linked to a third promoter.
  • the sequence encoding the engineered interleukin is operably linked to a first promoter
  • the sequence encoding the activator receptor and the inhibitory receptor is operably linked to a second promoter.
  • the sequence of the engineered interleukin, the first and/or inhibitory receptor is operably linked to the same promoter.
  • the membrane tethered interleukin and its receptor are expressed from the same polynucleotide. In some embodiments, the membrane tethered interleukin and its receptor are expressed from separate polynucleotides. In some embodiments, the membrane tethered interleukin and its receptor are expressed from the same vector. In some embodiments, the membrane tethered interleukin and its receptor are expressed from separate vectors. In some embodiments, the vectors is a viral vector. In some embodiments, the expression of the interleukin and its receptor are constitutive.
  • the expression of the interleukin and its receptor are inducible.
  • the membrane tethered interleukin is operably linked to a nuclear factor of activated T-cells (NFAT) promoter or a functional portion or functional variant thereof.
  • NFAT promoter as used herein means one or more NFAT responsive elements linked to a minimal promoter of any gene expressed by T-cells.
  • both receptors are encoded by a single vector.
  • the first, second, and/or third vector comprises an shRNA, for example a B2M shRNA.
  • both receptors are encoded by a single vector.
  • the vector comprises an shRNA, for example a B2M shRNA.
  • the engineered interleukin, and the first and inhibitory receptors are encoded by a single vector.
  • Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, inter alia, encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and/or self-cleaving peptides.
  • Illustrative self-cleaving peptides include T2A, P2A, E2A and F2A self-cleaving peptides.
  • the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 653).
  • the P2A self- cleaving peptide comprises a sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 654).
  • the E2A self-cleaving peptide comprises a sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 655).
  • the F2A self- cleaving peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 656).
  • the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 657). Any of the foregoing can also include an N terminal GSG linker.
  • a T2A self-cleaving peptide can also comprise a sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 658), which can be encoded by a sequence of GGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCC TGGCCCC (SEQ ID NO: 659).
  • the vector is an expression vector, i.e. for the expression of the engineered interleukin, the first and/or inhibitory receptor in a suitable cell.
  • Vectors derived from retroviruses such as the 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 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 low immunogenicity.
  • the expression of natural or synthetic nucleic acids encoding receptors is typically achieved by operably linking a nucleic acid encoding the receptor or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for Docket No.: 061250-559001WO replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the polynucleotides encoding the receptors can be cloned into a number of types of vectors.
  • the polynucleotides 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, bacterial artificial chromosome (BAC) vector and sequencing vectors.
  • the expression vector may be provided to cells, such as immune cells, 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. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), 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.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 basepairs (bp) upstream of the start site, although a number of promoters have recently been shown to 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. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • tk thymidine kinase
  • CMV immediate early cytomegalovirus
  • EF-1 ⁇ Elongation Growth Factor-1 ⁇
  • simian virus 40 SV40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV MoMuLV promoter
  • an avian leukemia virus promoter an Epstein-Barr virus immediate early promoter
  • Rous sarcoma virus promoter a Rous sarcoma virus promoter
  • U6 a U6 promoter
  • human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • NFAT promoter as used herein means one or more NFAT responsive elements linked to a minimal promoter of any gene expressed by T-cells.
  • the NFAT responsive elements may comprise, e.g., NFATl, NFAT2, NFAT3, and/or NFAT4 responsive elements.
  • the NFAT promoter (or functional portion or functional variant thereof) may comprise any number of binding motifs, e.g., at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs.
  • the minimal promoter of a gene expressed by T- cells is a minimal human IL-2 promoter.
  • the NFAT promoter comprises six NFAT binding motifs. See, e.g., US Patent No.8,556,882, which is incorporated by reference in its entirety and particularly for pertinent parts relating to NFAT promoters. [0512] Further, the disclosure should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated as part of the disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be 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 markers and reporter 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 neo and the like.
  • Reporter genes are used for identifying potentially transfected or transduced 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 assayed 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.
  • the polynucleotide encoding the engineered interleukins, activator receptor, and/or inhibitory receptor described herein is mRNA.
  • Engineered Cells or Immune Cells of the Present Disclosure [0516]
  • the present disclosure relates to an immune cell for treating a cancer, the immune cell including a membrane tethered cytokine and one or both of an inhibitor receptor including an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non-cancerous cell and an activator receptor including an extracellular ligand binding domain specific to an activator antigen expressed by a cancer cell.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one SEQ ID NO: 1 to SEQ ID NO: 22.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 27 or SEQ ID NO: 28.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 29, 30, 1046, 1046, and 1058.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 31 or SEQ ID NO: 32.
  • the present disclosure relates to an immune cell, wherein the cytokine is an interleukin.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one SEQ ID NO: 1 to SEQ ID NO: 22.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 27 or SEQ ID NO: 28.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 29, 30, 1046, 1046, and 1058.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 31 or SEQ ID NO: 32.
  • the present disclosure relates to an immune cell, wherein the interleukin is selected from an IL-12, an IL-2, an IL-15, an IL-18, and an IL-21.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one SEQ ID NO: 1 to SEQ ID NO: 22.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 27 or SEQ ID NO: 28.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 29, 30, 1046, 1046, and 1058.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 31 or SEQ ID NO: 32.
  • the present disclosure relates to an immune cell, wherein the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 1.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one SEQ ID NO: 1 to SEQ ID NO: 22.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 29, 30, 1046, 1046, and 1058.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 31 or SEQ ID NO: 32.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one SEQ ID NO: 1 to SEQ ID NO: 22.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 27 or SEQ ID NO: 28.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 29, 30, 1046, 1046, and 1058.
  • the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to either of SEQ ID NO: 31 or SEQ ID NO: 32.
  • the present disclosure relates to an immune cell, wherein the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one or both of SEQ ID NO: 23 or SEQ ID NO: 24.
  • the cytokine includes a fusion protein including an IL-12A polypeptide and an IL-12B polypeptide.
  • the present disclosure relates to an immune cell, wherein the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to each of SEQ ID NO: 23 and SEQ ID NO: 24.
  • the cytokine includes a fusion protein including an IL-12A polypeptide and an IL-12B polypeptide.
  • the present disclosure relates to an immune cell, wherein the cytokine includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 64.
  • the cytokine includes a fusion protein including an IL-12A polypeptide and an IL-12B polypeptide.
  • the fusion protein includes a linker separating the IL-12A polypeptide and the IL-12B polypeptide.
  • the linker includes a polypeptide sequence of one or more of (GS)n (SEQ ID NO: 41), (GSGGS)n (SEQ ID NO: 42), (GGGS)n (SEQ ID NO: 43), (GGGGS)n (SEQ ID NO: 44), (GGGGGS)n (SEQ ID NO: 45), or (GGGGGGS)n (SEQ ID NO: 46), where n is an integer of at least one (and generally from 3 to 10), and/or GSGSSRGGSGSGGSGGGGSK (SEQ ID NO: 47), GGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 48), or (G4Q)4 (SEQ ID NO: 49).
  • the linker includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 7.
  • the present disclosure relates to an immune cell, wherein the cytokine is tethered to the immune cell's membrane via a transmembrane domain operably linked to the cytokine.
  • the transmembrane region includes a PDGFRb transmembrane region, a B7 transmembrane region, a CD25 transmembrane region, a CD137 transmembrane region, a B7 transmembrane region, or a CD19 transmembrane region.
  • the present disclosure relates to an immune cell, wherein the cytokine is tethered to the immune cell's membrane via a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 8.
  • the present disclosure relates to an immune cell, wherein the cytokine is tethered to the immune cell's membrane via a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 55, 56, 57, 1048, 1076, 1077, or 1086.
  • the present disclosure relates to an immune cell, wherein the membrane tethered cytokine further includes a hinge domain.
  • the hinge domain includes a (G4Q)2, (G4Q)5, (G4Q)10, (G4Q)10-CD25_full, CD25_hinge, IgG4, EGF3, or EGF7 hinge region.
  • the hinge domain includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 6.
  • the present disclosure relates to an immune cell, wherein the membrane tethered cytokine further includes a linker domain.
  • the linker includes a polypeptide sequence of one or more of (GS)n (SEQ ID NO: 41), (GSGGS)n (SEQ ID NO: 42), (GGGS)n (SEQ ID NO: 43), (GGGGS)n (SEQ ID NO: 44), (GGGGGS)n (SEQ ID NO: 45), or (GGGGGGS)n (SEQ ID NO: 46), where n is an integer of at least one (and generally from 3 to 10), and/or GSGSSRGGSGSGGSGGGGSK (SEQ ID NO: 47), GGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 48), or (G4Q)4 (SEQ ID NO: 49).
  • the linker domain includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 7.
  • the present disclosure relates to an immune cell, wherein the membrane tethered cytokine further is expressed by a polynucleotide including one or both of a constitutive promoter and an inducible promoter. In some cases, the membrane tethered cytokine further is expressed by a polynucleotide including both of a constitutive promoter and an inducible promoter.
  • the inducible promoter is an activation induced promoter.
  • the activation induced promoter includes one or more nuclear factor of activated cells response elements (NFAT RE).
  • the activation induced promoter includes 1, 2, 3, 4, 5, 6, or more NFAT REs.
  • the constitutive promotor includes one or both of a eukaryotic translation elongation factor 1 alpha (EF1a) promoter or a TATA box promoter.
  • EF1a eukaryotic translation elongation factor 1 alpha
  • the membrane tethered cytokine further includes an intracellular domain.
  • the intracellular domain is capable of providing a stimulatory signal.
  • the intracellular domain includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 12.
  • the intracellular domain includes a PDGFRB, B7, CD137, CD27, OX40, Dap10, DAP12, MyD88, MyD88/CD40, CD3e Intracellular domain.
  • the membrane tethered cytokine includes an PDGFRB transmembrane and intracellular domain.
  • the PDGFRB transmembrane and intracellular domain includes a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1086.
  • the present disclosure relates to an immune cell, wherein the membrane tethered cytokine further includes a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus
  • the present disclosure relates to an immune cell, wherein the immune cell further expresses a receptor for the cytokine.
  • the receptor is selected from an IL-12 Docket No.: 061250-559001WO receptor (e.g., one or both of IL-12R-beta-1 and IL-12R-beta-2), an IL-2 receptor (e.g., one or both of L-2R-alpha and IL-2R-beta), an IL-18 receptor (e.g., one or both of IL-18R-alpha and IL-18R beta), or an IL-21 receptor.
  • the receptor for the cytokine binds to the membrane tethered cytokine and when bound transmits an intracellular signal consistent with natural cytokine binding.
  • the present disclosure relates to an immune cell, wherein the immune cell expresses the inhibitor receptor including an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non-cancerous cell.
  • the inhibitor antigen is an HLA class I allele.
  • the HLA class I allele includes HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.
  • the HLA-A allele includes an HLA-A*02 allele, an HLA-A*03 allele, or HLA-A*11 allele.
  • the HLA-B allele includes an HLA-B*07 allele.
  • the HLA-C allele includes an HLA-C*07 allele. In some cases, wherein the HLA class I allele includes an HLA-E allele. In some cases, the inhibitor antigen is an HLA-A*02 allele and the extracellular ligand binding domain of the inhibitor receptor includes a set of complementarity-determining region (CDR) sequences underlined in any one of SEQ ID NO: 153 to SEQ ID NO: 165.
  • CDR complementarity-determining region
  • the inhibitor antigen is an HLA-A*02 allele and the extracellular ligand binding domain of the inhibitor receptor includes a heavy chain variable (VH) region and a light chain variable (VL) region as disclosed in any one of SEQ ID NO: 153 to SEQ ID NO: 165 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto, wherein the VH and VL are separated in SEQ ID NO: 153 to SEQ ID NO: 165 by the GGGGSGGGGSGGGGSGG (SEQ ID NO: 152) linker.
  • VH heavy chain variable
  • VL light chain variable
  • the inhibitor antigen is an HLA-A*02 allele and the extracellular ligand binding domain of the inhibitor receptor includes an antibody fragment or a single chain Fv antibody fragment (scFv) as disclosed in any one of SEQ ID NO: 153 to SEQ ID NO: 165 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor antigen is an HLA-A*03 allele and the extracellular ligand binding domain of the inhibitor receptor includes a set of complementarity- determining region (CDR) sequences underlined in any one of the SEQ ID NOs in Table 13 or Table 14.
  • CDR complementarity- determining region
  • the inhibitor antigen is an HLA-A*03 allele and the extracellular ligand binding domain of the inhibitor receptor includes a heavy chain variable (VH) region and a light chain variable (VL) region as disclosed in any one of SEQ ID NO: 1099 to SEQ ID NO: 1110 or SEQ ID NO: 1125-1138, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto, wherein the VH and VL are separated in SEQ ID NO: 1099 to SEQ ID NO: 1110 or SEQ ID NO: 1125-1138 by the GGGGSGGGGSGGGGSGG (SEQ ID NO: 152) linker.
  • VH heavy chain variable
  • VL light chain variable
  • the inhibitor antigen is an HLA-A*03 allele and the extracellular ligand binding domain of the inhibitor receptor includes an antibody fragment or a single chain Fv antibody Docket No.: 061250-559001WO fragment (scFv) as disclosed in any one of SEQ ID NO: 1099 to SEQ ID NO: 1110 or SEQ ID NO: 1125-1138 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor antigen is an HLA-A*011 allele and the extracellular ligand binding domain of the inhibitor receptor includes a set of complementarity-determining region (CDR) sequences underlined in any one of SEQ ID NO: 1139 to SEQ ID NO: 1147.
  • CDR complementarity-determining region
  • the inhibitor antigen is an HLA-A*011 allele and the extracellular ligand binding domain of the inhibitor receptor includes an antibody fragment or a single chain Fv antibody fragment (scFv) as disclosed in any one of SEQ ID NO: 1139 to SEQ ID NO: 1147 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor antigen is an HLA-A*02 allele and the inhibitor antigen includes HLA-A*02 and the inhibitory receptor includes a sequence of: (SEQ ID NO: 121) or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor antigen is an HLA-A*02 allele and the inhibitor antigen includes HLA-A*02 and the inhibitory receptor includes a sequence of (SEQ ID NO: 123) or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor antigen is an HLA-A*03 allele and the inhibitor antigen includes HLA-A*03 and the inhibitory receptor includes a sequence of: (SEQ ID NO: 1045) or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor antigen is an HLA-A*011 allele and the inhibitor antigen includes HLA-A*011 and the inhibitory receptor includes a sequence of: (SEQ ID NO: 1045) or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the present disclosure relates to an immune cell, wherein the inhibitor receptor includes a leukocyte immunoglobulin like receptor B1 (LILRB1) intracellular domain, includes an LILRB1 hinge, and/or an LILRB1 transmembrane domains, or functional variants thereof.
  • LILRB1 leukocyte immunoglobulin like receptor B1
  • the LILRB1 intracellular domain, the LILRB1 hinge, and/or the LILRB1 transmembrane domains include a polypeptide sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any sequence disclosed in Table 11.
  • the inhibitor receptor includes a leukocyte immunoglobulin like receptor B1 (LILRB1) intracellular Docket No.: 061250-559001WO domain or a functional variant thereof.
  • the LILRB1 intracellular domain includes the sequence of SEQ ID NO: 636, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the inhibitor receptor includes an LILRB1 hinge and an LILRB1 transmembrane domains, or functional variants thereof.
  • the LILRB1 transmembrane domain includes the sequence of SEQ ID NO: 640, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 hinge domain includes the sequence of SEQ ID NO: 639, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 hinge and LILRB1 transmembrane domains include the sequence of SEQ ID NO: 641, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 transmembrane and LILRB1 intracellular domains include the sequence of SEQ ID NO: 642, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the LILRB1 hinge, LILRB1 transmembrane, and LILRB1 intracellular domains include the sequence of SEQ ID NO: 643 or SEQ ID NO: 644, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the present disclosure relates to an immune cell, wherein the inhibitor receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the present disclosure relates to an immune cell, wherein the immune cell expresses the activator receptor including an extracellular ligand binding domain specific to an activator antigen expressed by a cancer cell. In some cases, binding of the activator antigen by the activator receptor activates or promotes activation of the immune cell. In some cases, the activator antigen is selected from the antigens listed in Table 9.
  • the extracellular ligand binding domain specific to an activator antigen includes an antibody listed in Table 9, includes an antibody fragment of an antibody listed in Table 9, includes a heavy chain variable (VH) region and/or a light chain variable (VL) region of an antibody listed in Table 9, or includes a set of complementarity-determining region (CDR) sequences of an antibody listed in Table 9.
  • VH heavy chain variable
  • VL light chain variable
  • CDR complementarity-determining region
  • the activator antigen is selected from the group consisting of epidermal growth factor receptor (EGFR), mesothelin (MSLN), CEA cell adhesion molecule 5 (CEA), transferrin receptor (TFRC), HLA-E, erb-b2 receptor tyrosine kinase 2 (HER2), mesothelin (MSLN), PSMA, CD33, CD19 molecule (CD19), CLL-1, CD53, SPN, ITGA4, SELPLG, and CLEC12A or a peptide antigen thereof.
  • the activator receptor includes extracellular ligand binding domain specific to an activator receptor listed in Table 10, includes an scFv of an activator receptor listed in Table 10, includes a heavy chain variable (VH) region and/or a light chain variable (VL) region Docket No.: 061250-559001WO of activator receptor listed in Table 10, or includes a set of complementarity-determining region (CDR) sequences of activator receptor listed in Table 10.
  • VH heavy chain variable
  • VL light chain variable
  • the activator receptor includes a CD28 co-stimulatory domain including an amino acid sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 65) or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor includes a 4-1BB co-stimulatory domain including an amino acid sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 616) or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor includes a CD3 ⁇ activation domain including an amino acid sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 610) or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor includes a CD28 co-stimulatory domain, a 4-1BB costimulatory domain, and a CD3 ⁇ activation domain.
  • the activator receptor includes an amino acid sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ ID NO: 618) or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.
  • the activator receptor includes a sequence of one of SEQ ID NO: 983 to SEQ ID NO: 1044, as set forth in Table 10, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor receptor and the activator receptor are expressed by a single polynucleotide.
  • the single polynucleotide includes one or both of a constitutive promoter and an inducible promoter.
  • the constitutive promotor includes a eukaryotic translation elongation factor 1 alpha (EF1a) promoter.
  • the coding sequence for the inhibitor receptor precedes the coding sequence for the activator receptor in the single polynucleotide.
  • the coding sequence for the activator receptor precedes the coding sequence for the inhibitor receptor in the single polynucleotide.
  • the single polynucleotide encodes a self-cleaving peptide between the coding sequence for the inhibitor receptor and the coding sequence activator receptor.
  • the self-cleaving peptide is selected from the T2A, P2A, E2A and F2A self-cleaving peptides.
  • the T2A self- cleaving peptide includes a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 653)
  • the P2A Docket No.: 061250-559001WO self-cleaving peptide includes a sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 654)
  • the E2A self-cleaving peptide includes a sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 655)
  • the F2A self-cleaving peptide includes a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 656)
  • the T2A self-cleaving peptide includes a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 657), or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the inhibitor receptor is expressed by a first polynucleotide and the activator receptor is expressed by a second polynucleotide.
  • the first polynucleotide and/or the second polynucleotide includes one or both of a constitutive promoter and an inducible promoter.
  • the constitutive promotor includes a eukaryotic translation elongation factor 1 alpha (EF1a) promoter.
  • the present disclosure relates to an immune cell including the membrane tethered cytokine, the receptor including an extracellular ligand binding domain specific to an inhibitor antigen expressed by a non-cancerous cell, and the activator receptor including an extracellular ligand binding domain specific to an activator antigen expressed by a cancer cell.
  • the immune cell is a T cell.
  • the T cell is a CD8+ CD4- T cell or a CD8- CD4+ T cell.
  • the T cell is a cytotoxic T cell.
  • the immune cell is a natural killer (NK) cell.
  • the present disclosure relates to an immune cell, wherein the immune cell is modified to reduce or eliminate expression of the B2M gene product.
  • the expression of the B2M gene product is reduced or eliminated using a short hairpin RNA (shRNA).
  • shRNA includes a first sequence, having from 5′ to 3′ end a sequence complementary to the B2M mRNA; and a second sequence, having from 5′ to 3′ end a sequence complementary to the first sequence, wherein the first sequence and second sequence form the shRNA.
  • the present disclosure relates to an immune cell, wherein the membrane tethered cytokine promotes T cell survival in vitro and in vivo, maintains selective killing of cancer cells ("logic-gated selectivity") when expressed with the inhibitor antigen and the activator receptor in vitro and in vivo, and/or enhances antigen dependent expansion in vitro and in vivo.
  • the present disclosure relates to an immune cell, wherein the membrane tethered cytokine provides increased activation, proliferation, and killing capacity while maintaining the selectivity of a logic gated dual receptor system.
  • the present disclosure relates to an immune cell, wherein the immune cell has enhanced antigen-dependent expansion compared to a cell lacking the membrane tethered cytokine. Docket No.: 061250-559001WO [0542] In embodiments, the present disclosure relates to an immune cell, wherein the immune cell maintains antigen-dependent expansion longer when compared to a cell lacking the membrane tethered cytokine. In some cases, the immune cell does not exhaust after continued antigen- dependent expansion. [0543] In embodiments, the present disclosure relates to an immune cell, wherein the immune cell proliferates at least 10% more rapidly than a cell lacking the membrane tethered cytokine.
  • the present disclosure relates to an immune cell, wherein the immune cell continues to proliferate in the absence of exogenous IL-2. [0545] In embodiments, the present disclosure relates to an immune cell, wherein the immune cell overcomes TGF- ⁇ immune suppression better than a cell lacking the membrane tethered cytokine. [0546] In embodiments, the present disclosure relates to an immune cell, wherein the immune cell expresses and/or secretes greater amounts of IFN ⁇ compared to a cell lacking the membrane tethered cytokine.
  • the present disclosure relates to an immune cell, wherein the immune cell expresses greater amounts of the transcription factor T-bet compared to a cell lacking the membrane tethered cytokine.
  • the present disclosure relates to an immune cell, wherein the cancer is acute myelogenous leukemia, adult T-cell leukemia/lymphoma, B-cell lymphoma, breast cancer, chronic lymphocytic leukemia, colorectal cancer, diffuse large B-cell lymphoma, gastric cancer, glioblastoma multiforme, head & neck, urothelial cancer, a hematological cancer, hematological malignancy, Hodgkin's lymphoma, leukemia, lymphoma, malignant melanoma, melanoma, multiple myeloma, neuroblastoma, non-small cell lung cancer, non-small cell lung carcinoma, ovarian cancer, pancreatic cancer, prostate cancer
  • Another aspect of the present disclosure is a method of killing a tumor cell comprising contacting the tumor cell with any herein-disclosed immune cell.
  • a further aspect of the present disclosure is a pharmaceutical composition, including a plurality any herein-disclosed immune cells and a pharmaceutically acceptable diluent, carrier, or excipient.
  • the pharmaceutical composition is comprised in a kit.
  • the pharmaceutical composition is used in a method for treating cancer.
  • An additional aspect of the present disclosure is a method of treating a cancer comprising administering to a subject in need therefore, an effective amount of any herein-disclosed immune cell.
  • kits including any herein-disclosed immune cell Docket No.: 061250-559001WO [0553] In some embodiments, the present disclosure relates to use 120 in a method for treating cancer.
  • Another aspect of the present disclosure is a polynucleotide or polynucleotide system, including one or more polynucleotides including one or more polynucleotide sequences encoding the membrane tethered cytokine, the inhibitor receptor, and the activator receptor. In some cases, the polynucleotide or polynucleotide system is comprised in a nanocarrier.
  • nanocarrier is capable of delivering the polynucleotide or polynucleotide system to an immune cell in vivo or ex vivo.
  • the nanocarrier is a lipid nanoparticle (LNP).
  • An additional aspect of the present disclosure is a a method of making an immune cell therapy, comprising transforming immune cells with including any herein-disclosed polynucleotide or polynucleotide system .
  • the method comprises contacting immune cells with any herein disclosed nanocarrier.
  • a host cell comprises any herein- disclosed polynucleotide or polynucleotide system.
  • any immune cell, polynucleotide, pharmaceutical composition, or method disclosed herein is applicable to any herein-disclosed immune cell, polynucleotide, pharmaceutical composition, or method.
  • any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
  • the present disclosure provides engineered cells comprising the engineered interleukin, receptors, vectors, and/or polynucleotides described herein.
  • the cell is an engineered immune cell for logic-gated tumor cell killing, comprising a receptor specific to an inhibitor antigen, and a recombinant interleukin and/or a polynucleotide encoding the recombinant interleukin.
  • An emerging strategy for discriminating tumor from normal cells relies on multiple markers rather than a single definitive marker.
  • engineered cells that integrate multiple signals can respond in nuanced ways to more complex antigen profiles.
  • the signal integration systems that underlie these cell therapy approaches, and other synthetic biological control circuits have been called logic gates, terminology borrowed from a branch of mathematics relevant to computing which was pioneered by the English mathematician George Boole in the mid-1800s.
  • the “NOT gate” involves use of specific antigens to protect normal tissues via inhibitory receptors.
  • the cell is an engineered immune cell for logic-gated tumor cell killing, comprising an inhibitory receptor specific to an inhibitor antigen, an activator receptor Docket No.: 061250-559001WO specific to an activator antigen, and a recombinant interleukin and/or a polynucleotide encoding the recombinant interleukin.
  • recombinant interleukin is an engineered interleukin of the disclosure.
  • the engineered interleukin is an IL-2 described herein.
  • the engineered interleukin is an IL-12 described herein.
  • the engineered interleukin is an IL-15 described herein.
  • the engineered interleukin is an IL-18 described herein.
  • the engineered interleukin is an IL-21 described herein.
  • the cell is an immune cell.
  • the cell is a T cell.
  • the cell is a cytotoxic T cell.
  • the immune cell is CD4+, CD8+, a gamma delta T cell, an invariant T cells, an iNK cell , a NK cell, a macrophage, or combinations thereof.
  • the immune cell is a gamma delta ( ⁇ ) T cell.
  • the immune cell is an invariant T cell.
  • the immune cell is an invariant natural killer T cell (iNKT cell).
  • the immune cell is a T cell.
  • the immune cell is a B cell.
  • the immune cell is a Natural Killer (NK) cell.
  • the immune cell is CD8-.
  • the immune cell is CD8+.
  • the immune cell is CD4+. In some embodiments, the immune cell is CD4-. In some embodiments, the immune cell is CD8-/CD4+. In some embodiments, the immune cell is a CD8+ CD4- T cell. [0562] In some embodiments, the immune cell is non-natural. In some embodiments, the immune cell is isolated. [0563] Different cell types can be obtained from appropriate isolation methods. The isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used.
  • the separation is affinity-or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10040846; and U.S. Pat. Appl. Pub. No.2006/0121005.
  • the immune cell is autologous.
  • the immune cells is isolated or derived from same subject who will receive the cell as part of a therapeutic regimen. It can be advantageous to modify autologous immune cells to have reduced expression and/or function of MHC class I with the blocker receptor is specific to an MHC class I antigen. Without wishing to be bound by theory, modification of autologous immune cells to have reduced expression and/or function of MHC class I reduces binding of the blocker receptor by MHC class I expressed by the immune cells, either in cis or in trans.
  • the immune cell is allogeneic. Allogeneic immune cells can be derived from a donor other than the subject to which the immune cells will be administered.
  • Allogeneic immune cells have been commonly referred to in cell therapy as “off-the-shelf” or “universal” because of the possibility for allogeneic cells to be prepared and stored for use in subjects of a variety of genotypes.
  • Methods of introducing and expressing genes into a cell are known in the art.
  • 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. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.
  • Any suitable genetic modification method can be used to genetically modify the cells to include such engineered interleukins, activator receptor, and/or inhibitory receptor, including, for example, any of the gene editing methods described herein.
  • the genetic Docket No.: 061250-559001WO modification method is a CRISPR, TALE, zinc finger, or Cas-CLOVER method described herein.
  • such engineered cells are produced using any one of the retroviral methods (e.g., lentiviral methods) provided herein.
  • such engineered cells are produced using any of the transposon/transposase systems described herein, e.g., a piggyBac method (e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases), a Sleeping Beauty method (e.g., Sleeping Beauty or Sleeping Beauty-like transposons and transposases), a Helraiser method (e.g., Helraiser and Helraiser-like transposons and transposases), and a Tol2 method (e.g., Tol2 and Tol2-like transposons and transposases).
  • a piggyBac method e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases
  • a Sleeping Beauty method e.g., Sleeping Beauty or Sleeping Beauty-like transposons and transposases
  • a Helraiser method e.g., Helraiser
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • 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 illustrative colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • MHC major histocompatibility complex
  • HLA-A HLA-A
  • HLA-B HLA-B
  • HLA-C HLA-C
  • MHC class I alleles are highly polymorphic and expressed in all nucleated cells.
  • MHC class I polypeptides encoded by HLA-A, HLA-B, and HLA-C and alleles thereof form heterodimers with ⁇ 2 microglobulin (B2M) and present in complex with antigens on the surface of cells.
  • an MHC class I gene or polypeptide may refer to any polypeptide found in the MHC or the corresponding gene encoding said polypeptide.
  • the immune cells of the disclosure are inactivated by an inhibitor ligand comprising an MHC class I polypeptide, e.g., HLA-A and alleles thereof.
  • HLA-A alleles can be, for example HLA-A*03, and/or any gene that Docket No.: 061250-559001WO encodes protein identical or similar to HLA-A*03 protein.
  • HLA-A and alleles thereof in the immune cells.
  • Reduced MHC Class I Polypeptide Expression the genetically engineered immune cells described herein are modified to reduce or eliminate expression of the B2M gene product.
  • the beta-2 microglobulin (B2M) gene encodes a protein that associates with the major histocompatibility complex (MHC) class I, i.e. MHC-I complex.
  • MHC major histocompatibility complex
  • the MHC-I complex is required for presentation of antigens on the cell surface.
  • the MHC -I complex is disrupted and non-functional when the B2M is deleted (Wang D et al. Stem Cells Transl Med.4:1234 ⁇ 1245 (2015)).
  • the B2M gene can be disrupted with high efficiency using gene editing techniques known in the art (Ren et al. Clin. Cancer Res. 23:2255-2266 (2017)). Reducing or eliminating B2M can reduce, or eliminate functional MHC I on the surface of the immune cell.
  • Illustrative compositions, methods, and means for reducing MHC Class I polypeptide expression, such as in allogeneic immune cell therapy, are described in U.S. Pat.
  • the expression and of function of B2M is reduced using gene editing systems.
  • the disclosure provides gene editing systems for editing an endogenous target gene in an immune cell.
  • the disclosure provides interfering RNAs specific to sequences of target genes.
  • Gene editing systems such as CRISPR/Cas systems, TALENs and zinc fingers can be used to generate double strand breaks, which, through gene repair mechanisms such as homology directed repair or non-homologous end joining (NHEJ), can be used to introduce mutations. NHEJ after resection of the ends of the break, or improper end joining, can be used to introduce deletions.
  • NHEJ homology directed repair or non-homologous end joining
  • the target gene comprises a gene encoding a subunit of the MHC-I complex.
  • Target gene sequences include, but are not limited to, promoters, enhancers, introns, exons, intron/exon junctions, transcription products (pre-mRNA, mRNA, and splice variants), and/or 3’ and 5’ untranslated regions (UTRs). Any gene element or combination of gene elements may be targeted for the purpose of genetic editing in the immune cells described herein. Modifications to the target genes can be accomplished using any method known in the art to edit the target gene that results in altered or disrupted expression or function the target gene or gene product.
  • a target gene is edited in the immune cells described herein using a nucleic acid guided endonuclease.
  • Illustrative nucleic acid guided endonucleases include Class 2 Type II endonucleases, such as CRISPR/Cas9. Docket No.: 061250-559001WO
  • RNA interference refers to the process of sequence-specific post- transcriptional gene silencing, mediated by double-stranded RNA (dsRNA).
  • Duplex RNAs such as siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA- directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat associated siRNA) and modified forms thereof are all capable of mediating RNA interference. These dsRNA molecules may be commercially available or may be designed and prepared based on known sequence information.
  • Pharmaceutical Compositions A further aspect of the present disclosure is a pharmaceutical composition, including a plurality any herein-disclosed immune cells and a pharmaceutically acceptable diluent, carrier, or excipient. In some cases, the pharmaceutical composition is comprised in a kit.
  • compositions comprising a plurality of immune cells comprising the engineered interleukin, a first and/or inhibitory receptor of the disclosure, and a pharmaceutically acceptable diluent, carrier or excipient.
  • Such 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; and preservatives.
  • the immune cell expresses the engineered interleukin and the inhibitory receptor. In some embodiments, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the immune cells express both the engineered interleukin and the inhibitory receptor. In some embodiments, at least 90% of the immune cells express both the engineered interleukin and the inhibitory receptor. [0585] In some embodiments, the immune cell expresses the engineered interleukin and the activator receptor.
  • At least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the immune cells express both the engineered interleukin and the activator receptor. In some embodiments, at least 90% of the immune cells express both the engineered interleukin and the activator receptor. [0586] In some embodiments, the immune cell expresses the engineered interleukin, the activator receptor, and the inhibitory receptor. In some embodiments, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the immune cells express the engineered interleukin, the activator receptor, and the inhibitory receptor.
  • the immune cells express the engineered interleukin, the activator receptor, and the inhibitory receptor.
  • Methods of Using Engineered Interleukins [0587] The disclosure provides methods of killing tumor cells using an engineered cell of the disclosure. [0588] In some embodiments, the method comprises contacting the tumor cell with an immune cell or the pharmaceutical composition of the disclosure. [0589] In some embodiments, the immune cell with the engineered interleukin shows enhanced proliferation compared to immune cells without the interleukin. [0590] In some embodiments, the immune cell with interleukin shows enhanced tumor cell killing compared to immune cells without the interleukin.
  • the immune cell with interleukin shows enhanced activation compared to immune cells without the interleukin.
  • Methods of Treatment Another aspect of the present disclosure is a method of killing a tumor cell comprising contacting the tumor cell with any herein-disclosed immune cell.
  • An additional aspect of the present disclosure is a method of treating a cancer comprising administering to a subject in need therefore, an effective amount of any herein-disclosed immune cell.
  • the compositions and methods described herein can be used in a method for treating diseases. In some embodiments, they are for use in treating hyperproliferative disorders, such as cancer, in a subject in need thereof.
  • the methods described herein can reduce the risk of the developing diseases, conditions, and disorders as described herein.
  • the method comprises administering to the subject a plurality of the immune cells or the pharmaceutical composition of the disclosure.
  • the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, Docket No.: 061250-559001WO glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypopharynx cancer, larynx cancer, nasopharynx cancer,
  • the tumor is a solid tumor.
  • the cancer is the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.
  • NSCLC non-small-cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • GBM glioblastoma
  • gastrointestinal cancer renal cancer
  • renal cancer and renal cell carcinoma
  • the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.
  • the cancer is characterized by the loss of MHC-class I expression.
  • the MHC-class I is expressed differentially in various cell types or tissues.
  • the engineered cells and pharmaceutical compositions described herein are administered to a subject or patient having a particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy.
  • engineered cells and pharmaceutical compositions are administered to a subject, such as a subject having or at risk for the disease or condition.
  • the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in cancer expressing an antigen recognized by the engineered T cells.
  • Methods for administration of cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions.
  • kits and articles of manufacture comprising the polynucleotides and vectors encoding the engineered interleukins and receptors described herein, and immune cells comprising the engineered interleukins and receptors described herein.
  • the kit comprises articles such as vials, syringes, and instructions for use.
  • the kit comprises a polynucleotide or vector comprising a sequence encoding one or more engineered interleukins and/or receptors of the disclosure.
  • the kit comprises a plurality of immune cells comprising the engineered interleukins, first and inhibitory receptors as described herein.
  • the plurality of immune cells comprises a plurality of T cells.
  • the plurality of immune cells comprises a plurality of NK cells.
  • the kit further comprises instructions for use.
  • Example 1 describes materials and methods for the examples that follow.
  • Target cells were engineered to express (A) EGFR, CEA or MSLN only, EGFR, CEA or MLSN and HLA-A*02 or H-2Db, or (B) HLA-A*02 or H-2Db only.
  • T cells were transduced with a construct encoding an EGFR, CEA or MSLN activating receptor, a HLA-A*02 or H-2Db inhibitory receptor, and a B2Msh silencing module.
  • Membrane-tethered IL-2, IL-12, or IL-18 Docket No.: 061250-559001WO constructs (mt-IL 2, mt-IL-12 or mt-IL-18) were designed on separate constructs and co-transduced with the construct described above.
  • the membrane tethered interleukin was operably linked to a constitutive promotor (eukaryotic translation elongation factor 1 alpha (EF1a)) or an inducible promoter (NFAT response elements (NFAT RE)).
  • EF1a eukaryotic translation elongation factor 1 alpha
  • NFAT RE NFAT response elements
  • T cells transduced with a construct encoding a EGFR, CEA or MSLN activating receptor, HLA-A*02 or H-2Db inhibitory receptor, and a B2Msh silencing module and treated with exogenous, soluble IL- 2, IL-12, or IL-18.
  • Cell Staining and Supernatant Quantification [0605] Cell Trace fluorescence was used to label 1) non-transduced T cells (UTDs), 2) T cells transduced with a construct encoding a EGFR, CEA or MLSN activator receptor, HLA-A*02 or H-2Db blocker receptor, and a B2Msh silencing module, or 3) T cells co-transduced with a construct encoding a EGFR, CEA or MSLN activator receptor, HLA-A*02 or H-2Db blocker receptor, and a B2Msh silencing module and a second construct encoding membrane-tethered cytokine (either IL-2, IL-12, or IL-18).75,000 A target cells (EGFR+, MSLN+ or CEA +) or AB target cells (EGFR+, MSLN+ or CEA + and HLA-A*02+or H-2Db+) were co-cultured in 96-well cell-
  • T cells treated with exogenous, soluble IL-2, IL-12, or IL- 18 were used as positive controls.
  • the cell mixture was stained with anti-CD4, anti-CD8, activator receptor, blocker receptor, and anti-IL-2, anti- IL-12 or anti-IL-18 antibodies.
  • the absolute number of viable T cells and their cell division profile was measured using flow cytometry.
  • the supernatant was collected and measured for proinflammatory cytokine concentration, including IL-2, IL-12, IL-18, TNFa, IFN ⁇ , IL-6, Granzyme B, and GM-CSF.
  • IL-2 Starvation Assay Human PBMCs were thawed and transfected with IL-2 mutein constructs using the Piggybac system and a Lonza 4D nucleofector, followed by TransActTM (Miltenyi Biotec) stimulation 24 hours after transfection. The cells were then transferred to a 24-well G-Rex vessel and supplemented with 300 IU/mL IL-2 every 48-72 hours. Five days after transfection, the cells were spun down at 500 RCF for 5 minutes, media was aspirated, and the cells were resuspended in cell culture media (X-VIVO 15 plus 1% human serum) containing zero soluble IL-2, at a density of 1 million cells per mL.
  • TransActTM Miltenyi Biotec
  • T cells Three groups of T cells: 1) non-transduced T cells (UTDs), 2) T cells transduced with a construct encoding a EGFR, CEA or MSLN activator, HLA-A*02 or H-2Db blocker receptor, and a B2Msh silencing module, or 3) T cells co-transduced with a construct encoding a EGFR, CEA or MSLN activator, HLA-A*02 or H-2Db blocker receptor, and a B2Msh silencing module and a second construct encoding a membrane-tethered cytokine (either IL-2, IL-12, or IL-18) were co- cultured with fluorescence labeled
  • a target cells EGFR+, CEA+ or MSLN+
  • AB target cells EGFR+, CEA+ or MSLN+ and HLA-A*02+or H-2Db+
  • B target cells HLA-A*02+ or H- 2
  • T cells treated with exogenous, soluble IL-2, IL-12, or IL-18 were used as positive controls.
  • the fluorescence images of target cells were collected every 4 hours, and the specific killing was calculated as the reduction in the percentage of total fluorescence surface area compared to the UTD control.
  • IL-2 Computational modeling [0608] Structural modeling of the IL-2/IL2R complex was performed using Pyrosetta and the (Alford, et al., (2017) J Chem Theory Comput. 13(6): 3031–3048) score function. Using the “fastrelax” protocol, five cycles of backbone minimization and rotamer optimization brought the first asymmetric unit atomic coordinates in the PDB 2ERJ template structure to a local energy minimum.
  • IL-2 JNL mutein screen For each IL-2 mutein construct, 2 million Jurkat cells were transfected with 4 micrograms of DNA encoding membrane-tethered IL-2, using the Neon electroporation system.16 hours later the cells were assessed for cell count and viability, after which 100k cells (per stain) were removed for flow cytometric analysis. The cells were washed and stained with either (1) anti-IL-2 polyclonal antibody followed by secondary antibody, (2) streptavidin-conjugated recombinant CD25 protein, Docket No.: 061250-559001WO or (3) streptavidin-conjugated recombinant CD132 plus unlabeled CD122 protein.
  • Flow cytometric analysis was performed on a FLOW CYTOMETRYCanto II (BD), and the percent positive population for each mutein binding to CD25 and CD122/CD132 was normalized to the percent of cells positive for IL-2 as measured by polyclonal anti-IL-2 binding.
  • Mouse Xenograft Study [0610] Animals were implanted subcutaneously with 5e4 firefly luciferase (+), MSLN(+) HLA- A*02(-) MS751 cells (“tumor”; left flank) and 5e4 firefly luciferase (+), MSLN(+) HLA-A*02(+) MS751 cells (“normal”; right flank).
  • mice subcutaneously implanted with 5e4 firefly luciferase (+), EGFR (+), H-2Db (-) MS751 tumor cells. After eleven days of growth, T cells were infused at the indicated concentrations. Tumor growth was measured by bioluminescence imaging (BLI) twice a week. T cells were harvested at indicated time points for further characterization.
  • Example 2 Exogenous Soluble Interleukins [0611] This example shows the addition of exogenous, soluble interleukins induces proliferation while maintaining selectivity of logic-gated T-cells.
  • T-cells were engineered to express a CEA activating receptor alone or in combination with the HLA-A-*02 inhibitory receptor and cultured with H508 cells expressing CEA alone or CEA and HLA-A*02 at a ratio of 1:3.
  • Exogenous IL-2 was added at the indicated doses between 0-90 ng/ml.
  • the addition of exogenous IL-2 increased specific killing of A target cells by immune cells expressing the CAR alone or the dual receptor system.
  • the addition of exogenous IL-2 increased the killing of AB targets by immune cells expressing the CAR alone but did not in immune cells expressing the logic gated dual receptor system (FIG.1).
  • the bottom data curve that intersects the vertical line at hour 50 is the CEA receptor alone without exogenous IL-2.
  • the remaining data curves that have ⁇ 50% specific killing percentage were supplemented with exogenous IL-2 of various concentrations; the CEA activator receptor expressed alone supplemented with 3.3 IU/ml of IL-2 had the highest specific killing percentage.
  • the bottom data curve that intersects with the vertical line at hour 50 is the CEA receptor alone without exogenous IL-2.
  • Primary T-cells were engineered to express a CEA activating receptor alone or in combination with a HLA-A-*02 inhibitory receptor and cultured with H508 cells expressing CEA alone or CEA and HLA-A*02 at a ratio of 1:3.
  • Exogenous IL-12, IL-15, or IL-21 was added at the indicated doses between 0-90 ng/ml.
  • engineered immune cells were co-cultured with A target cells and the bottom data curve that intersects with the vertical line at hour 50 was not treated with exogenous IL-12.
  • the group supplemented with 0.4ng/ml had the highest percentage of specific killing at hour 50.
  • FIG.3A is a schematic illustrating membrane tethered interleukin in the dual receptor system.
  • Each engineered T-cell will express an antigen dependent activator receptor, an antigen dependent blocker receptor and an antigen independent membrane tethered interleukin that stimulates the endogenous interleukin receptor.
  • FIG. 4 is a schematic illustrating the structural rationale for each mt-interleukin design.
  • Mt- interleukins that bind the common cytokine receptor ⁇ chain ( ⁇ c) may include a flexible hinge to engage the ⁇ chain. Examples of interleukin receptors that contain the ⁇ chain are IL-1, IL-4, IL-7, IL-9, IL-15 and IL-21.
  • mt-IL-12 and related cytokines include a longer, more rigid Docket No.: 061250-559001WO hinge to access the more distal binding region of the receptor.
  • the construct may also include a linker connecting the IL12A and IL12B subunits.
  • Example 4 Generation of T cells expressing Membrane Tethered Cytokines [0615] This example describes how T cells engineered to express membrane tethered cytokines along with a EGFR, CEA or MSLN activator receptor, HLA-A*02 or H-2Db blocker receptor, and a B2M short hairpin against the B2M gene (B2Msh) were generated.
  • a single construct encoding a EGFR, CEA or MSLN activator receptor, HLA-A*02 or H-2Db blocker receptor, and a B2M short hairpin against the B2M gene (B2Msh) was delivered to primary T cells via lentiviral infection.
  • These constructs could also be delivered by DNA vectors, RNA vectors (including mRNA), by CRISPR (or other genome editing proteins), by adenovirus or other recombinant viruses (e.g., alpha virus, vaccinia virus, retrovirus, AAV or herpes virus).
  • a second construct encoding a membrane-tethered cytokine (either mt-IL-2 or mt-IL-12) was delivered to primary T cells via lentivirus infection.
  • These constructs could also be delivered by DNA vectors, RNA vectors (including mRNA) by adenovirus or other recombinant viruses (e.g., alpha virus, vaccinia virus, retrovirus, AAV or herpes virus).
  • adenovirus or other recombinant viruses e.g., alpha virus, vaccinia virus, retrovirus, AAV or herpes virus.
  • Example 5 Membrane Tethered Interleukins [0616] This example shows that expression of membrane tethered interleukins (mem-IL or mt- IL) on T cells results in increased activation, proliferation and killing capacity while maintaining the selectivity of the logic gated dual receptor system.
  • IFN ⁇ secretion [0617] shows T cells expressing membrane tethered interleukins (mt-IL-2 or mt-IL- 12) secrete higher levels of IFN ⁇ compared to the control group (UTD) (FIG.5).
  • Primary T-cells were engineered to express mt-IL-2 or mt-IL-12 as described in Example 1 and then IFN ⁇ levels were measured in the supernatant.
  • Expression of mt-IL2 increased IFN ⁇ secretion by greater than 2 fold compared to the UTD group.
  • Expression of mt-IL-12 increased IFN ⁇ secretion by greater than 4 fold compared to UTD group.
  • T-bet expression shows that mt-IL-2 or mt-IL-12 expressed on T cells is active and sufficient to drive IFN ⁇ secretion.
  • T-bet expression shows T cells expressing mt-IL-2 or mt-IL-12 express higher levels of the transcription factor T-bet compared to the T cells not expressing membrane tethered interleukins (FIG.6). Higher expression of T-bet can be indicative of T cell activation.
  • Primary T-cells were Docket No.: 061250-559001WO engineered to express mt-IL-2 or mt-IL-12 as described in Example 1 and then T-bet expression was analyzed via FLOW CYTOMETRY.
  • T-bet expression resultsed in increased T-bet expression based on the shift right of the dark solid line (mt-IL+) compared to the light solid line (mt-IL-) or the light dotted line (UTD).
  • mt-IL+ dark solid line
  • mt-IL- light solid line
  • UTD light dotted line
  • Human PBMCs were thawed and transfected with a mt-IL-2 construct.
  • the PBMCs were cultured for 5 days post transfection and then switched to cell culture media with exogenous IL-2 (300 IU/mL) or to cell culture media with no exogenous IL-2.
  • This example shows that T cells expressing mt- IL-2 are able to survive upon removal of exogenous IL-2.
  • HLA-A*02 inhibitory receptor
  • CEA activating receptor
  • mt-IL-12 mt-IL-12
  • Human PBMCs were thawed and transfected with a construct encoding mt-IL-12 and a construct encoding a CEA activating receptor and an HLA- A*02 inhibitory receptor.
  • FLOW CYTOMETRY analysis of HLA-A*02 tetramer and IL-12 expression shows that ⁇ 50% of transfected PBMCs (upper right quadrant) are dual positive for both mt-IL-12 and the HLA-A*02 inhibitory receptor (FIG.8).
  • Expression profile characterization of PBMCs transfected with an inhibitory receptor (HLA-A*02), an activating receptor (CEA) and mt-IL-2 [0621]
  • This example shows engineered PBMCs can express a mt-IL-2, a CEA activating receptor and an HLA-A*02 inhibitory receptor.
  • Human PBMCs were thawed and transfected with a construct encoding a mt-IL-2 and a construct encoding a CEA activating receptor and a HLA- A*02 inhibitory receptor.
  • FLOW CYTOMETRY analysis of HLA-A*02 tetramer and IL-2 Docket No.: 061250-559001WO expression shows that ⁇ 50% of transfected PBMCs (upper right quadrant) are dual positive for both mt-IL-2 and the HLA-A*02 inhibitory receptor (FIG.9).
  • T cells engineered to express a CEA activating receptor, HLA-A*02 inhibitory receptor, B2M shRNA and mt-IL-12 retain their ability to kill CEA expressing cells and are still inhibited from killing HLA-A*02 expressing cells (FIG.10).
  • Target cells were engineered to express (A) CEA only, (AB) CEA and HLA-A*02, or (B) HLA-A*02 only.
  • T cells were transduced with a construct encoding a CEA activator receptor, HLA-A*02 inhibitor receptor, and a B2M short hairpin against the B2M gene (B2Msh).
  • mt-IL-12 was designed on a separate construct and co-transduced (FIG. 11A). This group was compared to engineered T cells not expressing mt-IL-12 but that were supplemented with exogenous, soluble IL-12. mt-IL-12 boosted the killing of A target cells (CEA only) by engineered immune cells compared to engineered immune cells alone or engineered immune cells supplemented with exogenous IL-12 indicating that killing is enhanced with mt-IL-12.
  • Engineered T cells expressing mt-IL-12, CEA activating receptor, and HLA-A*02 inhibitory receptor showed reduced killing of AB target cells (FIG.10).
  • T cells expressing mt-IL-12 a CEA activating receptor and an HLA-A*02 inhibitory receptor have increased killing capacity but maintain the selectivity of the logic gated dual reporter receptor system.
  • This example shows T cells engineered to express a CEA activating receptor, HLA-A*02 inhibitory receptor and membrane tethered IL-12 retain their ability to kill CEA expressing cells and are still inhibited from killing HLA-A*02 expressing cells.
  • Target cells were engineered to express (A) CEA only or (AB) CEA and HLA-A*02. T cells were transduced with a construct encoding a CEA activator receptor, HLA-A*02 inhibitor receptor, and a B2Msh.
  • mt-IL-12 was designed on a separate construct and co-transduced (FIG. 11A). Expression of the HLA-A*02 inhibitor receptor and the mt-IL-12 was verified via Flow cytometry (FIG.11B). This group was compared to engineered T cells that express a secreted IL-12 and engineered T cells that do not express mt-IL-2. Secreted and mt-IL-12 was expressed under the control of the EF1a promoter.
  • Engineered T cells expressing mt-IL-12, CEA activating receptor, and HLA-A*02 inhibitory receptor showed increased killing of A target cells (solid circle solid line) compared to engineered immune cells expressing a CEA activating receptor, and HLA-A*02 inhibitory receptor expressing alone (solid square solid line) (FIG. 11C).
  • Engineered T cells expressing membrane tethered IL-12, CEA activating receptor, and HLA-A*02 inhibitory receptor showed reduced killing of AB target cells (empty circle dashed line) compared to A target cells which shows the selective killing power of the logic gated dual receptor system, e.g., NOT gate”, is maintained with Docket No.: 061250-559001WO expression of mt-IL-12. Expression of the secreted IL-12 showed a similar specific killing percentage of A target cells (solid triangle solid line) as mt-IL-12 engineered immune cells.
  • FIG.11C shows that engineered immune cells expressing mt-IL-12, CEA activating receptor, and HLA-A*02 inhibitory receptor with shRNA has a higher selectivity window (18 vs 11.7) than engineered immune cells expressing no mt-IL-12 or secreted IL-12.
  • the selectivity window is the ratio of ET50 for AB divided by ET 50 for A and demonstrates changes in specificity in cell killing and cell sparing for various engineered T cells.
  • the same engineered immune cells tested in FIG. 11 were next used to test antigen dependent proliferation. For this experiment, T cells were transduced with a construct encoding a CEA activator receptor, HLA-A*02 inhibitor receptor, and a B2Msh.
  • mt-IL-12 was designed on a separate construct and co-transduced (FIG.11A). Engineered immune cells expressing a secreted IL-12 were also used in this experiment. Additionally, untransduced (UTD) T cells and T cells treated exogenously with IL-12 were tested in this experiment. Target cells were engineered to express CEA only (tumor cells) or CEA and HLA-A*02 (“normal” cells). [0626] Engineered T cells were treated with CellTrace Violet to track proliferation. CellTrace Violet is a fluorescent stain that labels cells and allows the measurement of proliferation. The level of fluorescence in cell populations indicates the number of generations, i.e., the lower fluorescence level the more proliferation.
  • Engineered immune cells expressing CEA activator receptor, HLA-A*02 inhibitor receptor, and a B2Msh with mt-IL-12 or secreted IL-12 also showed antigen dependent proliferation (FIG.12B). This shows that engineered immune cells expressing mt-Il-12 or secreted IL-12 retain their ability to undergo antigen induced proliferation when exposed to cancer cells expressing the antigen.
  • T cells engineered to express a CEA activating receptor, HLA-A*02 inhibitory receptor and mt-IL-2 have increased killing capacity of A target cells (CEA only) compared to T cells expressing CEA activating receptor, HLA-A*02 inhibitory receptor and supplemented with exogenous IL-2 (FIG.13). Additionally, T cells engineered to express a CEA activating receptor, HLA-A*02 inhibitory receptor and mt-IL-2 retain the function of the logic gated dual receptor system to not kill AB target cells.
  • Target cells were engineered to express (A) CEA only or (AB) CEA and HLA-A*02.
  • T cells were transduced with a construct encoding a CEA activator receptor, HLA-A*02 inhibitor receptor, and a B2Msh.
  • mt-IL-2 (C125A) was designed on a separate construct and co-transduced. The C125A mutation may reduce interleukin aggregation which could result in increased signaling.
  • This group was compared to engineered T cells treated with 100IU of exogenous, soluble IL-2.
  • mt-IL-2 boosted the killing of A cells while preserving the selectivity against killing of AB cells (FIG. 13).
  • Example 6 Screen for functional IL-2 muteins [0629] This example shows the identification of IL-2 mutations that may have reduced affinity for CD25 but still bind to IL2R. A computational model was used to select IL-2 attenuation mutations to de-emphasize T regulatory cell (CD25high) interactions in trans and promote cis- biding in T-effector cells. Each IL-2 mutation was modeled in Rosetta and scored for self, common ⁇ chain IL2R ⁇ and CD25 interactions.
  • Example 7 In vitro characterization of mt-IL-2 muteins [0631] This example shows the validation of IL-2 mutations that retain affinity for IL2R, have reduced affinity for CD25, and induce T cell proliferation. To test each IL-2 mutein construct identified in Example 6, 2 million Jurkat cells were transfected with DNA encoding the mt-IL-2 mutein.
  • FLOW CYTOMETRY analysis was performed with either (1) anti- IL2 polyclonal antibody followed by secondary antibody, (2) streptavidin-conjugated recombinant CD25 protein, or (3) streptavidin-conjugated recombinant CD132 plus unlabeled CD122 protein.
  • the percent positive population for each mutein binding to CD25 and CD122/CD132 was normalized to the percent of cells positive for IL-2 as measured by polyclonal anti-IL-2 binding.
  • Flow cytometry analysis revealed a group of mutations maintained high expression (detected by an anti-IL2 mAB) and reduced CD25 affinity (determined by a reduction in detection with soluble CD25) (FIG. 15).
  • the PBMCs were cultured for 5 days post transfection and then switched to cell culture media with exogenous IL-2 (300 IU/mL) or without exogenous IL-2.
  • Analysis of the percentage of mt-IL-2 positive cells shows that IL-2 mutations with reduced CD25 affinity induce proliferation in a low IL-2 environment (FIG.16B).
  • Expression of any of the mt- IL-2 muteins resulted in a similar increase in the percentage of IL-2+ T cells compared to T cells treated with exogenous human IL-2. This example shows that the mt-IL-2 muteins identified in the screen are functional when expressed and drive T cell proliferation.
  • Example 8 Characterization of membrane tethered Interleukin under control of Inducible Promoter
  • This example shows that constitutive or inducible expression of membrane tethered interleukins on T cells results in enhanced antigen dependent expansion of T cells, while maintaining the efficacy and selectivity of the logic gated dual receptor system, e.g., “NOT gate” system.
  • T cells were engineered to co-express an MSLN activator receptor and HLA-A*02 blocker receptor from one construct and further express mt-IL-12 from a second construct.
  • the mt-IL-12 construct was either constitutively expressed via an EF1a promoter (pEF1a) Docket No.: 061250-559001WO (FIG.
  • HLA-A*02 tetramer was used to detect expression of the HLA-A*02 inhibitor receptor and an IL-12 P70 specific antibody was used to detect cell surface IL-12 ⁇ heterodimer expression.
  • Expression of the HLA-A*02 blocker receptor and mt-IL-12 operably linked to the EF1a promoter was confirmed in primary T cells (FIG.17C). To test the functionality of the activation induced NFAT RE promoter, after transfection with the two constructs, the T cells were stimulated with CD3/CD28 beads for 24hrs and then the expression of mt-IL-12 was quantified.
  • an inducible mt-IL-12 construct was further tested in T cells. For this experiment, T cells were transduced with constructs encoding constitutive mt-IL-12 (EF1a) or activation induced mt-IL-12 (4x NFAT or 6x NFAT). To further test the inducible construct the transfected T cells were treated overnight with TransActTM (Miltenyi Biotec) which comprises CD3-CD28 beads.
  • T cells were washed out after 24hrs and then expression of mt-IL-12 was quantified each day for 4 days.
  • Treatment with 1:100 of TransActTM resulted in an initial peak of mt-IL-12 expression in the 4x NFAT and 6x NFAT groups that receded after TransAct was washed out, suggesting mt-IL-12 expression is inducible and reversible (FIG. 18).
  • expression of mt-IL-12 in the EF1a group was independent of T cell stimulation by TransActTM (FIG. 18).
  • TransActTM treated groups are indicated by the solid line with the inverted triangle and the untreated group is indicated by the solid line with the square (FIG. 18).
  • mt-IL-12 cleavage was tested in the constitutive and activation inducible mt-IL-12. If the mt-IL-12 is cleaved or otherwise unable to be membrane tethered, the IL-12 becomes soluble and released (“shed”) into a culture medium (when in vitro) or into an animal’s bloodstream (when in vivo)
  • mt-IL-12 EF1a A constitutively expressed mt-IL-12 was also tested in this experiment (mt-IL-12 EF1a). Immune cells were activated with TransActTM as described above, with wash out, and then IL-12 cleavage was measured in the media at day 1 and day 4.
  • Cleaved and shed mt-IL-12 was be detected in the media via the cytometric bead array assay (CBA).
  • CBA cytometric bead array assay
  • UTD T cells showed little to no IL-12 in the media suggesting little to no cleavage of IL-12 was taking place.
  • Expression of the secreted IL-12 showed a large amount of soluble IL-12 as expected.
  • Expression of constitutive mt- IL-12 resulted in some cleavage of IL-12 (more than UTD but less than secreted IL-12) indicated by the ⁇ 100pg/ml of IL-12p70 in the media.
  • MSLN activator receptor HLA-A*02 blocker receptor and B2M shRNA
  • MSLN activator receptor HLA-A*02 blocker receptor and B2M shRNA
  • a second construct encoding mt-IL- 12 EF1a
  • MSLN activator receptor HLA-A*02 blocker receptor and B2M shRNA and a second construct encoding mt-IL-124xNFAT
  • CAR MSLN activator receptor alone
  • Transfected primary donor T cells were co-cultured with MSLN(+)HLA-A*02(-) MS751 (A cells/tumor cells) or MSLN(+)HLA-A*02(+) MS751 (AB cells/normal cells) for 96 hours with E:T ratios from 1:81 to 3:1.
  • Coculture of primary T cells expressing the MSLN CAR alone with A (solid square solid line) or AB target cells (empty square dashed line) results in very little difference in specific killing percentage illustrated by the similar slope and curves of the two lines.
  • the reduced killing of AB target cells illustrates the selectivity of the dual receptor system.
  • FIG.21 shows the ET50 for each of the constructs and for each co-culture experiment.
  • FIG.21 also include the selectivity window which is the ratio of ET50 for AB and ET for A (“ET50 AB/A”) which is a measure of specific killing of target cells.
  • MSLN CAR alone had an ET50 AB/A of 2 or 1.3 from donor 1 and 2, respectively.
  • the low ET50 AB/A indicates indiscriminate killing of A and AB target cells.
  • ET50 AB/A from the MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA group was greater than 33 for donor 1 and greater than 15 for donor 2.
  • the higher ET50 AB/A with the dual receptor system is illustrative of its selective killing of A tumor cells but sparing of AB “normal” cells. Expression of either of the mt-IL-12 constructs (constitutive or activation induced) resulted in a higher ET50 AB/A than MSLN activator receptor alone.
  • This example shows Docket No.: 061250-559001WO that constitutive or activation induced expression of mt-IL-12 maintains the selective killing capacity of the dual receptor system when expressed in combination with the dual receptor system.
  • antigen dependent activation of primary T cells expressing MSLN activator receptor, HLA-A*02 blocker receptor and mt-IL-12 was tested.
  • Constitutive or activation induced expression of mt-IL-12 was tested in this experiment.
  • Primary T cells from two donors were used in this experiment.
  • MSLN(+) HLA-A*02(-) MS751 target cells were co-cultured with primary T cells from each donor for 17 days and the number of T cells was quantified at the indicated times.
  • MSLN activator receptor CAR
  • MSLN activator receptor HLA-A*02 blocker receptor and B2M shRNA
  • MSLN activator receptor HLA-A*02 blocker receptor and B2M shRNA
  • mt-IL-12 EF1a promoter
  • 2e4 MS751 target cells per well were plated in each well of a 96 well plate and incubated for 24hrs.
  • TGF ⁇ Transforming growth factor ⁇
  • TGF ⁇ Transforming growth factor ⁇
  • mt-IL-12 constitutive or activation-induced
  • MSLN tumor cells
  • HLA-A*02 normal cells
  • Engineered immune cells expressing MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA that also underwent repeated antigen challenges was used as a control.
  • mt-IL-12 constructs were tested in an in vivo xenograft model.
  • 5e4 MSLN(+) HLA-A*02(-) MS751 cells were engrafted in the left flank of mice (“tumor”) and 5e4 MSLN(+) HLA-A*02(+) MS751 (“normal”) cells were engrafted on the right flank of mice.
  • T cell groups were infused into mice eleven days after cell engraftment: 1) untreated (UTD), 2) MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA, 3) MSLN activator receptor, HLA-A*02 blocker receptor, B2M shRNA and constitutively expressed mt-IL-12 (EF1a), or 4) MSLN activator receptor, HLA-A*02 blocker receptor, B2M shRNA and activation inducible mt-IL-12 (NFAT). Expression of the HLA-A02*02 blocker and mt-IL-12 was confirmed via FLOW CYTOMETRY (FIG.25). TransActTM was used to stimulate the expression of the activation inducible mt-IL-12.
  • T cells infused on Day 11 were 7.5e4 or 7.5e5.
  • Tumor growth was measured by bioluminescence imaging twice a week and T cells were collected every 7 days for characterization from Day 3 to Day 25 (FIG. 26A).
  • Infusion of T cells expressing mt-IL-12 (constitutive or activation induced) at 7.5e4 and 7.5e5 resulted in a ⁇ 10x increase in efficacy of killing target cells compared to untreated T cells or T cells expressing MSLN activator receptor and HLA-A*02 blocker receptor (FIG. 26B).
  • the number of dual positive (Blocker + and hCD3+) and hCD3+ T cells increased in groups expressing mt-IL- 12 over the course of the in vivo study in mice infused with 7.5e4 and 7.5e5; thereby showing that mt-IL-12 boosts antigen dependent expansion of T cells in vivo. Circulating IFN ⁇ and IL-12 was measured in the blood samples collected from the same mice. Analysis of IFN ⁇ showed that engineered immune cells expressing the dual receptor system, B2M shRNA and either the constitutive or activation induced mt-IL-12 increased IFN ⁇ levels suggesting increased immune cell activation (FIG.27C-27D).
  • T cells were engineered to express 1) mouse EGFR (mEGFR) activator receptor (CAR); 2) mEGFR CAR and H-2Db blocker receptor; 3) mEGFR CAR, H-2Db blocker receptor and activation induced mt-IL-12 (4x NFAT); or 4) mEGFR CAR, H-2Db blocker receptor and activation induced mt-IL-12 (6x NFAT).
  • Untransduced (UTD) T cells were used as a control in this experiment. Each engineered T cell was first tested in an in vitro cell kill assay.
  • mt-IL12 and H-2Db blocker receptor Prior to the cell kill assay expression of the mEGFR activator receptor, mt-IL12 and H-2Db blocker receptor was verified via FLOW CYTOMETRY (FIG. 28). The function of the activation induced mt-IL-12 was confirmed by treating with TransActTM. Target cells were engineered to express mEGFR+H-2Db- tumor cells (A) or mEGFR+ H-2Db+ “normal” cells. Engineered immune cells and target cells were mixed at the indicated Effector and Target cell ratio (E:T). Analysis of cell kill data showed that engineered immune cells expressing the dual receptor system and activation induced mt-IL-12 maintain the selectivity and potency of engineered immune cells expressing only the dual receptor system (FIG.29).
  • Tumors were established by subcutaneous engraftment of 5e4 mEGFR(+)H-2Db(-) MS751 cells (FIG. 30A). After eleven days of growth, T cells were infused at the indicated concentrations. Tumor growth was measured by bioluminescence imaging (BLI) twice a week. Survival of mice infused with the indicated doses of T cells is shown in FIG.30B. All four animals infused with 1.2E7 mEGFR CAR succumbed within 5 days post T cell infusion, with >15% weight loss. BLI imaging revealed that mice infused with T cells expressing mEGFR CAR alone showed little to no reduction in tumor burden at any of the infusion doses (light gray thick solid line).
  • mice treated with Tmod + mt-IL-12 displayed no overt signs of toxicity caused by expression of the cytokine, the level of mt-IL-12 shedding and IFN ⁇ secretion was measured in blood samples taken from the experiments described in FIG. 30. Blood samples were harvested on Day 2, Day 9, Day 16, Day 23 and Day 30 from each group of mice.
  • the groups that were measured are 1) mEGFR activator receptor (CAR) (solid square solid line); 2) mEGFR CAR and H-2Db blocker receptor (empty circle solid line); 3) mEGFR CAR, H-2Db blocker receptor and activation induced mt-IL-12 (4x NFAT) (empty inverted triangle dashed line); or 4) mEGFR CAR, H-2Db blocker receptor and activation induced mt-IL-12 (6x NFAT) (solid triangle dashed line).
  • UTD T cells solid circle solid line
  • Analysis of circulating IL-12p70 shows very little shedding of IL-12 in the 1.2e7 infusion group.
  • the 1.2e6 showed increased levels of circulating IL-12 compared to D2 across all groups except UTD (FIG. 31).
  • Flow cytometry analysis of the T cells post infusion shows that there is an increased number of engineered immune cells expressing the EGFR activator receptor and the H-2Db blocker receptor at day 30 post infusion in the 1.2e7 and the 3.79e6 groups (FIG.32A).
  • Analysis of the 3.79e6 showed that both the mt-IL124xNFAT and 6xNFAT groups had the highest increase in the number of immune cells suggesting the mt-IL-12 confers a greater antigen dependent expansion in vivo than the dual receptor system alone.
  • a similar trend was seen when the number of hCD3 cells was analyzed.
  • Example 9 Characterization of Membrane Tethered Interleukin-18
  • mt-IL-18 membrane tethered IL-18
  • T cells were engineered to co-express MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA from one construct and to further express mt-IL-18 from a second construct.
  • the mt-IL-18 was constitutively expressed via an EF1a promoter (pEF1a) (FIG. 34A).
  • HLA-A*02 tetramer was used to detect expression of the HLA- A*02 inhibitor receptor and an IL-18 specific antibody was used to detect cell surface mt-IL-18 expression. Transduction with the two constructs resulted in robust expression of the HLA-A*02 blocker and mt-IL-18 (FIG.34B).
  • mt-IL-18 the ability of mt-IL-18 to promote proliferation in vitro was tested. For this experiment primary T cells from two donors were either untreated (FIG.
  • T cells 35, UTD dashed line), transduced with an MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA (dark colored line) or transduced with an MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA, and mt-IL-18 (light colored line).
  • Each group of T cells was grown in XVIVO-15 media supplemented with 300IU/ml of IL-2. On day 11 post transduction, media was replaced with fresh media with IL-2 and this was repeated every 2-3 days for the remainder of the experiment.
  • Expression of mt-IL-18 with an MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA resulted in increased T cell survival from Day 16- Day 24 (FIG.
  • T cells engineered to express an MSLN activator receptor, HLA- A*02 blocker receptor and B2M shRNA, and mt-IL-18 retain their ability to kill MSLN + cells and are still inhibited from killing HLA-A*02 expressing cells (FIG.36).
  • T cells from two donors were engineered to express a MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA with or without mt-IL-18.
  • Target cells were engineered to express (A) MSLN only or (AB) MSLN and HLA-A*02.
  • T cells were transduced with a construct encoding a MSLN activator receptor, HLA-A*02 inhibitor receptor, and a B2Msh.
  • Mt-IL-18 was designed on Docket No.: 061250-559001WO a separate construct and co-transduced. This group was compared to engineered T cells that do not express mt-IL-18.
  • T cells and target cells were grown at different Effector:Target (E:T) ratios ranging from 1:9 to 27:1.
  • E:T Effector:Target
  • Expression of mt-IL-18 enhanced killing of A target cells (dark circle solid line compared to dark circle dashed line).
  • Expression of mt-IL-18 maintained the selectivity of the dual receptor system (hollow circle solid line compared to hollow circle dashed line).
  • T cells co-expressing the dual receptor system with mt-IL-18 to selectively kill A target cells and spare AB target cells shows that the selective killing properties of the dual receptor system are maintained when mt-IL-18 is expressed in primary T cells (FIG.36).
  • MSLN activator receptor HLA- A*02 blocker receptor
  • mt-IL-18 antigen dependent activation of T cells expressing MSLN activator receptor, HLA- A*02 blocker receptor and mt-IL-18 was tested.
  • MSLN(+) HLA-A*02(-) MS751 target cells were co-cultured with T cells for 17 days and the number of T cells was quantified at the indicated times (shown in FIG. 37A and 37B).
  • T cells from two donors were transduced with either 1) MSLN activator receptor (CAR) alone, 2) MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA, or 3) MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA and constitutively expressed mt-IL-18 (EF1a).
  • CAR MSLN activator receptor
  • HLA-A*02 blocker receptor and B2M shRNA constitutively expressed mt-IL-18
  • the antigen challenge transfer step was repeated on day 7, 10, and 14 with 10% of T cells from the previous well moved to a new well of MS751 cells.
  • Expression of mt-IL-18 enhanced antigen-dependent expansion of engineered T cells expressing MLSN activator receptor and HLA-A*02 blocker receptor (FIG.37A).
  • Expression of mt-IL-18 was able to partially overcome the immunosuppressive effects of the addition of 10ng/ml of that was added each time T cells were plated or replated with targets (FIG.37B).
  • the mt-IL-18 construct was tested in an in vivo xenograft model (FIG. 38A).
  • mice 5e4 MSLN(+) HLA-A*02(-) MS751 cells were engrafted in the left flank of mice (“tumor”) and 5e4 MSLN(+) HLA-A*02(+) MS751 (“normal”) cells were engrafted on the right flank of mice.
  • T cell groups were infused into mice eleven days after tumor cell engraftment: 1) untreated (UTD), 2) MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA, or 3) MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA, and constitutively expressed mt-IL-18 (EF1a).
  • 7.5e4 T cells were infused eleven days after cell engraftment. Tumor growth was measured by bioluminescence imaging (BLI) and T cells were collected for characterization on intervals from Day 3 to Day 25. Infusion of T cells expressing mt-IL-18 reduced tumor cell burden based on BLI measurements (FIG. 38B). Additionally, analysis of T cells isolated at the indicated time points showed that expression of mt-IL-18 resulted Docket No.: 061250-559001WO in increased expansion of total T cells and T cells which express the dual receptor system (FIG. 38C). Blocker expression was quantified using HLA-A*02 tetramer and human T cells were quantified using anti-human CD3 antibody.
  • Example 10 Characterization of Different Promoters for mt-IL Expression and expression of intracellular signaling domain
  • This example describes the development of different expression vectors for membrane tethered interleukins. See, FIG. 39A – 39D.
  • the membrane tethered interleukin was expressed constitutively under the control of the EF1a promoter or expressed upon activation via NFAT response element promoters (NFAT RE 4x or 6x) in a reverse direction to avoid the baseline activation by the 5’ LTR.
  • TATA box promoter to control a lower level of constitutive expression of the membrane tethered cytokine.
  • Another variation of the vector further incorporates an NFAT RE upstream of the TATA box promoter in a forward direction (FIG. 39B and FIG. 39D).
  • the membrane- tethered interleukin will be constitutively expressed under the control of the TATA box and expression will be increased upon activation and binding to the NFAT RE.
  • the present disclosure is not limited to these promoters and envisions any promoter can be incorporated into the vector to control the expression of the membrane tethered interleukin.
  • Example 11 Characterization of membrane tethered interleukins fused with intracellular signaling domains.
  • This example describes the development of different membrane tethered interleukins fused to intracellular signaling domains, see FIG. 39A to FIG. 39D. In the examples described above, the membrane tethered interleukins were expressed with a transmembrane domain.
  • intracellular signaling domains are attached to the C terminus of the transmembrane domain to act signaling nodes. These intracellular domains can be incorporated into any of the vectors used to express the membrane tethered interleukin.
  • the following intracellular signaling domains have been fused to membrane tethered interleukins: B7, CD137, CD27, OX40, DAP10, DAP12, MyD88, MyD88/CD40, CD3e and PDGFRb intracellular Docket No.: 061250-559001WO signaling domains or transmembrane and intracellular signaling domains.
  • the functionality of each of the fusion proteins can be tested in the assay described above.
  • Example 12 Characterization of repeated antigen challenges and TGF ⁇ immune suppressive effects on immune cells expressing membrane tethered interleukins [0652] This example shows that both activation induced and constitutively expressed membrane tethered interleukins enhance antigen-dependent expansion of engineered immune cells upon repeated antigen challenges and partially overcome TGF ⁇ inhibition.
  • MSLN activator receptor alone (dark line solid square) (SEQ ID NO: 1169) 2) MSLN activator receptor, HLA-A*02 blocker receptor (SEQ ID NO: 1167) and B2M shRNA (dark line solid circle) 3) MSLN activator receptor, HLA-A*02 blocker receptor, B2M shRNA and mt- IL-18 in a single vector (FIG.
  • Primary T cells were transduced with a construct encoding the activator, blocker receptors and B2M shRNA, and a construct encoding the membrane tethered interleukin, except for mt-IL-18 (FIG.40A).
  • the activator and blocker receptors were expressed under the control of a EF1a promoter.
  • the membrane tethered IL-12 constructs were expressed under the control of a 6X NFAT promoter, whereas IL7R ⁇ dimer was expressed under the control of a EF1a promoter.
  • MSLN activator receptor, HLA-A*02 blocker receptor, B2M shRNA, and mt-IL-18 were constructed as a single vector (labeled as BAC) with mt-IL-18 at the 3’ of MSLN activator receptor separated by a self-cleaving peptide (2A peptide) (FIG. 40B), such that the expression of the blocker receptor, activator receptor and mt-IL-18 was controlled by an EF1a promoter.
  • MSLN(+)HLA-A*02(-) MS751 target cells were used in this experiment. [0654] The experiment was run similarly as in FIG.22.
  • the antigen challenge transfer step was repeated on day seven, eleven, fourteen, eighteen, and twenty-one with 10% of T cells from the previous well moved to a new well of MS751 cells (dark arrows). Cumulative expansion of immune cells was measured every time T cells were transferred to new targets. Analysis of the cumulative fold expansion shows that expression of any of the membrane tethered interleukins with the MSLN activator receptor, HLA- A*02 blocker receptor and B2M shRNA increases antigen dependent expansion of immune cells compared to immune cells only expressing the MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA (FIG. 40C).
  • MSLN activator receptor, HLA-A*02 blocker receptor and B2M shRNA and IL7Ra dimer resulted in less antigen dependent expansion than any of the immune cells expressing a membrane tethered interleukin, but more antigen dependent expansion compared to immune cells only expressing the MSLN activator receptor and HLA-A*02 inhibitor receptor.
  • TGF ⁇ has been shown to reduce T cell proliferation.
  • Immune cells engineered to Docket No.: 061250-559001WO express MSLN activator receptor, HLA-A*02 inhibitor receptor and B2M shRNA, and IL7Ra dimer (dark line with X) did not rescue antigen dependent expansion upon treatment with TGF ⁇ (FIG.40C).
  • This example shows that both activation induced and constitutively expressed membrane tethered interleukins enhance antigen-dependent expansion of engineered immune cells upon repeated antigen challenges when expressed in the dual receptor system described herein. Expression of IL7Ra dimer with MSLN activator receptor, HLA-A*02 inhibitor receptor and B2M shRNA also increases antigen-dependent expansion of engineered immune cells upon repeated antigen challenges.
  • Example 13 Development of Constitutively Active Interleukin Receptor Constructs [0657] This example describes the development of constitutively active interleukin constructs that, when compared to wildtype interleukin receptors, lead to changes in signaling properties. This example describes the development of a modified, constitutively active IL7 receptor. However, the present disclosure contemplates the use of any cytokine receptor, or domain thereof, that drives immune cell activation and survival.
  • IL7R ⁇ Native IL7 receptor alpha
  • IL7R ⁇ Native IL7 receptor alpha
  • binding of IL7 to the extracellular domains of both receptors results in dimerization which activates JAK1/Jak3 signaling to activate STA5 phosphorylation.
  • Stat5 is the primary downstream signaling node activated by the IL-7 receptor.
  • SEQ ID NO: 1172 To generate the constitutively active IL7R construct the IL7R ⁇ transmembrane and intracellular domain (SEQ ID NO: 1172) was fused to an extracellular protein tag (FIG.41A).
  • the extracellular tag can be any protein or fragment thereof that maintains the proper protein folding of the constitutively active interleukin receptor transmembrane and intracellular domains to which is to operatively linked.
  • the current experiments tested both an extracellular EGFR domain (ECD) (SEQ ID NO: 1170) and a CD33 ECD (SEQ ID NO: 1171) .
  • JNL cells were transduced with a construct encoding an HLA-A*02 blocker receptor and a CEA activator receptor. These JNL cells were further transduced with a second construct encoding the constitutively active IL7R ⁇ construct (FIG. 41A). Expression of the HLA-A*02 blocker receptor and the IL7R ⁇ construct was confirmed via FACs (FIG. 41B).
  • JNL cells were grown without the addition of exogenous IL2 and proliferation was measured on day two, day five, day seven, day ten and day thirteen. Analysis of cell count found the untransduced JNL cells and JNL cells expressing a HLA-A*02 blocker receptor and a CEA activator receptor only were dead by day two and day five respectively (FIG. 41C). Surprisingly, JNL cells expressing a CEA activator receptor, a HLA-A*02 blocker receptor and the constitutively active IL7R ⁇ construct continued to proliferate at day 13 (FIG.41C). This shows that the constitutively active IL7R ⁇ construct is functional and is capable of driving immune cell proliferation in the absence of exogenous IL2.
  • JNL cells were engineered to express a CEA activator receptor, a HLA-A*02 blocker receptor (SEQ ID NO: 1167) with or without the constitutively active IL7R ⁇ construct.
  • Engineered JNL cells were co-cultured with target cells that were CEA(+)HLA-A*02(-), target cells that were CEA(+)HLA-A*02(+), or targets that were CEA(- )HLA-A*02(-).
  • Engineered JNL cells and target cells were cultured at the indicated effector: target (E:T) cell ratios.
  • Co-culture of the engineered JNL cells with or without the constitutively active IL7R ⁇ construct with target cells that were CEA(+)HLA-A*02(-) resulted in nearly 100% specific killing of target cells.
  • Co-culture of the engineered JNL cells with or without the constitutively active IL7R ⁇ construct with target cells that were CEA(+)HLA-A*02(+) resulted in reduced killing of target cells (FIG.41D). The reduced killing of non-target cells is illustrated by the right shift of the curve.
  • JNL cells were engineered to express a MSLN activator receptor (SEQ ID NO: 1169), an HLA-A*02 blocker receptor (SEQ ID NO: 1167) with or without the constitutively active IL7R ⁇ construct. Expression of the HLA-A*02 blocker receptor and the IL7R ⁇ construct was confirmed via FACs (FIG.42A).
  • the engineered immune cells were then co-cultured with target cells that were either Docket No.: 061250-559001WO MSLN(+)HLA-A*02(-) or MSLN(+)HLA-A*02(+).
  • Engineered JNL cells and target cells were cultured at the indicated effector: target (E:T) cell ratios.
  • Co-culture of the engineered JNL cells with or without the constitutively active IL7R ⁇ construct with target cells that were MSLN(+)HLA-A*02(-) resulted in nearly 100% specific killing of target cells.
  • Co-culture of the engineered JNL cells with or without the constitutively active IL7R ⁇ construct with target cells that were MSLN(+)HLA-A*02(+) resulted in reduced killing of target cells (FIG. 42B). The reduced killing of non-target cells is illustrated by the right shift of the curve.
  • Embodiment 1 An engineered immune cell for logic-gated tumor cell killing, comprising: a) an inhibitory receptor specific to an inhibitor antigen, and b) a recombinant interleukin and/or a polynucleotide encoding said recombinant interleukin.
  • the immune cell of Embodiment 1, wherein the interleukin is an interleukin selected from an interleukin in Table 1 to Table 5, or a mutein thereof, optionally comprising a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a polypeptide sequence disclosed in Table 1 to Table 5. Docket No.: 061250-559001WO Embodiment 3.
  • the immune cell of Embodiment 1, wherein the interleukin is an interleukin 2 (IL- 2).
  • Embodiment 4 The immune cell of Embodiment 3, wherein the interleukin 2 (IL-2) has a C125A mutation relative to SEQ ID NO: 1.
  • Embodiment 6 The immune cell of Embodiment 1, wherein the interleukin is an interleukin 15 (IL-15).
  • Embodiment 6 wherein the IL-15 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 28) (human IL-15 no SS)).
  • Embodiment 8 The immune cell of Embodiment 1, wherein the interleukin is an interleukin 12 (IL-12).
  • IL-12 is a single-chain IL-12 comprising an IL-12 alpha (IL-12a) polypeptide segment and an IL-2 beta (IL-12b) polypeptide segment.
  • IL-12b polypeptide segment comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKE FGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAC PAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYP DTWSTP
  • Embodiment 11 The immune cell of Embodiment 9, wherein the single-chain IL-12 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKE FGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAC PAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYP DTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSS WSEWASVPCSGSGSSRGGSGSGGSGGGGSKRNLPVATPDPGMFPCLHHSQNL
  • Embodiment 12 The immune cell of Embodiment 1, wherein the interleukin is an interleukin 18 (IL-18).
  • Embodiment 13 The immune cell of Embodiment 12, wherein the IL-18 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to: MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQ GNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEM NPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDR SIMFTVQNED (SEQ ID NO: 29).
  • Embodiment 14 The immune cell of Embodiment 12, wherein the IL-18 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGM AVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEG YFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 30).
  • Embodiment 15 Embodiment 15.
  • Embodiment 16 The immune cell of Embodiment 12, wherein the IL-18 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGM AVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEG YFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDGGGGQGGGGQGGQGGQGGQGG GGQGG GGQGGQGG GGQGGQGGQGGGGPQIISFFLALTSTALLFLLFFLTLRFSVVKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 1047).
  • Embodiment 17 The immune cell of Embodiment 1, wherein the interleukin is an interleukin 21 (IL-21).
  • Embodiment 18 The immune cell of Embodiment 17, wherein the IL-21 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to: HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLL QKMIHQHLSSRTHGSEDS (SEQ ID NO: 32) (human IL-21).
  • Embodiment 19 Human IL-21.
  • Embodiment 20 The immune cell of Embodiment 19, wherein the tethered interleukin comprises a transmembrane domain, optionally a transmembrane domain selected from Table 8, or a functional variant thereof, optionally comprising a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a polypeptide sequence disclosed in Table 8.
  • Embodiment 21 Embodiment 21.
  • Embodiment 23 Embodiment 23.
  • the immune cell of Embodiment 21, wherein the hinge comprises an amino acid sequence set forth in SEQ ID NO: 33 ((G4Q)2), SEQ ID NO: 34 ((G4Q)5), SEQ ID NO: 35 ((G4Q)10), SEQ ID NO: 36 ((G4Q)10-CD25_full), SEQ ID NO: 37 (CD25_hinge), SEQ ID NO: 38 (IgG4), SEQ ID NO: 39 (EGF3), or SEQ ID NO: 40 (EGF7). Docket No.: 061250-559001WO Embodiment 24.
  • the immune cell of Embodiment 20 wherein the transmembrane domain comprises a PDGFRb, B7, CD25, or CD137 transmembrane domain.
  • Embodiment 25 The immune cell of Embodiment 20, wherein the transmembrane domain comprises an amino acid sequence set forth in SEQ ID NO: 55 (PDGFRb), SEQ ID NO: 56 (B7), SEQ ID NO: 57 (CD25), or SEQ ID NO: 1048 (CD137).
  • Embodiment 26 The immune cell of any one of Embodiments 1 to 25, wherein the interleukin further comprises a linker.
  • Embodiment 27. The immune cell of Embodiment 26, wherein the linker consists essentially of glycine (G) and glutamine (Q).
  • Embodiment 28 The immune cell of Embodiment 27, wherein the linker is a GS or a (G4Q)4 linker.
  • Embodiment 29 The immune cell of Embodiment 29.
  • Embodiment 30. The immune cell of any one of Embodiments Embodiment 19-29, wherein the tethered interleukin is a tethered IL-12.
  • Embodiment 31. The immune cell of Embodiment 30, wherein the tethered IL-12 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NOs: 64.
  • the immune cell of Embodiment 30, wherein the tethered interleukin is a tethered IL-2.
  • Embodiment 33. The immune cell of Embodiment 32, wherein the tethered IL-2 comprises a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NOs: 15-21.
  • Embodiment 34. The immune cell of any one of Embodiments 1-5, or 19-29, wherein the interleukin is an IL-2 (optionally a soluble IL-2 or a tethered IL-2) and wherein the IL-2 comprises a modification that decreases binding to CD25.
  • the immune cell of Embodiment 34 wherein the IL-2 comprises one or more amino acid substitutions C125A, G27C and F78C, E62Q, P65R, E62M, F42V, or R38D.
  • Embodiment 36 The immune cell of Embodiment 34 or Embodiment 35, wherein the interleukin is a fusion protein comprising: a) the IL-2 (optionally a soluble IL-2 or a tethered IL-2), and b) a CD25 or a fragment thereof. Docket No.: 061250-559001WO Embodiment 37.
  • Embodiment 38 The immune cell of Embodiment 19, wherein the tethered interleukin is a tethered IL-21.
  • Embodiment 39 The immune cell of any one of Embodiments 1 to 38, wherein the cell is a T cell.
  • Embodiment 40 The immune cell of Embodiment 39, wherein the T cell is a cytotoxic T cell.
  • Embodiment 41 The immune cell of any one of Embodiments 1 to 38, wherein the cell is a NK cell.
  • Embodiment 42 The immune cell of any one of Embodiments 1-41, wherein the interleukin acts in cis on an IL-2 receptor in the immune cell.
  • Embodiment 43 The immune cell of any one of Embodiments 1-41, wherein the interleukin acts in cis on an IL-2 receptor in the immune cell.
  • Embodiment 44. The immune cell of any one of Embodiments 1-43, wherein the inhibitory receptor selectively binds an inhibitor antigen, wherein binding of the inhibitor antigen by the inhibitory receptor inhibits activation of the immune cell.
  • Embodiment 45. The immune cell of Embodiment 44, wherein the inhibitor antigen is an HLA class I allele comprising HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.
  • Embodiment 46. The immune cell of Embodiment 45, wherein the inhibitor antigen is an HLA- A*02 allele.
  • Embodiment 48. The immune cell of any one of Embodiments 44 to 47, wherein the inhibitor antigen is not expressed by a cancer cell and is expressed by a non-cancerous cell.
  • Embodiment 49. The immune cell of any one of Embodiments 1-48, further comprising an activator receptor that selectively binds an activator antigen, wherein binding of the activator antigen by the activator receptor activates or promotes activation of the immune cell.
  • the immune cell of Embodiment 49 wherein the activator antigen is selected from the group consisting of transferrin receptor (TFRC), epidermal growth factor receptor (EGFR), CEA cell adhesion molecule 5 (CEA), CD19 molecule (CD19), erb-b2 receptor tyrosine kinase 2 (HER2), and mesothelin (MSLN), or a peptide antigen thereof. Docket No.: 061250-559001WO Embodiment 51.
  • the immune cell of Embodiment 50, wherein the activator antigen is selected from antigens in Table 9.
  • Embodiment 52 wherein the activator antigen is selected from antigens in Table 9.
  • Embodiment 53. A pharmaceutical composition, comprising a plurality of the immune cells of any one of Embodiments 1-52.
  • Embodiment 54. A method of killing a tumor cell comprising contacting the tumor cell with an immune cell of any one of Embodiments 1-52 or the pharmaceutical composition of Embodiment 53.
  • Embodiment 55 The method of Embodiment 54, wherein the immune cell with interleukin proliferates at least 10% more rapidly than immune cells without the interleukin.
  • Embodiment 54 wherein the immune cell with interleukin shows enhanced tumor cell killing compared to immune cells without the interleukin.
  • Embodiment 57 The method of Embodiment 54, wherein the immune cell with interleukin shows enhanced activation compared to immune cells without the interleukin.
  • Embodiment 58 A method of treating cancer, comprising administering to a subject a plurality of the immune cells of any one of Embodiments 1-52 or the pharmaceutical composition of Embodiment 53.
  • Embodiment 59 A kit comprising the immune cell of any one of Embodiments 1-52 or the pharmaceutical composition of Embodiment 53.

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Abstract

L'invention concerne des interleukines modifiées et des cellules immunitaires comprenant des interleukines modifiées. L'invention concerne également des compositions pharmaceutiques comprenant de telles interleukines modifiées, des vecteurs d'expression et des cellules hôtes pour la fabrication de telles interleukines modifiées et des procédés d'utilisation de telles interleukines modifiées et de telles cellules immunitaires comprenant des interleukines modifiées dans le traitement de cancers.
PCT/US2024/052114 2023-10-20 2024-10-18 Interleukines modifiées et utilisations associées Pending WO2025085846A1 (fr)

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WO2022040444A1 (fr) * 2020-08-20 2022-02-24 A2 Biotherapeutics, Inc. Compositions et méthodes de traitement de cancers egfr positifs
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