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WO2024251290A1 - Cellules immunitaires modifiées et leurs utilisations - Google Patents

Cellules immunitaires modifiées et leurs utilisations Download PDF

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Publication number
WO2024251290A1
WO2024251290A1 PCT/CN2024/098303 CN2024098303W WO2024251290A1 WO 2024251290 A1 WO2024251290 A1 WO 2024251290A1 CN 2024098303 W CN2024098303 W CN 2024098303W WO 2024251290 A1 WO2024251290 A1 WO 2024251290A1
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cell
peptide
cells
seq
domain
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Inventor
Yanni LIN
Shuyang He
Feifei GUO
Xiaocui Zheng
Rong Wang
Chuan FENG
Ying Shu
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Suzhou Cure Genetics Biosciences Co Ltd
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Suzhou Cure Genetics Biosciences Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4231—Cytokines
    • A61K40/4232—Tumor necrosis factors [TNF] or CD70
    • 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
    • A61K40/4261—Proteoglycans, e.g. glypican, brevican or CSPG4
    • 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]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/56—Kidney
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • This disclosure relates to engineered molecular biology, cell biology, and immuno-oncology.
  • CD70 is an inducible costimulatory molecule expressed on activated T cells, B cells, and dendritic cells. Its ligand, CD27, plays a critical role in the activation and survival of T cells, making the CD70-CD27 interaction pivotal for immune responses.
  • CD27 ligand that plays a critical role in the activation and survival of T cells, making the CD70-CD27 interaction pivotal for immune responses.
  • aberrant expression of CD70 has been observed in various malignancies, including renal cell carcinoma, non-Hodgkin lymphoma, and glioblastoma, among others. This aberrant expression makes CD70 a compelling target for cancer therapy, as it is associated with poor prognosis and contributes to tumor immune evasion.
  • the therapeutic potential of targeting CD70 lies in its restricted expression in normal tissues and high prevalence in tumors. There is an urgent need to develop safe and effective CD70-targeted cancer therapies due to the limited treatment options for cancers that express this antigen and the potential for such therapies to improve patient outcomes significantly.
  • Interleukin-15 is a cytokine that plays a crucial role in the activation and proliferation of natural killer (NK) cells and CD8+T cells, both of which are vital components of the immune system's response to tumors.
  • NK natural killer
  • CD8+T CD8+T cells
  • IL-15 has garnered significant attention for its potential to enhance anti-tumor immunity.
  • IL-15 therapy faces several limitations that hinder its widespread clinical application. The primary challenges include its short in vivo half-life and limited bioavailability . IL-15 therapeutic options with optimized bioavailability are urgently needed.
  • compositions and methods disclosed herein address these need and provide related advantages.
  • engineered natural killer T (NKT) cells expressing a chimeric antigen receptor (CAR) targeting CD70 wherein endogenous CD70 expression on cell surface of the NKT cell is reduced or eliminated.
  • the endogenous CD70 expression of the NKT cell is reduced or eliminated by gene editing.
  • the engineered NKT cell is CD70 negative.
  • the CAR targeting CD70 comprises a CD70-binding domain selected from the group consisting of an anti-CD70 scFv, an anti-CD70 VHH, a full-length or truncated CD27 peptide, and any combinations thereof.
  • the CD70-binding domain is an anti-CD70 scFv or an anti-CD70 VHH.
  • the anti-CD70 scFv or anti-CD70 VHH is humanized.
  • the anti-CD70 scFv comprises a light chain variable region (VL) and a heavy chain variable region (VH) , wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, and the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 2.
  • the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15.
  • the NKT cell also expresses an exogenous IL-15 peptide.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28.
  • the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28, with a N72D substitution.
  • the IL-15 peptide further comprises an IL-15R ⁇ peptide or a functional fragment thereof.
  • the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • the IL-15 peptide further comprises a sushi domain of IL-15R ⁇ .
  • the sushi domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • the IL-15 peptide is connected to the CAR through a self-cleaving peptide.
  • the self-cleaving peptide is a 2A peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26.
  • the IL-15 peptide comprises a signal peptide at its N-terminus.
  • the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 27 and 50-52.
  • the CAR targeting CD70 further comprises (1) a hinge domain; (2) a transmembrane domain; (3) a co-stimulatory domain; and/or (4) an intracellular signaling domain.
  • the engineered NKT cells comprise (1) the hinge domain of CD8, CD28, IgG1, or IgG4; (2) the transmembrane domain of CD8 ⁇ , CD28, CD3 ⁇ , or CD4; (3) the co-stimulatory domain of CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, FceRI ⁇ , BTLA, GITR, HVEM, CD2, NKG2C, LIGHT, or DAP12; and/or (4) the activation domain of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FceRI ⁇ , FceRI ⁇ , immunoglobulin ⁇ , immunoglobulin ⁇ , bovine leukemia virus gp30, EB virus LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus, or DAP-12,
  • the hinge domain is a CD8 hinge domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 19;
  • the transmembrane domain is a CD8 transmembrane domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 20;
  • the co-stimulatory domain is a 4-1BB co-stimulatory domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 21; and/or (4) the intracellular
  • the CAR targeting CD70 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-33.
  • NKT cells expressing a peptide having a CAR targeting CD70 connected to an IL-15 peptide through a self-cleaving peptide, wherein endogenous CD70 expression on cell surface of the NKT cell is reduced or eliminated, wherein the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-49 and 56-62.
  • the engineered NKT cells constitute at least 30%, at least 50%, or at least 70%of the cell population. In some embodiments, at least 90%, at least 70%, at least 50%, at least 30%, or at least 10%of NKT cells of the cell population have reduced CD70 expression. In some embodiments, at least 90%of the NKT cells of the cell population have reduced CD70 expression. In some embodiments, the engineered NKT cells are activated. In some embodiments, the cell population can persist in vivo for at least 7 days, at least 14 days, at least 21 days, at least 28 days, or at least 35 days. In some embodiments, the cell population can persist in vivo for at least 35 days.
  • provided herein are methods for preparing the engineered NKT cells disclosed herein, comprising sequentially performing the following steps: (i) reducing or eliminating the expression of endogenous CD70 of a NKT cell; and (ii) introducing into the NKT cell an expression construct encoding a CAR targeting CD70.
  • the engineered NKT cell has enhanced proliferation efficiency.
  • the interval between step (i) and step (ii) is at least 48 hours or at least 72 hours. In some embodiments, the interval between step (i) and step (ii) ranges between about 48 to about 72 hours.
  • step (i) is achieved based on the CRISPR-Cas based system, Base Editor, Prime Editor, CRISPRi, ZFN, zinc finger transcriptional repressor, TALEN, TALE repressor, meganuclease, mega-TAL, RNA-guided endonuclease, RNA editing system, ADAR, RNA interference, antisense oligonucleotides, antisense RNA, microRNA, siRNA, shRNA, or protein expression blocker (PEBL) .
  • CRISPR-Cas based system Base Editor, Prime Editor, CRISPRi, ZFN, zinc finger transcriptional repressor, TALEN, TALE repressor, meganuclease, mega-TAL, RNA-guided endonuclease, RNA editing system, ADAR, RNA interference, antisense oligonucleotides, antisense RNA, microRNA, siRNA, shRNA, or protein expression blocker (PEBL) .
  • compositions comprising the engineered NKT cells disclosed herein or the population of cells disclosed herein, and a pharmaceutically acceptable carrier.
  • provided herein are methods of promoting macrophage polarization towards the M1 type, comprising contacting a macrophage with the engineered NKT cells disclosed herein, the population of cells disclosed herein, or the pharmaceutical compositions disclosed herein.
  • provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the engineered NKT cells disclosed herein, the population of cells disclosed herein, or the pharmaceutical compositions disclosed herein. In some embodiments of the methods provided herein, further comprising administering an additional therapy to the subject. In some embodiments, the subject is a human.
  • provided herein are uses of the engineered NKT cells disclosed herein or the population of cells disclosed herein in cancer treatment. In some embodiments, provided herein are uses of the engineered NKT cells disclosed herein or the population of cells disclosed herein in the preparation of a medicament for treating cancer.
  • the cancer is renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung cancer, cervical cancer, breast cancer, ovarian cancer, colorectal cancer, endometrial cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, or mesothelioma, or metastatic cancers thereof.
  • the cancer is renal cell carcinoma or metastatic renal cell carcinoma, or lung cancer or metastatic lung cancer.
  • the renal cell carcinoma is refractory metastatic clear cell renal cell carcinoma.
  • the cancer is CD70 positive cancer.
  • the CAR targeting CD70 comprises (1) an anti-CD70 scFv comprising a VL and a VH, wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, and the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 2; or (2) an anti-CD70 VHH having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:
  • non-naturally occurring peptides comprising, from N terminus to C terminus, a signal peptide (SP) and an IL-15 peptide.
  • SP signal peptide
  • the SP is an IL-4 SP, an IgK SP, or a GM-CSF SP.
  • the SP is an IL-4 SP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50.
  • the SP is an IgK SP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 51.
  • the SP is a GM-CSF SP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 52.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28.
  • the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28, with a N72D substitution.
  • the IL-15 peptide further comprises an IL-15R ⁇ peptide or a functional fragment thereof.
  • the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • the IL-15 peptide further comprises a sushi domain of IL-15R ⁇ .
  • the sushi domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • the IL-15 peptide further comprises an Fc domain.
  • peptides provided herein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-55.
  • nucleic acids encoding the peptides disclosed herein.
  • vectors comprising the nucleic acids disclosed herein.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.
  • provided herein are genetically engineered cells expressing the peptides disclosed herein. In some embodiments, provided herein are genetically engineered cells comprising the nucleic acids disclosed herein, or the vectors disclosed herein.
  • the synthetic receptor is selected from the group consisting of a CAR, a TCR, a chimeric TCR, a TRuC, a TAC, an AbTCR, a STAR, and a chimeric CD3 ⁇ receptor.
  • the synthetic receptor is a CAR.
  • the synthetic receptor comprises an antigen-binding domain targeting a tumor antigen.
  • the tumor antigen is CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, Her3, GD2, MAGE, FOLR1, GPC3, Mesothelin, PSMA, ROR1, EGFR, MUC1, or NY-ESO-1.
  • the synthetic receptor is a CAR targeting CD70 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-33, a CAR targeting FOLR1 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 69, or a CAR targeting GPC3 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70.
  • the cell is an immune cell.
  • the immune cell is a leukocyte selected from the group consisting of a T cell, a NK cell, a NKT cell, a B cell, a plasma cell, a dendritic cell, a neutrophil, a monocyte, a macrophage, a granulocyte, a mast cell, a lymphocyte, a leukocyte, and a peripheral blood mononuclear cell.
  • the immune cell is a T cell, a NK cell, or a NKT cell.
  • compositions comprising the cells disclosed herein, or the nucleic acids disclosed herein, and a pharmaceutically acceptable carrier.
  • provided herein are methods of increasing IL-15 level in a subject in need thereof comprising administering a therapeutically effective amount of the cells disclosed herein, or the nucleic acids disclosed herein, or the pharmaceutical compositions disclosed herein to the subject.
  • methods of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the cells disclosed herein, or the nucleic acids disclosed herein, or the pharmaceutical compositions disclosed herein to the subject.
  • the subject is a human.
  • provided herein are uses of the cells disclosed herein or the nucleic acids disclosed herein in cancer treatment. In some embodiments, provided herein are uses of the cells disclosed herein or the nucleic acids disclosed herein for the preparation of a medicament for the treatment of cancer.
  • the cancer is a hematological cancer or a solid tumor.
  • FIG. 1 provides a schematic illustration of the structure of an Armored CAR-NKT cell.
  • FIG. 2 provides a schematic illustration of the structure of a CAR-IL-15 construct.
  • TM transmembrane domain
  • Co-stim co-stimulatory domain.
  • the CAR-IL-15 molecule includes a CD70 targeting CAR and a secretory IL-15 linked to the CAR through a 2A peptide, wherein the CD70 targeting CAR includes CD70 binding domain (e.g., an anti-CD70 scFv or VHH) , a hinge (e.g., a CD8 hinge domain) , a transmembrane domain (e.g., a CD8 transmembrane domain) , a co-stimulatory domain (e.g., a 4-1BB co-stimulatory domain) , a signaling domain (e.g., a CD3 ⁇ signal domain) .
  • CD70 binding domain e.g., an anti-CD70 scFv or VHH
  • a hinge e.g., a CD
  • FIG. 3 provides results of cell characterization analysis using flow cytometry at the endpoint of cell preparation, detecting NKT purity, CAR expression, CD4/CD8 ratio, and gene knockout rate.
  • FIG. 4 provides ELISA results showing the IL-15 levels in the supernatants collected during the preparation of CAR-NKT cells from two different healthy donors (labeled as 1172W and 2041W) , demonstrating efficient IL-15 secretion when co-expressed with CAR using different 2A peptides.
  • FIG. 5 provides results of flow cytometry showing high expression of CD70 on stimulated iNKT cells, with lower expression before stimulation.
  • FIG. 6 shows the cell counts of NKT, CD70 targeting CAR-NKT (CD70 CAR) , and CD19 targeting CAR-NKT (CD19 CAR) .
  • CD70 CAR-NKT cells prepared without CD70 knockout failed to expand.
  • FIGs. 7A-7B provide results showing CD70%in NKT cells at different time points after stimulation (FIG. 7A) , and CD70 expression on day 8 after stimulation, wherein the NKT cells had either no CD70 knockout (first row) or CD70 knockout (second row) (FIG. 7B) .
  • the NKT cells were prepared from three healthy donors. 02W-NKT: XW0102002W-NKT; 23W-NKT: XC11123W-NKT; 19W-NKT: XC11119W-NKT.
  • FIGs. 8A-8C provide results showing the effect of CD70 knockout and the efficiency of knockout.
  • FIG. 8A shows the fold increase in the number of CAR-NKT cells between day 3 and day 5 after virus transduction with CAR-IL-15 molecules targeting CD70, either with CD70 gene knockout (KO-CAR) or without knockout (NKT-CAR) . Cells were from three healthy donors.
  • FIG. 8B shows the knockout efficiency (where high efficiency was reflected by low CD70 expression) at different time points after electroporation at different times (e.g., D4 K1: electroporation knockout on day 4 of culture, 1 day after electroporation) .
  • FIG. 8A shows the fold increase in the number of CAR-NKT cells between day 3 and day 5 after virus transduction with CAR-IL-15 molecules targeting CD70, either with CD70 gene knockout (KO-CAR) or without knockout (NKT-CAR) . Cells were from three healthy donors.
  • FIG. 8B shows the knockout efficiency
  • 8C shows the knockout efficiency for cells from another healthy donor, wherein the electroporation was conducted on day 6 (D6) of culture using different voltages.
  • the knockout efficiency was measured at different time points after electroporation (e.g., K1: 1 day after electroporation) .
  • FIGs. 9A-9D provide results showing in vitro cytotoxic activity and cytokine secretion of Armored CAR-NKT cells.
  • FIG. 9A shows the killing of double negative target cells 786-O-- (CD70-CD1d-) by NKT and CD70 CAR-NKT at different effector to target ratios (E: T) .
  • FIG. 9B shows the killing of double positive target cells 786-O++ (CD70+CD1d+) at different E: T ratios.
  • FIGs. 9C-9D show the levels of IFN- ⁇ (FIG. 9C) and IL-15 (FIG. 9D) detected in the supernatant of the NKT or CAR-NKT cells after co-culture with target cells or culture alone (NC) .
  • FIG. 10A-10B provide results showing tumor inhibition effects of Armored CAR-NKT cells in mice bearing 786-O tumors.
  • FIG. 10A shows changes in tumor volume over time after intravenous infusion of different doses of Armored CAR-NKT, compared to PBS and NKT cells.
  • FIG. 10B shows tumor volume in different study groups on day 54 after infusion of Armored CAR-NKT cells.
  • FIG. 11 provides results showing the levels of IFN- ⁇ in the serum of mice from different study groups at various time points (7/21/35/49 days after infusion of Armored CAR-NKT cells) .
  • FIG. 12 provides results showing the proportion of NKT cells in the blood samples of mice from different study groups at various time points (7/14/21/28/35 days after infusion of Armored CAR-NKT cells) , where the high-dose group of CAR-NKT cells showed significant expansion and long persistence time in mice (more than 35 days) .
  • FIG. 13 provides results showing changes in mouse body weight during the study.
  • FIG. 14A-B provide results showing in vitro killing and secretion of cytokine of Armored CAR-NKT cells.
  • FIG. 14A shows the killing of target cells 786-O++and 786-O--by CAR-NKT cells with different anti-CD70 antigen-binding domains. The effector cells and target cells were co-cultured for 24 hours before evaluating target cell killing.
  • FIG. 14B shows the concentrations of IFN- ⁇ , IL-15, IL-6, Granzyme B, IL-2, IL-10, GM-CSF, TNF- ⁇ , and IL-4 in the supernatant after co-culturing effector cells with target cells at a 1: 1 ratio for 48 hours.
  • FIGs. 15A-C provide results showing in vitro cytotoxicity (FIG. 15A) and cytokine secretion (FIGs. 15B-15C) of CAR-NKT cells with different CD70 antigen-binding domains against CD70+lung cancer cell line NCI-H460.
  • FIGs. 16A-C provide results showing in vitro cytotoxicity (FIG. 16A) and cytokine secretion (FIGs. 16B-16C) of CAR-NKT cells with different CD70 antigen-binding domains against CD70+ovarian cancer cell line SK-OV-3.
  • FIG. 17 provides results showing changes in tumor volume in mice bearing 786-O tumors over time after intravenous infusion of different CD70 CAR-IL-15 NKT cells containing different CD70 antigen binding domains, compared to PBS and NKT control groups. Two dosages were used, respectively 5x10 ⁇ 6 cells/mouse (5E6) and 1x10 ⁇ 7 cells/mouse (1E7) .
  • FIG. 18 provides flow cytometry results showing number of human iNKT cells with different CD70 antigen-binding domains in mouse blood on day 7 after treatment.
  • FIG. 19 provides flow cytometry results showing persistence of CD70 CAR-positive NKT cells with different CD70 antigen-binding domains in mouse blood after treatment.
  • FIG. 20 provides results showing changes in mouse body weight during the in vivo experiment with CD70 CAR-NKTs.
  • FIG. 21 provides results showing changes in mouse food consumption during the in vivo experiment with CD70 CAR-NKTs.
  • FIG. 22 provides results showing CAR copy numbers in different tissues/organs of the mice at the endpoint of the in vivo experiment with CD70 CAR-NKTs.
  • FIGs. 23A-B provide results showing persistence of CD70 CAR-NKT cells expressing IL-15 or not.
  • FIG. 23A shows changes in the cell viability of CAR-NKT wo IL-15, CAR-NKT-IL-15, and negative control NKT cultured in cytokine-free medium for 6 days.
  • the positive control groups labeled “0.5 ng/ml IL15” and “5 ng/ml IL15” were CAR-NKT wo IL-15 cells with 0.5 ng/ml or 5 ng/ml recombinant IL-15 protein added to the culture medium, respectively.
  • FIG. 23B shows cell expansion of CAR-NKT wo IL-15, CAR-NKT-IL-15, and CAR-T when co-cultured with Raji cells. The Raji cells were added on day 0 and day 6.
  • FIGs. 24A-F provide results showing in vivo tumor infiltration capacity of Armored CAR-NKT cells.
  • FIG. 24A shows changes in tumor volume over time after the mice bearing 786-O tumors received intravenous infusion of different doses of Armored CAR-NKT. PBS and NKT cells were used as control.
  • FIG. 24B shows survival curves of mice after infusion of the Armored CAR-NKT cells.
  • FIG. 24C shows the number of CAR-NKT cells in the peripheral blood of mice at different time points after the treatment of Armored CAR-NKT cells.
  • FIGs. 24D-E show the total number of CAR copies in the DNA extracted from various organs of the mice (FIG. 24D) and the proportion of CAR copy numbers in different organs within the mice (FIG. 24E) .
  • FIG. 24F shows the proportion of human CD45 positive cells in the tumor (left) and peripheral blood (right) of the mice.
  • FIGs. 25A-C provide results showing the cytotoxicity of Armored CAR-NKT co-cultured with 786-O++ (FIG. 25A) , Jurkat (FIG. 25B) , and K562 (FIG. 25C) cells.
  • FIG. 26 provides results showing the polarization effect of T, NKT, and Armored CAR-NKT on macrophages.
  • FIGs. 27A-B provide results of flow cytometry showing enhanced stemness of Armored CAR-NKT cells.
  • FIG. 27A shows changes in the proportion of CD27-positive cells in CAR-NKT (left) and changes in the proportion of CD27+CD28+double-positive cells in CAR-NKT (right) .
  • FIG. 27B shows changes in the proportion of CCR7-positive cells in CD27-positive CAR-NKT (left) and changes in the proportion of CCR7-positive cells in CD27-negative CAR-NKT (right) .
  • FIG. 28 provides results showing cell expansion kinetics in peripheral blood at different time points after infusion of Armored CAR-NKT in a clinical study for treating clear cell renal cell carcinoma (ccRCC) patients.
  • VCN vector copy number
  • LOD limit of detection
  • LLOQ lower limit of quantification.
  • FIGs. 29A-D provide results showing the change over time in the proportion of CD70+cells in T cells in three patients, 0101 (FIG. 29A) , 0102 (FIG. 29B) , and 0103 (FIG. 29C) , as well as the corresponding changes in vector copy number (VCN) at respective time points, and the number of peripheral-blood CD70-positive T cells before lymphodepletion (LD) and after cell therapy at the latest time points (FIG. 29D) .
  • W week.
  • FIGs. 30A-B provide results showing characterization of CAR-NKT cells before and after infusion.
  • FIG. 30A shows the proportion of CD27+cells in CAR-NKT cells in drug product (DP) and in patient peripheral blood at the time of maximum CAR transgene copies (PB, Tmax) , reflecting significantly increased expression of CD27 in Armored CAR-NKT after infusion.
  • FIG. 30B shows the memory phenotypes of CD27+and CD27-CAR-NKT cells in peripheral blood of patient 0106 on day 12 post drug infusion.
  • FIG. 31 provides ELISA results showing IL-15 concentrations in supernatant during CD70 CAR-IL-15 NKT manufacture from two healthy donors labeled 1172W and 2041W, wherein different signal peptides (SP) were used in the IL-15 expression sequence.
  • SP signal peptides
  • FIG. 32 provides ELISA results showing IL-15 secretion when CD70 CAR-IL-15 NKTs were co-cultured with CD70+target tumor cell lines or alone (CAR-NKT only control) , wherein different SPs were used in the IL-15 expression sequence. NKTs were used as control group.
  • FIG. 33 provides ELISA results showing IL-15 concentrations in supernatant during FOLR1 CAR-IL-15 NKT manufacture, wherein different SPs were used in the IL-15 expression sequence.
  • FIG. 34 provides ELISA results showing IL-15 secretion when FOLR1 CAR-IL-15 NKTs were co-cultured with FOLR1+ (SKOV3) , FOLR1- (SKOV3-FOLR1 KO) target tumor cell lines or alone (Control) , wherein different SPs were used in the IL-15 expression sequence. NKT cells were used as control group.
  • FIG. 35 provides ELISA results showing IL-15 concentrations in supernatant during GPC3 CAR-IL-15 T manufacture, wherein different SPs were used in the IL-15 expression sequence.
  • FIG. 36 provides ELISA results showing IL-15 secretion when GPC3 CAR-IL-15 Ts were co-cultured with GPC3+ (HEPG2 and PLC/PRF/5) or GPC3- (786-O++) target tumor cell lines or alone (Control) , wherein different SPs were used in the IL-15 expression sequence. T cells were used as control group.
  • FIG. 37 provides results showing changes in tumor volume in mice bearing 786-O tumors over time after intravenous infusion of CD70 CAR-IL-15 NKTs carrying different SPs for IL-15, compared to PBS and NKT control groups.
  • FIGs. 38A-C provide results showing in vivo persistence of FOLR1 CAR-IL-15 NKTs carrying different SPs for IL-15 in tumor-free mice (FIG. 38A) or mice bearing SK-OV-3 tumors (FIG. 38B) and plasma IL-15 in mice bearing SK-OV-3 tumors (FIG. 38C) .
  • CD70 is a member of the tumor necrosis factor (TNF) family.
  • the term “CD70 peptide” as used herein include the functional variants and/or fragments of wildtype full length CD70, which can be of natural origin or synthetically made.
  • Exemplary CD70 genes can be found in Genebank under accession number: NM_001252.5.
  • An exemplary amino acid sequence of CD70 protein can be referred to in UniProtKB/Swiss-Prot: P32970.2.
  • CD70 expressing means that the cell has detectable CD70 expression. In some embodiments, the cell has detectable CD70 expression on its surface.
  • CD70-expressing refers to a cancer or tumor having cells with detectable CD70 expression.
  • Aperson of ordinary skill in the art can readily determine whether a cancer or tumor has CD70 expression using any methods known and available in the art, including, for example, immunohistochemistry (IHC) , immunocytochemistry (ICC) , an enzyme-linked immunosorbent assay (ELISA) , flow cytometry (FACS) , etc.
  • IHC immunohistochemistry
  • ICC immunocytochemistry
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • CD27 is the ligand for CD70, which is also a member of the TNF super family.
  • the full-length amino acid sequence of human CD27 protein can be found, for example, in UniProtKB/Swiss-Prot: P26842.2 (SEQ ID NO: 29) .
  • the term “CD27 peptide” as used herein include the functional variants and/or fragments of wildtype full length CD27, which can be of natural origin or synthetically made.
  • a truncated CD27 peptide only includes the extracellular domain of CD27, for example, the amino acid fragment from position 20 to 191 of SEQ ID NO: 29.
  • Interleukin-15 is a T cell growth factor, primarily produced by monocytes and macrophages, and IL-15 mRNA is expressed in tissues such as the heart, lung, kidney, especially the placenta, and muscle.
  • the main mechanism of the IL-15 signaling pathway is that IL-15 binds to the IL-15R ⁇ subunit on the cell, IL-15 remains on the cell surface, forms an immunological synapse with the nearby effector NK cells or T cells'IL-2R/IL-15R ⁇ - ⁇ c, activating JAK1/JAK3 and STAT3/STAT5 pathways, Syk kinase and phospholipase C (PLC) ⁇ , Lck kinase and Shc, leading to the activation of PI3K/Akt and Ras/Raf/MAPK signaling cascades.
  • PLC phospholipase C
  • the full-length amino acid sequence of human IL-15 can be referred to in UniProtKB/Swiss-Prot: P40933.1.
  • the amino acid sequence of mature human IL-15 is shown as SEQ ID NO: 28 (corresponding to the amino acid fragment from position 49 to 162 in UniProtKB/Swiss-Prot: P40933.1) .
  • the amino acid sequence of the signal peptide of wild-type human IL-15 is shown as SEQ ID NO: 27 (corresponding to the amino acid fragment from position 1 to 29 in UniProtKB/Swiss-Prot: P40933.1) .
  • IL-15 peptide as used herein include the functional variants and/or fragments of wildtype full length IL-15, which can be of natural origin or synthetically made.
  • An IL-15 peptide can also include a signal peptide.
  • An IL-15 peptide is typically secretory, meaning that it is produced and released into the extracellular environment by cells. This secretion process can involve transport of IL-15 through the endoplasmic reticulum and Golgi apparatus, and finally its release outside the cell.
  • IL-15R ⁇ peptide as used herein include the functional variants and/or fragments of wildtype full length IL-15R ⁇ , which can be of natural origin or synthetically made.
  • the IL-15R ⁇ peptide can be the sushi domain of a full-length IL-15R ⁇ or a function variant thereof.
  • An IL-15R peptide can also include a signal peptide.
  • a “signal peptide” or “SP” refers to a short, typically 15-30 amino acid long sequence found at the N-terminus of a newly synthesized protein, whose primary function is to direct the nascent protein to the secretory pathway, which includes the endoplasmic reticulum (ER) and, subsequently, the Golgi apparatus, for secretion out of the cell or for integration into cellular membranes.
  • the secretory pathway which includes the endoplasmic reticulum (ER) and, subsequently, the Golgi apparatus, for secretion out of the cell or for integration into cellular membranes.
  • ER endoplasmic reticulum
  • Golgi apparatus the Golgi apparatus
  • peptide, ” “polypeptide, ” “protein, ” and their grammatical equivalents as used interchangeably herein refer to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids.
  • a peptide, polypeptide, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • nucleotide e.g., DNA, RNA, DNA, and RNA.
  • nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • variant refers to a different protein or peptide having one or more (such as, for example, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to the reference protein or reference peptide.
  • the changes to an amino acid sequence can be amino acid substitutions.
  • the changes to an amino acid sequence can be conservative amino acid substitutions.
  • the changes to an amino acid sequence can be amino acid deletions.
  • a variant can be a fragment of the reference protein or peptide.
  • a functional variant of a protein or peptide maintains the basic structural and functional properties of the reference protein or peptide.
  • amino acids can be divided into classes based on common side chain properties, including 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and aromatic: Trp, Tyr, Phe.
  • Conservative amino acid substitutions can involve the exchange of a member of one of these classes with another member of the same class.
  • non-conservative substitutions can involve the exchange of a member of one of the above classes for a member from another class.
  • amino acid residues can also encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. Synthetic, rare, or modified amino acid residues having known similar physiochemical properties to those of an above-described grouping can be used as a “conservative” substitute for a particular amino acid residue in a sequence. For example, a D-Arg residue may serve as a substitute for a typical L-Arg residue.
  • a substitution with a small and hydrophobic residue means substituting one amino acid with a residue (s) that is found in both of the above-described classes or other synthetic, rare, or modified residues that are known in the art to have similar physiochemical properties to such residues meeting both definitions) .
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art.
  • two nucleic acids or polypeptides provided herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • vector refers to a vehicle that is used to carry genetic material (e.g., a nucleic acid sequences) , which can be introduced into a host cell, where it can be replicated and/or expressed.
  • vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
  • Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • the term “synthetic receptor” refers to an engineered cell surface protein or protein complex comprising (1) a target-binding domain (or antigen-binding domain) that can specifically bind a target molecule, and (2) a functional domain that can activate a signaling pathway in the engineered cell.
  • the target-binding domain comprises an extracellular domain.
  • the functional domain comprises an intracellular domain.
  • the synthetic receptor further includes a transmembrane sequence.
  • the synthetic receptor can be a protein complex that comprises proteins expressed from exogenous nucleic acids.
  • the synthetic receptor can also be a protein complex that comprises at least one protein that is exogenously expressed, and at least one protein that is endogenously expressed.
  • the engineered cell is typically an immune cell, such as a T cell, a national killer (NK) cell, a B cell, a macrophage, etc., and the functional domain can activate the immune cell, either directly or indirectly.
  • exemplary synthetic receptors include, such as,chimeric antigen receptors ( “CAR” ) , T cell receptors ( “TCR” ) , chimeric TCRs, TCR receptor fusion constructs ( “TRuC” ) , T cell antigen couplers ( “TAC” ) , antibody TCR receptors ( “AbTCR” ) , synthetic T cell Receptor and Antigen Receptors ( “STAR” ) and chimeric CD3 ⁇ receptors.
  • CAR chimeric antigen receptors
  • TCR T cell receptors
  • TCR receptor fusion constructs “TRuC” )
  • T cell antigen couplers “TAC”
  • antibody TCR receptors “AbTCR”
  • STAR synthetic T cell Receptor and Antigen
  • CAR Chimeric Antigen Receptor
  • ACAR typically includes an antigen-binding domain (e.g., tumor-specific antigens and/or tumor-associated antigens) , a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.
  • antigen-binding domain e.g., tumor-specific antigens and/or tumor-associated antigens
  • transmembrane domain when used in connection with a synthetic receptor (e.g., a CAR) refers to the structural domain that traverses the cell membrane, connecting to the intracellular signaling domain, playing a role in signal transmission.
  • hinge or “hinge domain” when used in connection with a synthetic receptor (e.g., a CAR) refers to the connecting region between the antigen-binding domain and the transmembrane region.
  • co-stimulatory domain when used in connection with a synthetic receptor (e.g., a CAR) refers to the intracellular structural domain that can provide co-stimulatory signals, which are necessary for an effective lymphocyte response to antigens.
  • intracellular signaling domain when used in connection with a synthetic receptor (e.g., a CAR) refers to the structural domain located inside the cell that can transmit signals for initiating T cell activation upon antigen recognition.
  • CAR-expressing T cells are referred to as “CART” or “CAR-T” cells.
  • CAR expressing NKT cells are referred to as “CAR NKT” or “CAR-NKT” cells.
  • target binding domain or “antigen binding domain” refers to a domain that specifically binds to the target or the antigen.
  • the target binding domain or antigen binding domain can be an antibody, a ligand, or a different moiety that specifically binds to the target or antigen.
  • the target binding domain or antigen binding domain can be of natural origin, synthetic origin, semi-synthetic origin, or recombinant origin.
  • CD70 binding domain can be an anti-CD70 antibody or an antigen-binding fragment thereof, a CD70 ligand (e.g., a CD27 peptide) , or a different moiety that can specifically bind to CD70, such as those described in, e.g., Olaleye et al., Biomolecules 2021, 11 (12) , 1791; Simeon&Chen, Protein &Cell 2018, volume 9, pages 3–14, incorporated herein by reference.
  • a CD70 ligand e.g., a CD27 peptide
  • a cell that is engineered to “target” an antigen, protein or any other target molecule means that the cell is engineered to express a target binding domain or antigen binding domain that specifically binds the antigen, protein or target molecule.
  • a binding moiety e.g. antibody
  • a target molecule e.g. antigen
  • a binding moiety that specifically binds a target molecule can be identified, for example, by immunoassays, ELISAs, SPR (e.g., Biacore) , or other techniques known to those of skill in the art.
  • a specific reaction will be at least twice background signal or noise and can be more than 10 times background.
  • a binding moiety that specifically binds a target molecule can bind the target molecule at a higher affinity than its affinity for a different molecule.
  • an antibody typically refers to a peptide molecule that can specifically recognize and/or neutralize a specific antigen.
  • an antibody may include at least two heavy (H) chains and two light (L) chains composed of immunoglobulin, interconnected by disulfide bonds, and includes any molecule containing its antigen-binding portion.
  • the term “antibody” includes monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies (e.g., dAb) , single-chain antibodies (e.g., scFv) , and antigen-binding antibody fragments (e.g., Fab, Fab', and (Fab) 2 fragments) .
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, and any antigen-binding antibody fragments and their derivatives described in this application.
  • Each heavy chain can consist of a variable region of the heavy chain (VH) and a constant region of the heavy chain.
  • Each light chain can consist of a variable region of the light chain (VL) and a constant region of the light chain.
  • VH and VL regions can further be distinguished into highly variable regions known as complementarity-determining regions (CDRs) , which are interspersed with more conserved regions called framework regions (FRs) .
  • CDRs complementarity-determining regions
  • Each VH and VL can consist of three CDRs and four FR regions, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • single-chain antibody can be composed of the variable region of the heavy chain and the variable region of the light chain of said antibody, or comprise antibody that is connected by a linker.
  • VHH refers to the single antigen-binding fragment of heavy-chain-only antibodies (HcAb) produced by camelid animals (such as llamas, alpacas) or sharks.
  • VHH or nanobodies are single variable regions of heavy chains, consisting of 3 highly variable regions (CDR1, CDR2, CDR3) and 4 framework regions (FR1, FR2, FR3, and FR4) that separate the highly variable regions.
  • Immune effector cells refer to cells that are of hematopoietic origin and play a direct role in the immune response against a target, such as a pathogen, a cancer cell, or a foreign substance.
  • Immune effector cells include, but are not limited to, T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, basophils, lymphocytes, leukocytes, and peripheral blood mononuclear cells (PBMCs) .
  • NKT cells refer to lymphoid lineage cells that possess both NK cell and T cell physiologic and functional characteristics. Morphologically, NKT cells are very similar to conventional T cells. Anatomically, a small number of NKT cells are found in almost all locations where T and NK cells are found, including peripheral blood, spleen, liver, thymus, bone marrow, and lymph nodes. A subset of NKT cells that carry a so-called “semi-invariant” ⁇ TCR, which recognizes glycolipid antigens presented by CD1d, are referred to as invariant NKT ( “iNKT” ) .
  • NKT cells In humans, the TCR of NKT cells almost always contains V ⁇ 24/J ⁇ 18, paired with a TCR ⁇ chain containing V ⁇ 11. Many (but not all) subsets of mouse NKT cells express the NK co-stimulatory molecule NK1.1, while many human NKT cells express the human homolog of NK1.1, NKR-P1c (CD161c) . NKT cells in both species also express NK inhibitory receptors. For example, human NKT cells express both KIRs and the inhibitory CD94/NKG2 receptors, while mouse NKT cells express members of the Ly-49 family. Human NKT cells also express the activating NK receptor NKG2D.
  • NKT cells used as cell therapy are often “activated” or “stimulated” in vitro before administration, which helps ensure that the cells are in an optimal state for therapeutic efficacy.
  • Common methods for activating NKT cells in vitro use, for example, glycolipid antigens, such as ⁇ -Galactosylceramide ( ⁇ -GalCer) , cytokines, such as IL-2, IL-15, and/or IL-12, or antigen-presenting cells (APCs) , such as dendritic cells (DCs) or monocytes.
  • glycolipid antigens such as ⁇ -Galactosylceramide ( ⁇ -GalCer)
  • cytokines such as IL-2, IL-15, and/or IL-12
  • APCs antigen-presenting cells
  • DCs dendritic cells
  • a population of cells that “consisting essentially of” NKT cells refers to a group of cells that predominantly comprises NKT cells, but may include other cells as long as they do not materially affect the fundamental characteristics of the NKT cell population. This means that while the primary components of the population are NKT cells, the presence of a small number of other cell types that do not significantly alter the function or properties of the NKT cells is permissible.
  • Permissible additional cells include, but are not limited to, supporting cells, such as antigen-presenting cells (APCs) that do not outnumber or functionally dominate the NKT cells, and which support the activation and function of NKT cells, minor contaminants, such as small numbers of other lymphocytes or immune cells that do not significantly impact the primary activities or therapeutic potential of the NKT cells, etc.
  • supporting cells such as antigen-presenting cells (APCs) that do not outnumber or functionally dominate the NKT cells, and which support the activation and function of NKT cells, minor contaminants, such as small numbers of other lymphocytes or immune cells that do not significantly impact the primary activities or therapeutic potential of the NKT cells, etc.
  • APCs antigen-presenting cells
  • exogenous and its grammatical equivalents as used herein are intended to mean that the referenced molecule is introduced into the host cell.
  • the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid.
  • the term refers to introduction of the encoding nucleic acid in an expressible form into the cell.
  • endogenous and its grammatical equivalents as used herein refer to a referenced molecule that is naturally present in the host cell.
  • the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid naturally contained within the cell.
  • genetic engineering or its grammatical equivalents when used in reference to a cell is intended to mean alteration of the genetic materials of the cell that is not normally found in a naturally occurring cell. Genetic alterations include, for example, modifications introducing expressible nucleic acids, other nucleic acid additions, nucleic acid mutations/alterations, nucleic acid deletions and/or other functional disruption of the cell’s genes. Such modifications can be done in, for example, coding regions and functional fragments thereof of a gene. Additional modifications can be done in, for example, non-coding regulatory regions in which the modifications alter expression of a gene.
  • Cas protein also known as “CRISPR associated protein” generally refers to a class of enzymes that can use CRISPR sequences as guides to recognize and cleave specific DNA strands.
  • Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12) , Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Cs
  • Cas9 protein or “Cas9 nuclease, ” also known as Csn1 or Csx12, typically refers to a class of proteins in type II CRISPR/Cas systems that are involved in both crRNA biogenesis and the destruction of invasive DNA.
  • Cas9 proteins generally include the RuvC nuclease domain and the HNH nuclease domain, each cleaving different strands of the double-stranded DNA molecule.
  • Cas9 proteins have been described in different bacterial species such as Streptococcus thermophiles, Listeria innocua, and Streptococcus pyogenes.
  • Streptococcus pyogenes Cas9 protein its amino acid sequence can be referred to in SwissProt under accession number Q99ZW2; Neisseria meningitides Cas9 protein, its amino acid sequence seen in UniProt under entry number A1IQ68; Streptococcus thermophilus Cas9 protein, its amino acid sequence seen in UniProt under entry number Q03LF7; Staphylococcus aureus Cas9 protein, its amino acid sequence seen in UniProt under entry number J7RUA5.
  • the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a material that is suitable for drug administration to an individual along with an active agent without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.
  • the pharmaceutical compositions disclosed herein can comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof.
  • a buffer system a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof.
  • preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 19 th edition, 1995.
  • treat and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action, intervention and/or measure that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated.
  • administer and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art.
  • the therapeutic can be a compound, a polypeptide, an antibody, an antibody-drug conjugate, or a cell.
  • Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject.
  • Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV) , intramuscular (IM) , or intraperitoneal (IP) ; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.
  • oral dosage forms such as tablets, capsules, syrups, suspensions
  • injectable dosage forms such as intravenous (IV) , intramuscular (IM) , or intraperitoneal (IP)
  • transdermal dosage forms including creams, jellies, powders, or patches
  • buccal dosage forms inhalation powders, sprays, suspensions, and rectal suppositories.
  • an effective amount refers to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject.
  • an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects.
  • subject refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • a human subject who needs the treatment may be a human subject having, at risk for, or suspected of having a disease.
  • a subject having a disease can be identified by routine medical examination, e.g., a physical examination, alaboratory test, an organ functional test, a CT scan, or an ultrasound.
  • a subject suspected of having any of such a disease can show one or more symptoms of the disease.
  • a subject at risk for the disease can be a subject having one or more of the risk factors for that disease.
  • a subject can be a human.
  • a subject can have a particular disease or condition.
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .
  • the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
  • the term “about” encompasses the exact number recited.
  • “about” means within plus or minus 10%of a given value or range.
  • “about” means that the variation is within a range of0.5%to 10%above or below a specified value, for example, within a range of0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%above or below the specified value.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • nucleotides, nucleic acids, nucleosides, and amino acids used herein is consistent with International Union of Pure and Applied Chemistry (IUPAC) standards (see, e.g., bioinformatics. org/smsylupac. html) .
  • IUPAC International Union of Pure and Applied Chemistry
  • Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOS. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to Uniprot (https: //www. uniprot. org/) , GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • CD70 is a ligand of the tumor necrosis factor (TNF) superfamily, whose expression is strictly regulated under physiological conditions: it is transiently expressed only on antigen-activated T cells and B cells, mature dendritic cells, and a unique group of antigen-presenting cells. Its unique receptor, CD27, plays a role in the co-stimulatory pathway, leading to signals for proliferation, differentiation, and survival.
  • TNF tumor necrosis factor
  • CD27 is a member of the TNF receptor superfamily and is constitutively expressed on naive T cells, memory B cells, NK cells, hematopoietic stem cells (HSC) , and progenitor cells.
  • CD27 is a transmembrane phosphoglycoprotein expressed on CD4+and CD8+T cells, its expression increases upon T cell activation, and it sheds from the cell surface after activation to form soluble CD27 (sCD27) .
  • CD70 (CD27L) is the only ligand for CD27.
  • CD27 Upon binding to CD70, CD27 associates with TNF receptor-associated factors (TRAFs) , producing intracellular signals that enhance the survival and activation of T, B, and NK cells through the activation of Traf2 and Traf5 signaling as well as the NF- ⁇ B pathway.
  • TNF receptor-associated factors Upon binding to CD70, CD27 associates with TNF receptor-associated factors (TRAFs) , producing intracellular signals that enhance the survival and activation of T, B, and NK cells through the activation of Traf2 and Traf5 signaling as well as the NF- ⁇ B pathway.
  • CD27 signaling can either enhance T cell function or lead to T cell dysfunction.
  • Overexpression of CD70 on tumor cells has been described in a variety of hematological malignancies and cancers. It has been reported that CD70 is expressed in primary and metastatic tumor resections of renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung cancer, cervical cancer, breast cancer, ovarian cancer, and mesothelioma, and is associated with reduced survival rates.
  • CD70 In metastatic specimens of lung cancer, pancreatic cancer, and osteosarcoma, the expression of CD70 is even higher, indicating the importance of CD70 in disease progression (Flieswasser et al., Cancers (Basel) . 2019 Oct 22; 11 (10) : 1611) . Therefore, therapies targeting CD70 have significant potential in combating both early and late-stage cancers. For instance, in phase I clinical trials using CD70-targeting antibodies, a response rate of 92%was observed in patients with acute myeloid leukemia (Riether et al., Nat. Med. 2020; 26 (9) : 1459-1467) . ADC (antibody-drug conjugate) compounds targeting CD70 have been developed and are undergoing clinical evaluation for hematological tumors and solid tumors.
  • ADC antibody-drug conjugate
  • TME Tumor heterogeneity and suppressive tumor microenvironment
  • engineered NKT cells expressing a CD70-targeting CAR, wherein the expression of endogenous CD70 in the engineered NKT cell is reduced or eliminated.
  • the expression of endogenous CD70 in the engineered NKT cell is reduced or eliminated compared to the parent cell from which the engineered NKT cell is derived from.
  • engineered NKT cells expression a CD70 targeting CAR, wherein the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the endogenous CD70 expression of the NKT cell is reduced or eliminated by gene editing.
  • the engineered NKT cell is CD70 negative.
  • the CAR targeting CD70 includes a CD70 binding domain.
  • the CD70 binding domain is an anti-CD70 scFv.
  • the CD70 binding domain is an anti-CD70 VHH.
  • the CD70 binding domain is a CD27 peptide that is full-length or truncated CD27.
  • engineered NKT cells expressing a CAR targeting CD70 wherein the CAR includes an anti-CD70 scFv, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the anti-CD70 scFv is humanized scFv.
  • Exemplary humanized anti-CD70 scFv includes a light chain variable region (VL) and a heavy chain variable region (VH) , connected by a suitable linker.
  • An exemplary humanized anti-CD70 scFv can be represented as NH2-VL-Linker-VH-*or NH2-VH-Linker-VL-*, where*represents the connection location to other parts of CAR; NH2-represents the N-terminus of scFv.
  • the anti-CD70 scFv comprises a VL and a VH, wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, and the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 2.
  • the light chain variable region and heavy chain variable region of humanized anti-CD70 scFv are connected by a peptide linker (e.g., a linker composed of glycine and serine) .
  • a peptide linker e.g., a linker composed of glycine and serine
  • the amino acid sequence of the exemplary peptide linker is shown as SEQ ID NO: 17.
  • engineered NKT cells expressing a CAR targeting CD70 wherein the CAR includes a humanized anti-CD70 scFv comprising a VL and a VH, wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, and the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 2, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the CAR includes a humanized anti-CD70 scFv comprising a VL and a VH
  • the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 9
  • engineered NKT cells expressing a CAR targeting CD70 wherein the CAR includes an anti-CD70 VHH, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the anti-CD70 VHH is a camelid VHH.
  • the anti-CD70 VHH is a humanized VHH.
  • Exemplary anti-CD70 scFv or anti-CD70 VHH includes antibodies against CD70 disclosed in the following patent literature: CN116063524A, CN116023490A, CN115989033A, CN115925951A, CN115850484A, CN114262377A, CN109293778B, CN109021106B, CN111139223B, CN110592023B, CN103596979B, TW202308699A, TW1785009B, JP7212468B2, WO2023072307A1, WO2022238963A1, WO2022262100A1, WO2022262101A1, WO2022262099A1, WO2022143951A1, WO2023061063A1, WO2022105914A1, WO2022078344A1, US8663642B2, US8337838B2, US7641903B2, with their entire contents incorporated herein by reference.
  • the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15. In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 3. In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4 (VHH 1E) .
  • the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5. In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6 (VHH 1G) . In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 7.
  • the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 8. In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 9. In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 10.
  • the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11. In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 12 (VHH L2) . In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 13.
  • the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 13. In some embodiments, the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 15.
  • engineered NKT cells expressing a CAR targeting CD70, wherein the CAR includes an anti-CD70 VHH having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the CAR includes an anti-CD70 VHH having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • engineered NKT cells expressing a CAR targeting CD70 wherein the CAR includes an a CD27 peptide, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the CD27 peptide can be full length CD27.
  • the CD27 peptide can be a truncated C27.
  • the CD27 peptide is the extracellular domain of CD27.
  • the CD70 targeting CAR comprises a CD70 binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and/or intracellular signaling domain.
  • engineered NKT cells expressing a CAR targeting CD70, wherein the CAR includes a CD70 binding domain, ahinge domain, a transmembrane domain, a co-stimulatory domain, and/or intracellular signaling domain; wherein the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the transmembrane domain can be derived from one or more proteins selected from the following: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , a cytokine receptor, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ R, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM, and a combination thereof.
  • the transmembrane domain can be derived from one or more proteins selected from the following: CD8, CD28, CD27, CD7, TRAC, TRBC, CD3 ⁇ , CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, and BTLA.
  • the CD70 targeting CAR provided herein includes a CD28 transmembrane domain.
  • the CD70 targeting CAR provided herein includes a CD8 transmembrane domain.
  • the CD8 transmembrane domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 20.
  • the co-stimulatory domain can be derived from one or more proteins selected from the following: 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, CD80, CD86, OX40, CD226, DR3, SLAM, CDS, ICAM, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, CD30L, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin- ⁇ receptor, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, KIR2DS2, CD83 ligand, CD40 and MyD88, and a combination thereof.
  • 4-1BB 4-1BB, CD28, CD137, CD27, CD2, CD7
  • the co-stimulatory domain can be derived from one or more proteins selected from the following: CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, CD2, NKG2C, LIGHT, and DAP12.
  • the CD70 targeting CAR provided herein includes a CD28 co-stimulatory domain.
  • the CD70 targeting CAR provided herein includes a 4-1BB co-stimulatory domain.
  • the 4-1BB co-stimulatory domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 21.
  • the intracellular signaling domain can be derived from one or more proteins selected from the following: CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, CD66d, CD5, CD22, FcR ⁇ , FcR ⁇ , FcR ⁇ , FceRI ⁇ , FceRI ⁇ , Fc ⁇ RI (CD64) , Fc ⁇ RIIa (CD32) , Fc ⁇ RIIIa (CD16) , bovine leukemia virus (BLV) gp30, Epstein-Barr virus (EBV) LMP2A, Simian immunodeficiency virus (SIV) PBj14 Nef, Kaposi’s sarcoma-associated herpesvirus (KSHV) K1, DAP10, DAP12, and a domain containing at least one immunoreceptor tyrosine-based activation motif (ITAM) , and a combination thereof.
  • TAM immunoreceptor tyrosine-based activation motif
  • the intracellular signaling domain can include one or more of the following: CD3 ⁇ activation domain, CD3 ⁇ activation domain, CD3 ⁇ activation domain, Fc ⁇ RI ⁇ activation domain, Fc ⁇ RI ⁇ activation domain, immunoglobulin ⁇ activation domain, immunoglobulin ⁇ activation domain, bovine leukemia virus gp30 activation domain, EB virus LMP2A activation domain, simian immunodeficiency virus PBj14Nef activation domain, Kaposi's sarcoma herpesvirus activation domain, DAP-12 activation domain, and immune receptor tyrosine activation motif (ITAM) .
  • the CD70 targeting CAR provided herein includes a CD3 ⁇ activation domain.
  • the CD3 ⁇ signaling domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 22.
  • the hinge domain can be derived from one or more proteins selected from the following: CD8, CD28, IgG (e.g., IgG1, IgG2, IgG4) , 4-1BB, CD4, CD27, CD7, PD-1, CH2, CH3, and a combination thereof.
  • the CD70 targeting CAR provided herein includes a CD8 hinge.
  • the CD8 hinge has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 19.
  • engineered NKT cells expressing a CAR targeting CD70 wherein the CAR includes a CD70 binding domain, a hinge domain, atransmembrane domain, a co-stimulatory domain, and an intracellular signaling domain; wherein the CD70 binding domain is selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations; the hinge domain is derived from one or more proteins selected from the following: CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, and PD-1; the transmembrane domain is derived from one or more proteins selected from the following: CD8, CD28, CD27, CD7, TRAC, TRBC, CD3 ⁇ , CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, and BTLA; the co-stimulatory domain is derived from one or more proteins selected from the following: 4-1BB, CD28,
  • engineered NKT cells expressing a CAR targeting CD70 wherein the CAR includes a CD70 binding domain, a hinge domain, atransmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, wherein the CD70 binding domain is selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations; the hinge domain is derived from one or more proteins selected from the following: CD8, CD28, IgG1, and IgG4; the transmembrane domain is derived from one or more proteins selected from the following: CD8 ⁇ , CD28, CD3 ⁇ , and CD4; the co-stimulatory domain is derived from 4-1BB, CD28, or both; and the intracellular signaling domain includes the CD3 ⁇ activation domain; and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the CAR includes a CD70 binding domain, a hinge domain, atransmembrane domain, a co-
  • engineered NKT cells expressing a CAR targeting CD70 wherein the CAR includes a CD70 binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain; wherein the CD70 binding domain is selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations; the hinge domain is the CD8 hinge domain; the transmembrane domain is the CD8 transmembrane domain; the co-stimulatory domain is the 4-1BB co-stimulatory domain; and the intracellular signaling domain is the CD3 ⁇ activation domain; and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the amino acid sequence of the exemplary CD8 hinge domain is shown as SEQ ID NO: 19 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 19.
  • the amino acid sequence of the exemplary CD8 transmembrane domain is shown as SEQ ID NO: 20 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 20.
  • the amino acid sequence of the exemplary 4-1BB co-stimulatory domain is shown as SEQ ID NO: 21 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 21.
  • the amino acid sequence of the exemplary CD3 ⁇ activation domain is shown as SEQ ID NO: 22 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 22.
  • engineered NKT cells expressing a CAR targeting CD70, wherein the CAR includes a CD70 binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain, and wherein the amino acid sequence of the CAR targeting CD70 is shown as SEQ ID NO: 30 to 33 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity with SEQ ID NO: 30 to 33; and wherein the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the CD70 targeting CAR has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 30. In some embodiments, the CAR has the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CD70 targeting CAR has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 31. In some embodiments, the CAR has the amino acid sequence of SEQ ID NO: 31.
  • the CD70 targeting CAR has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 32. In some embodiments, the CAR has the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CD70 targeting CAR has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 33. In some embodiments, the CAR has the amino acid sequence of SEQ ID NO: 33.
  • the engineered NKT cells disclosed herein are further engineered to express an exogenous IL-15 peptide.
  • the IL-15 peptide is wildtype IL-15 (SEQ ID NO: 28) .
  • the IL-15 peptide is a variant of the wildtype IL-15 with enhanced activity and binding affinity.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • the IL-15 peptide has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to wildtype IL-15 (SEQ ID NO: 28) . It is also understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the polypeptide half-life.
  • the IL-15 peptide is an IL-15 agonist or superagonist.
  • an IL-15 agonist or superagonist can simply identify an IL-15-agonist or-superagonist.
  • a list of known IL-15-agonist or -superagonist can be found in the US11273204B2, or Cai et al., Frontiers in Pharmacology 14 (2023) .
  • the IL-15 agonist or superagonist contains a substitution at L45, S51, or N72 in reference to wildtype IL-15 (SEQ ID NO: 28) .
  • the IL-15 agonist or superagonist contains a substitution selected from the group consisting of L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y and N72P, in reference to wildtype IL-15 (SEQ ID NO: 28) .
  • the IL-15 peptide contains a N72D substitution (SEQ ID NO: 71) .
  • the IL-15 peptide expressed on the engineered NKT cells disclosed herein is linked to an IL-15 receptor alpha (IL-15R ⁇ ) peptide, and/or an immunoglobulin fragment to form more stable and long-lasting complexes.
  • the IL-15 peptide expressed on the engineered NKT cells disclosed herein is linked to wildtype human IL-15R ⁇ , or a function variant thereof.
  • the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • the IL-15R ⁇ peptide has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to wildtype IL-15 (SEQ ID NO: 72) . It is also understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the polypeptide half-life.
  • the IL-15 peptide expressed on the engineered NKT cells disclosed herein is linked to an IL-15R ⁇ peptide that is a function fragment of the full-length IL-15R ⁇ .
  • the IL-15R ⁇ peptide can be the sushi domain of a full-length IL-15R ⁇ or a function variant thereof.
  • the sushi domain of IL-15R ⁇ refers to a domain beginning at the first cysteine residue (C1) after the signal peptide of IL-15R ⁇ and ending at the fourth cysteine residue (C4) after said signal peptide.
  • the sushi domain corresponding to a portion of the extracellular region of IL-15R ⁇ is necessary for its binding to IL-15 (Wei et al., J.
  • the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • the IL-15R ⁇ peptide has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to wildtype IL-15 sushi domain (SEQ ID NO: 73) .
  • natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the polypeptide half-life.
  • the engineered NKT cells disclosed herein that express an IL-15 peptide or an IL-15 peptide linked to an IL-15R ⁇ peptide further comprise an Fc domain. Accordingly, in some embodiments, the engineered NKT cells disclosed herein further express a fusion protein comprising an IL-15 peptide, an IL-15R ⁇ peptide, and an Fc peptide. In some embodiments, the Fc domain can be human IgG1 Fc domain.
  • the IL-15 peptide and IL-15R ⁇ peptide can be linked non-covalently such as in the complex disclosed in U.S. Pat. No. 8,124,084 B2. Said complex can be simply obtained by expressing both peptides in the same cell.
  • the IL-15 peptide and IL-15R ⁇ peptide can also be covalently linked in a fusion protein.
  • fusion protein refers to a protein created through the joining of two or more genes which originally coded for separate proteins. It is also known as a chimeric protein. Translation of this fusion gene results in a single peptide with functional properties deriving from each of the original proteins.
  • Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics.
  • a recombinant fusion protein is a protein created through genetic engineering of a fusion gene. This typically involves removing the stop codon from a cDNA sequence coding for the first protein, then appending the cDNA sequence of the second protein in frame through ligation or overlap extension PCR. That DNA sequence will then be expressed by a cell as a single protein.
  • the protein can be engineered to include the full sequence of both original proteins, or only a portion of either.
  • the IL-15 peptide comprises a signal peptide (SP) .
  • SP signal peptide
  • the SP can be located at the N-terminus.
  • the IL-15 peptide comprises, from N to C, a SP, and the mature IL-15, or a functional variant thereof (e.g., N72D mutant of mature IL-15) .
  • the IL-15 peptide comprises an SP that is the wild-type SP of human IL-15, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 27.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 27.
  • the IL-15 peptide comprises an SP that is the wild-type SP of human IL-4, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 50.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 50.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 53.
  • the IL-15 peptide comprises an SP that is the wild-type SP of human IgK, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 51.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 54.
  • the IL-15 peptide comprises an SP that is the wild-type SP of human GM-CSF, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 52.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 52.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 55.
  • the engineered NKT cells provided herein express an IL-15 peptide and an IL-15R ⁇ peptide (e.g., a sushi domain) covalently linked as a fusion protein.
  • the IL-15 peptide can be in a C-terminal or in an N-terminal position relative to IL-15R ⁇ peptide.
  • the IL-15 peptide and IL-15R ⁇ peptide (e.g., a sushi domain) can be linked by a peptide linker.
  • the peptide linker can be of a length sufficient to ensure that the fusion protein form proper secondary and tertiary structures. The length of the linker can vary without significantly affecting the biological activity of the fusion protein. In some embodiments, the linker comprises 2-30 amino acids.
  • the linker comprises 10-30 amino acids. In some embodiments, the linker comprises 15-30 amino acids. In some embodiments, the linker comprises 15-25 amino acids. In some embodiments, the linker comprises 18-22 amino acids.
  • the linkers allow the fusion to adopt a proper conformation (i.e., a conformation allowing a proper signal transducing activity through the IL-15Rbeta/gamma signaling pathway) . In some embodiments, the linkers (1) adopt a flexible extended conformation, (2) do not exhibit a propensity for developing ordered secondary structure which could interact with the functional domains of fusion proteins, and (3) have minimal hydrophobic or charged character which could promote interaction with the functional protein domains. Examples of linker sequences are described in U.S. Pat. Nos. 5,073,627; 5,108,910; and 11,273,204.
  • the genetically engineered NKT cells comprise one expression cassette that encodes both the CD70 targeting CAR and the IL-15 peptide. In some embodiments, the genetically engineered NKT cells comprise a first expression cassette that encodes the CD70 targeting CAR and a second expression cassette that encodes the IL-15 peptide. In some embodiments, the genetically engineered NKT cells comprise two separate nucleic acids each containing one expression cassette.
  • An “expression cassette, ” as used herein and understood in the art, is a distinct and continuous component of vector DNA, which includes regulatory sequences that can control the expression of a nucleotide sequence potentially carried by the expression cassette.
  • the regulatory sequences include, for example, transcriptional initiation (promoter) and termination sequences, enhancer, intron, origin of replication sites, polyadenylation sequences, peptide signal and chromatin insulator elements. Regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) . Simply put, the expression cassette directs the host cell’s machinery to make RNA and protein (s) encoded by the nucleotide sequence contained in the cassette. Thus, expression in cells from different organisms or species, such as mammalian cells, requires different regulatory sequences.
  • Cistrons within one expression cassette can be separated by, for example, an internal ribosomal entry site (IRES) or 2A element.
  • IRES refers to nucleotide sequences in an expression cassette which when transcribed into mRNA, can recruit ribosomes directly, without a previous scanning of untranslated region of mRNA by the ribosomes.
  • A2A element as understood in the art, encoding self-cleaving short peptides (about 20 amino acids) that provide a mechanism for subsequent separation of equimolarly produced polypeptides of interest.
  • Illustrative 2A self-cleaving peptides include P2A, E2A, F2A, and T2A.
  • the IL-15 peptide is expressed by a construct independent of the CAR expressing construct. In some embodiments, the IL-15 peptide is expressed by a separate expression cassette on the same construct expressing the CAR.
  • the genetically engineered NKT cells comprise a nucleic acid encoding both the CD70 targeting CAR and the IL-15 peptide, wherein the sequence encoding the CAR and the sequence encoding the IL-15 peptide are separated a cleavage site.
  • Exemplary cleavage sites include proteolytic cleavage sites or self-cleaving peptides cleavage sites such as T2A, P2A, E2A, or F2A.
  • the genetically engineered NKT cells express the IL-15 peptide connected to CAR as a fusion protein through a self-cleaving peptide.
  • the self-cleaving peptide is a 2A peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26.
  • the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 23. In some embodiments, the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24.
  • the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 25. In some embodiments, the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26.
  • the genetically engineered NKT cells express a fusion protein comprising the IL-15 peptide connected to the N-terminus of CAR, that is, an IL-15-cleavage site-CAR fusion. In other embodiments, the genetically engineered NKT cells express a fusion protein comprising IL-15 connected to the C-terminus of CAR, that is, a CAR-cleavage site-IL-15 fusion.
  • the terms “CAR/IL-15 construct” or “CAR/IL-15” are used to denote the fusion of IL-15 with CAR without specifying the exact positional relationship between the two.
  • CAR-IL-15 refers to the fusion constructs wherein the IL-15 peptide is located at the carboxyl (C) end of CAR.
  • IL-15-CAR refers to the fusion constructs wherein the IL-15 peptide is located at the amino (N) end of CAR.
  • engineered NKT cells expressing a CAR targeting CD70 and an IL-15 peptide, wherein the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • engineered NKT cells expressing a CAR targeting CD70 and an IL-15 peptide, the CD70 binding domain selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations; wherein the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • NKT cells expressing a CAR targeting CD70 and an IL-15 peptide, the CD70 binding domain being anti-CD70 scFv or anti-CD70 VHH, the IL-15 peptide is connected to CAR by a cleavage site, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • engineered NKT cells expressing a CAR targeting CD70 and an IL-15 peptide, the CD70 binding domain being anti-CD70 scFv or anti-CD70 VHH, the IL-15 peptide is connected to the C-terminus of CAR by a self-cleaving peptide, and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • engineered NKT cells expressing a CAR targeting CD70 and IL-15 peptide, the IL-15 peptide is connected to the C-terminus of CAR by a self-cleaving peptide, the CD70 binding domain being anti-CD70 scFv or anti-CD70 VHH; wherein the hinge domain includes hinge domains derived from proteins selected from the following: CD8, CD28, IgG1, and IgG4; the transmembrane domain includes transmembrane domains derived from proteins selected from the following: CD8 ⁇ , CD28, CD3 ⁇ , and CD4; the co-stimulatory domain includes co-stimulatory domains or their combinations derived from proteins selected from the following: 4-1BB and CD28; the intracellular signaling domain includes the CD3 ⁇ activation domain; and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the hinge domain includes hinge domains derived from proteins selected from the following: CD8, CD28, IgG1, and IgG4
  • engineered NKT cells expressing a CAR targeting CD70 and an IL-15 peptide
  • the IL-15 peptide is connected to the C-terminus of CAR by a self-cleaving peptide, the CD70 binding domain selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations; wherein the hinge domain is the CD8 hinge domain; the transmembrane domain is the CD8 transmembrane domain; the co-stimulatory domain is the 4-1BB co-stimulatory domain; the intracellular signaling domain is the CD3 ⁇ activation domain; and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • engineered NKT cells expressing a peptide comprising a CAR targeting CD70 and an IL-15 peptide, wherein the CAR includes a CD70 binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain; and the expression of endogenous CD70 in the NKT cell is reduced or eliminated.
  • the peptide comprising the CAR the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-49 and 56-62.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 34. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 35. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 36.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 37. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 38. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 39.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 40. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 41. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 42.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 44. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 45.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 46. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 47. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 48.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 49. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 56.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 57. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 58.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 59. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 60.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 61. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 62.
  • engineered NKT cells expressing a CD70-targeting CAR, and optionally also expressing an IL-15 peptide, wherein the expression of endogenous CD70 in the engineered NKT cell is reduced or eliminated.
  • the expression of endogenous CD70 in the engineered NKT cell is reduced or eliminated compared to the parent cell from which the engineered NKT cell is derived from.
  • a variety of methods for reduction or elimination of endogenous CD70 expression in NKT cells are known in the field.
  • the reduction or elimination of endogenous CD70 expression in NKT cells is achieved by using one of the following: CRISPR-Cas based system, Base Editor, Prime Editor, CRISPRi, zinc finger nucleases (ZFN) , zinc finger transcriptional repressor, TALEN, TALE repressor, meganuclease, mega-TAL, RNA-guided nuclease, RNA editing system, ADAR, RNA interference, antisense oligonucleotides, antisense RNA, microRNA, siRNA, shRNA, and protein expression blocker (PEBL) .
  • CRISPR-Cas based system Base Editor, Prime Editor, CRISPRi, zinc finger nucleases (ZFN) , zinc finger transcriptional repressor, TALEN, TALE repressor, meganuclease, mega-TAL, RNA-guided nuclease, RNA editing system, ADAR, RNA interference, antisense oligonucleotides, anti
  • the endogenous CD70 gene in the NKT cells is inactivated by gene editing.
  • the editing is achieved through CRISPR-Cas systems, such as the CRISPR-Cas base editing system, the prime editor system, or the CRISPR-associated transposase (CAST) system.
  • CRISPR-Cas systems such as the CRISPR-Cas base editing system, the prime editor system, or the CRISPR-associated transposase (CAST) system.
  • Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12) , Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and/or their homologs, or modified forms thereof.
  • the Cas protein is Cas9.
  • the CD70 gene is edited using the CRISPR-Cas9 system, with the sgRNA used referring to CN111909966B, the entire contents of which are incorporated herein by reference.
  • the sequence of the sgRNA used for CRISPR-Cas9 system knockout of CD70 is shown as SEQ ID NO: 16.
  • the expression of endogenous CD70 in the engineered NKT cell is reduced or eliminated by at least 30%compared to wild-type NKT cells in the same state.
  • naive NKT cells or NKT cells that have not been activated by specific antigen
  • the expression of endogenous CD70 in the naive engineered NKT cell is reduced or eliminated by at least 30%compared to naive wild-type NKT cells.
  • activated NKT cells or NKT cells that have been stimulated by specific antigen, and the expression of endogenous CD70 in the activated engineered NKT cell is reduced or eliminated by at least 30%compared to activated wild-type NKT cells.
  • the expression of endogenous CD70 is reduced by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more compared to wild-type NKT cells in the same state. In some embodiments, in the engineered NKT cell, the expression of endogenous CD70 is reduced by at least about 50%compared to wild-type NKT cells in the same state. In some embodiments, in the engineered NKT cell, the expression of endogenous CD70 is reduced by at least about 70%compared to wild-type NKT cells in the same state.
  • the expression of endogenous CD70 is reduced by at least about 80%compared to wild-type NKT cells in the same state. In some embodiments, in the engineered NKT cell, the expression of endogenous CD70 is reduced by at least about 90%compared to wild-type NKT cells in the same state. In some embodiments, in the engineered NKT cell, the expression of endogenous CD70 in the engineered NKT cell when activated is reduced by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more compared to wild-type NKT cells when activated.
  • the expression of endogenous CD70 is eliminated.
  • the CD70 gene in the NKT cell is completely knocked out, rendering the expression of CD70 undetectable, thus making the NKT cell CD70 negative.
  • engineered NKT cells with reduced expression of endogenous CD70 and expressing a CAR targeting CD70 are engineered NKT cells with reduced expression of endogenous CD70 expressing a CAR targeting CD70, wherein the CAR includes a CD70 binding domain selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations.
  • engineered NKT cells with reduced expression of endogenous CD70 expressing a CAR targeting CD70 wherein the CAR includes a CD70 binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain, the CD70 binding domain selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations.
  • NKT cells with reduced expression of endogenous CD70 expressing a CAR targeting CD70, wherein the CAR includes a CD70 binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain, the CD70 binding domain selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations; wherein the hinge domain includes hinge domains derived from proteins selected from the following: CD8, CD28, IgG1, and IgG4; the transmembrane domain includes transmembrane domains derived from proteins selected from the following: CD8 ⁇ , CD28, CD3 ⁇ , and CD4; the co-stimulatory domain includes co-stimulatory domains or their combinations derived from proteins selected from the following: 4-1BB and CD28; the intracellular signaling domain includes the CD3 ⁇ activation domain.
  • the NKT cells are CD70 negative.
  • NKT cells with reduced expression of endogenous CD70 expressing a CAR targeting CD70 and an IL-15 peptide, the CD70 binding domain being anti-CD70 scFv or anti-CD70 VHH, the secreted IL-15 is connected to the C-terminus of CAR by a self-cleaving peptide.
  • the NKT cells are CD70 negative.
  • NKT cells with reduced expression of endogenous CD70 expressing a CAR targeting CD70 and an IL-15 peptide, the IL-15 peptide is connected to the C-terminus of CAR by a self-cleaving peptide, the CD70 binding domain being anti-CD70 scFv or anti-CD70 VHH; wherein the hinge domain includes hinge domains derived from proteins selected from the following: CD8, CD28, IgG1, and IgG4; the transmembrane domain includes transmembrane domains derived from proteins selected from the following: CD8 ⁇ , CD28, CD3 ⁇ , and CD4; the co-stimulatory domain includes co-stimulatory domains or their combinations derived from proteins selected from the following: 4-1BB and CD28; the intracellular signaling domain includes the CD3 ⁇ activation domain.
  • the NKT cells are CD70 negative.
  • NKT cells with reduced expression of endogenous CD70 expressing a CAR targeting CD70 and an IL-15 peptide
  • the IL-15 peptide is connected to the C-terminus of CAR by a self-cleaving peptide, the CD70 binding domain selected from anti-CD70 scFv, anti-CD70 VHH, full-length or truncated CD27, and their combinations; wherein the hinge domain is the CD8 hinge domain; the transmembrane domain is the CD8 transmembrane domain; the co-stimulatory domain is the 4-1BB co-stimulatory domain; the intracellular signaling domain is the CD3 ⁇ activation domain.
  • the NKT cells are CD70 negative.
  • populations of cells that consist essentially of NKT cells, which comprise the engineered NKT cells described herein.
  • the cell population contains about 1x10 ⁇ 5 to about 1x10 ⁇ 7 cells, for example, about 1x10 ⁇ 5 cells, about 2x10 ⁇ 5 cells, about 3x10 ⁇ 5 cells, about 4x10 ⁇ 5 cells, about 5x10 ⁇ 5 cells, about 6x10 ⁇ 5 cells, about 7x10 ⁇ 5 cells, about 8x10 ⁇ 5 cells, about 9x10 ⁇ 5 cells, about 1x10 ⁇ 6 cells, about 2x10 ⁇ 6 cells, about 3x10 ⁇ 6 cells, about 4x10 ⁇ 6 cells, about 5x10 ⁇ 6 cells, about 6x10 ⁇ 6 cells, about 7x10 ⁇ 6 cells, about 8x10 ⁇ 6 cells, about 9x10 ⁇ 6 cells, or about 1x10 ⁇ 7 cells.
  • the cell population contains less than about 1x10 ⁇ 5 cells. In other embodiments, the cell population contains more than 1x10 ⁇ 7 cells.
  • cell populations provided consist substantially of NKT cells. In some embodiments, not less than about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%of the cell populations provided herein are NKT cells, including wild-type NKT cells and engineered NKT cells disclosed herein.
  • the engineered NKT cells disclosed herein make up at least 30%of the cell population.
  • the engineered NKT cells disclosed herein make up about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%of the cell population.
  • At least 90%of the NKT cells of the cell population have reduced expression of endogenous CD70.
  • about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%of the NKT cells of the population have reduced expression of endogenous CD70.
  • the CD70 negative engineered NKT cells disclosed herein make up at least 90%of the NKT cells of the cell population.
  • the CD70 negative engineered NKT cells disclosed herein about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%of the NKT cells of the population.
  • the engineered NKT cells make up at least 30%of the NKT cells of the cell population and at least 90%of the cell population have reduced expression of endogenous CD70.
  • the engineered NKT cells make up about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%of the NKT cells of the cell population, and about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%of the NKT cells of the cell population have reduced expression of endogenous CD70.
  • the engineered NKT cells make up at least 30%of the NKT cells of the cell population and the CD70 negative NKT cells make up at least 90%of the cell population.
  • the engineered NKT cells make up about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%of the NKT cells of the cell population, and the CD70 negative NKT cells make up about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%of the NKT cells of the cell population.
  • the NKT cells are in an activated state.
  • the NKT cells have been activated with a specific antigen, e.g., ⁇ -GalCer.
  • the NKT cells are in an unactivated state.
  • the NKT cells are in a naive state.
  • the cell population after the cell population is delivered in vivo, it can persist in the body for at least 7 days, at least 14 days, at least 21 days, at least 28 days, or at least 35 days. In some embodiments, when the cell population is delivered in vivo, it can persist in the body for at least 35 days or longer.
  • a cell population that can “persist” in vivo for a period of time means that the cell population remains detectable for such a period of time after it is administered to the subject.
  • the cell population can be detectable by assays available in the art including, for example, flow cytometry or qPCR.
  • the cell population continues to proliferate in vivo for at least 7 days, at least 14 days, at least 21 days, at least 28 days, or at least 35 days.
  • the methods comprising sequentially performing the following steps: (i) reducing or eliminating the expression of endogenous CD70 on the cell surface of a NKT cell; and (ii) introducing into the NKT cell an expression construct encoding a CAR targeting CD70 or a CAR/IL-15 construct.
  • the methods further comprise providing a cell population consisting essentially of NKT cells as the starting cell population for step (i) .
  • the NKT cells make up not less than about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%of the starting cell population.
  • the expression rate of CD70 in the starting cell population is at least 40%, namely, at least 40%of the starting cell population expresses CD70. In some embodiments, the expression rate of CD70 in the starting cell population in step (i) ranges from 40%to 90%. For example, the expression rate of CD70 in the starting cell population can be about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.
  • the starting cell population in step (i) is isolated from human peripheral blood. In some embodiments, the starting cell population in step (i) is isolated from the peripheral blood of a person suffering from a CD70 positive tumor (e.g., renal clear cell carcinoma) .
  • a CD70 positive tumor e.g., renal clear cell carcinoma
  • Step (i) of the methods disclosed herein reduces or eliminates the expression of endogenous CD70.
  • step (i) reduces or eliminates the surface expression of CD70.
  • the reduction or elimination of endogenous CD70 expression in NKT cells is achieved by using one of the following: CRISPR-Cas based system, Base Editor, Prime Editor, CRISPRi, zinc finger nucleases (ZFN) , zinc finger transcriptional repressor, TALEN, TALE repressor, meganuclease, mega-TAL, RNA-guided nuclease, RNA editing system, ADAR, RNA interference, antisense oligonucleotides, antisense RNA, microRNA, siRNA, shRNA, and protein expression blocker (PEBL) .
  • CRISPR-Cas based system Base Editor, Prime Editor, CRISPRi, zinc finger nucleases (ZFN) , zinc finger transcriptional repressor, TALEN, TALE repressor, meganucle
  • step (i) is achieved by gene editing.
  • the reduction or elimination of CD70 expression is based on CRISPR-Cas systems, ZFN, or TALEN.
  • the reduction or elimination of CD70 expression is based on CRISPR-Cas systems.
  • an expression construct encoding a CAR targeting CD70 or a CAR/IL-15 construct is introduced into the NKT cell.
  • Methods can be used to introduce expression constructs into the cells are well known in the art. Non-limiting examples include viral transduction, electroporation transfection, liposome delivery, polymer carriers, chemical carriers, lipid complexes, polymer complexes, dendrimer polymers, nanoparticles, emulsions, natural endocytosis or phagocytosis pathways, cell-penetrating peptides, microinjection, microneedle delivery, particle bombardment, etc.
  • electroporation transfection can be used, non-limiting examples of electroporation instruments include: Neon transfection system (Thermo Fisher Scientific) , Gemini instrument, and AgilePulse/CytoPulse instrument (BTX-Harvard apparatus) , 4D-Nucleofector system, Amaxa Nucleofector II, Nucleofector 202b instrument (Lonza) , CTX-1500A instrument (Celetrix) , MaxCyte GT or VLX instrument (MaxCyte) , GenePulser Xcell (Biorad) . Based on the manufacturer's guidance, pulse duration, intensity, intervals between pulses, the number of pulses can be modified to achieve optimal conditions of high transfection efficiency and low mortality rate.
  • Neon transfection system Thermo Fisher Scientific
  • Gemini instrument Gemini instrument
  • AgilePulse/CytoPulse instrument BTX-Harvard apparatus
  • 4D-Nucleofector system 4D-Nucleo
  • gene editing can be used for endogenous CD70 knockout.
  • CRISPR/Cas9-mediated gene editing can be used, and the Cas9 protein and sgRNA can be delivered to the NKT cells by electroporation. It was unexpectedly found by the inventors that within 72 hours after electroporation in step (i) , the proportion of CD70 negative cells in the obtained cell population continued to increase and reached a plateau at about 72 hours.
  • step (ii) e.g., on the 4th, 5th, or 6th day after cell activation
  • the voltage used in electroporation e.g., 1100V, 1200V, 1250V, 1300V, 1350V, or 1500V
  • the interval between step (i) and step (ii) is about 72 hours, the proportion of CD70 negative cells in the obtained cell population is maximized, minimizing the fratricide after CAR targeting CD70 is introduced, and resulting in a greater proportion of CAR positive and CD70 negative cells in the final product cell population.
  • the proportion of CAR positive and CD70 negative cells in the cell population can be increased by about 10%to about 500%when the interval about 72 hours after step (i) , for example, about 10%to about 450%, about 10%to about 400%, about 10%to about 350%, about 10%to about 300%, about 10%to about 250%, about 10%to about 200%, about 10%to about 150%, about 10%to about 100%, about 10%to about 80%, about 10%to about 60%, about 10%to about 50%, about 10%to about 40%, about 10%to about 40%, about 10%to about 20%, about 30%to about 500%, about 50%to about 500%, about 60%to about 500%, about 80%to about 500%, about 100%to about 500%, about 150%to about 500%, about 200%to about 500%, about 250%to about 500%, about 300%to about 500%, about 350%to about 500%, or about 400%to about 500%.
  • the proportion of CD70 negative cells in the obtained cell population increases from about 10%to about 100%, from about 20%to about 100%, from about 30%to about 100%, from about 40%to about 100%, from about 45%to about 100%, from about 50%to about 100%, from about 55%to about 100%, from about 60%to about 100%, from about 65%to about 100%, from about 70%to about 100%, from about 75%to about 100%, from about 80%to about 100%, about 10%to about 98%, from about 20%to about 98%, from about 30%to about 98%, from about 40%to about 98%, from about 45%to about 98%, from about 50%to about 98%, from about 55%to about 98%, from about 60%to about 98%, from about 65%to about 98%, from about 70%to about 98%, from about 75%to about 98%, or from about 80%to about 98%, about 10%to about 95%, from about
  • provided herein are method for preparing engineered NKT cells or cell populations as described herein, the method comprising: (i) reducing or eliminating the expression of endogenous CD70 of the NKT cells by delivering the agent for reducing endogenous CD70 to the NKT cells (e.g., Cas9 and gRNA) ; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after an interval of about 72 hours after the agent (e.g., Cas9 and gRNA) is delivered in step (i) .
  • the methods further comprise providing a cell population consisting essentially of wild-type NKT cells as the starting population for step (i) .
  • the methods further comprise providing a cell population substantially consisting of wild-type NKT cells as the starting population for step (i) .
  • sufficient reduction of endogenous CD70 is reached if step (ii) is performed at least 24 hours after step (i) . In some embodiments, sufficient reduction of endogenous CD70 is reached if step (ii) is performed at least 36 hours after step (i) . In some embodiments, sufficient reduction of endogenous CD70 is reached if step (ii) is performed at least 48 hours after step (i) . In some embodiments, sufficient reduction of endogenous CD70 is reached if step (ii) is performed at least 60 hours after step (i) .
  • a method for preparing engineered NKT cells or cell populations as described herein comprising: (i) reducing or eliminating the expression of endogenous CD70 of the NKT cells by delivering the agent for reducing endogenous CD70 to the NKT cells (e.g., Cas9 and gRNA) ; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after an interval of at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours after the agent (e.g., Cas9 and gRNA) is delivered in step (i) .
  • the agent e.g., Cas9 and gRNA
  • the interval between the delivery of the agent for reducing CD70 expression and the delivery of the agent for CD70 CAR or CD70 CAR-IL-15 expression is at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours. In some embodiments, the interval is at least 24 hours. In some embodiments, the interval is at least 36 hours. In some embodiments, the interval is at least 48 hours. In some embodiments, the interval is at least 60 hours. In some embodiments, the interval is at least 72 hours. In some embodiments, the interval between the delivery of the agent for reducing CD70 expression and the delivery of the agent for CD70 CAR or CD70 CAR-IL-15 expression is about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.
  • the interval is about 24 hours. In some embodiments, the interval is about 36 hours. In some embodiments, the interval is about 48 hours. In some embodiments, the interval is about 60 hours. In some embodiments, the interval is about 72 hours. In some embodiments, the interval between the delivery of the agent for reducing CD70 expression and the delivery of the agent for CD70 CAR or CD70 CAR-IL-15 expression ranges between 24 to 72 hours, 36 to 72 hours, 48 to 72 hours, or 60 to 72 hours. In some embodiments, the interval ranges between 24 to 72 hours. In some embodiments, the interval ranges between 36 to 72 hours. In some embodiments, the interval ranges between 48 to 72 hours. In some embodiments, the interval ranges between 60 to 72 hours.
  • provided herein are method for preparing engineered NKT cells or cell populations as described herein, the method comprising: (i) reducing or eliminating the expression of endogenous CD70 of the NKT cells by delivering Cas9 and gRNA targeting CD70 (e.g., SEQ ID NO: 16) to NKT cells; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after at least 72 hours after the Cas9 and gRNA are delivered in step (i) .
  • Cas9 and gRNA targeting CD70 e.g., SEQ ID NO: 16
  • provided herein are method for preparing engineered NKT cells or cell populations as described herein, the method comprising: (i) reducing or eliminating the expression of endogenous CD70 of the NKT cells by delivering Cas9 and gRNA targeting CD70 (e.g., SEQ ID NO: 16) to NKT cells; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after at least48 hours after the Cas9 and gRNA are delivered in step (i) .
  • Cas9 and gRNA targeting CD70 e.g., SEQ ID NO: 16
  • NKT cell populations e.g., those with a CD70 expression rate higher than 80%, higher than 85%, or higher than 90%
  • the cell population failed to proliferate when step (ii) was performed before step (i) ; whereas when step (i) was performed first, the CAR positive NKT cells increased by 0.6 to 1.5 times on the 3rd to 5th day after CAR introduction.
  • the proliferation efficiency of the cell population obtained by first performing step (i) then performing step (ii) was also significantly higher (e.g., increased by about 70%to 80%) than performing step (ii) before (i) . Therefore, overall, compared to the cell population obtained by first performing step (ii) then performing step (i) , the proliferation efficiency of the cell population obtained by first performing step (i) then performing step (ii) is significantly increased.
  • method for enhancing the proliferation efficiency of the engineered NKT cell or cell population as described herein comprising sequentially performing the following steps: (i) reducing or eliminating the expression of endogenous CD70 in NKT cells or cell populations consisting essentially of NKT cells; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15.
  • Methods disclosed herein further comprise providing a cell population consisting essentially of NKT cells as the starting population for step (i) .
  • Methods disclosed herein further comprise providing a cell population substantially consisting of NKT cells as the starting population for step (i) .
  • the expression rate of CD70 of the starting cell population in step (i) is at least 40%. In some embodiments, the expression rate of CD70 of the starting cell population in step (i) is 40%to 90%, for example, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.
  • the cell population in step (i) is isolated from human peripheral blood. In some embodiments, the cell population in step (i) is isolated from the peripheral blood of a person suffering from a CD70 positive tumor (e.g., renal clear cell carcinoma) .
  • a CD70 positive tumor e.g., renal clear cell carcinoma
  • Methods for isolating NKT cells from peripheral blood are known in the field, for example, the isolation and enrichment methods described in experimental section below.
  • step (i) is achieved by editing the gene of endogenous CD70.
  • the reduction or elimination of CD70 expression is based on CRISPR-Cas systems, ZFN, or TALEN.
  • methods for enhancing the proliferation efficiency of the engineered NKT cell or cell population as described herein comprising: (i) reducing or eliminating the expression of endogenous CD70 in the cells or the cell population; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after an interval of at least 72 hours after step (i) .
  • methods provided herein further comprise providing a cell population consisting essentially of or consisting substantially of wild-type NKT cells as the starting population for step (i) , where the expression rate of CD70 is at least 40%.
  • methods for enhancing the proliferation efficiency of the engineered NKT cell or cell population as described herein comprising: (i) reducing or eliminating the expression of endogenous CD70 in the cells or the cell population; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after an interval of at least48 hours after step (i) .
  • methods provided herein further comprise providing a cell population consisting essentially of or consisting substantially of wild-type NKT cells as the starting population for step (i) , where the expression rate of CD70 is at least 40%.
  • methods for enhancing the proliferation efficiency of the engineered NKT cell or cell population as described herein comprising: (i) inactivating the CD70 gene in the NKT cells or the cell population by editing with the Crispr-cas system; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after an interval of at least 72 hours after step (i) .
  • methods provided herein comprise providing a cell population consisting essentially of or consisting substantially of wild-type NKT cells as the starting population for step (i) , where the expression rate of CD70 is at least 40%.
  • methods for enhancing the proliferation efficiency of the engineered NKT cell or cell population as described herein comprising: (i) inactivating the CD70 gene in the NKT cells or the cell population by editing with the Crispr-cas system; and (ii) introducing into the NKT cells obtained in step (i) an expression construct for a CAR targeting CD70 or CAR/IL-15 after an interval of at least 48 hours after step (i) .
  • methods provided herein comprise providing a cell population consisting essentially of or consisting substantially of wild-type NKT cells as the starting population for step (i) , where the expression rate of CD70 is at least 40%.
  • kits for promoting macrophage polarization towards the M1 type comprising contacting a macrophage with the engineered NKT cell or cell population described herein.
  • kits for treating a CD70 positive tumor in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the engineered NKT cell or cell population as described above, for a duration sufficient to treat the CD70 positive tumor.
  • methods for treating a CD70 positive tumor in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition containing the engineered NKT cell or cell population as described above.
  • the engineered NKT cells or cell populations disclosed herein can be administered in any appropriate manner, for example, by parenteral or non-parenteral administration, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation.
  • parenteral or non-parenteral administration including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation.
  • it can be administered to a patient by arterial, intradermal, subcutaneous, intra-tumoral, intramedullary, intra-nodal, intramuscular, by intravenous (i. v. ) injection, or intraperitoneally.
  • the cells or cell populations of this disclosure are administered by i. v. injection.
  • the cells or cell populations of this disclosure are administered to the subject by intradermal injection or subcutaneous injection.
  • the cells or cell populations disclosed herein can be directly injected into tumors, lymph nodes, tissues, organs, or sites of infection, for example.
  • one treatment can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer.
  • the treatment process can also be repeated as in the case of chronic administration. Repeated administrations can be at the same dosage or different dosages.
  • the cells or cell populations disclosed herein can be administered in combination with at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is surgery, chemotherapy, immunotherapy, androgen blockade therapy, radiation therapy, or any combination thereof.
  • the chemotherapy is selected from sunitinib, sorafenib, pazopanib, axitinib, everolimus, lenvatinib, anlotinib, erlotinib, and their combinations; preferably axitinib, lenvatinib, cabozantinib, or their combinations.
  • the immunotherapy is selected from anti-PD-1 monoclonal antibodies (e.g., Nivolumab or Pembrolizumab) , anti-CTLA-4 monoclonal antibodies (e.g., Ipilimumab) , anti-PD-L1 monoclonal antibodies (e.g., Avelumab or Atezolizumab) , anti-VEGF monoclonal antibody (Bevacizumab) , and their combinations.
  • anti-PD-1 monoclonal antibodies e.g., Nivolumab or Pembrolizumab
  • anti-CTLA-4 monoclonal antibodies e.g., Ipilimumab
  • anti-PD-L1 monoclonal antibodies e.g., Avelumab or Atezolizumab
  • Bevacizumab anti-VEGF monoclonal antibody
  • the delivery of one therapy is still ongoing when the delivery of a second therapy starts, such that there is an overlap in terms of administration. This can be referred to as “concurrent” or “concomitant” administration or delivery.
  • the delivery of one therapy ends before the start of another therapy.
  • the treatment is more effective.
  • the first and second therapy are more effective (e.g., resulting in a greater or more durable therapeutic effect) when administered in combination than when administered alone as a single agent.
  • the delivery can result in a reduction in symptoms or other disease-related parameters to a greater extent than would be observed with the delivery of one therapy in the absence of the other.
  • an equivalent effect can be achieved with a lower dose of the first therapy or the second therapy, when used in combination than used separately.
  • the effect of the two therapies can be partially additive, fully additive, or greater than additive.
  • the second therapy is administered when the effect of the first therapy is still detectable.
  • provided herein are the uses of the engineered NKT cells or cell populations described herein in the manufacture of a medicament for treating CD70 positive tumors.
  • Provided herein are also the engineered NKT cells or cell populations described herein for use in treating CD70 positive tumors.
  • CD70 positive tumors include but are not limited to renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung cancer, cervical cancer, breast cancer, ovarian cancer, colorectal cancer, endometrial cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, or mesothelioma, or metastatic cancers thereof.
  • the CD70 positive tumor is renal cell carcinoma or its metastatic tumor. In some embodiments, the CD70 positive tumor is nasopharyngeal carcinoma or its metastatic tumor. In some embodiments, the CD70 positive tumor is glioblastoma or its metastatic tumor. In some embodiments, the CD70 positive tumor is melanoma or its metastatic tumor. In some embodiments, the CD70 positive tumor is lung cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is cervical cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is breast cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is ovarian cancer or its metastatic tumor.
  • the CD70 positive tumor is colorectal cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is endometrial cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is bladder cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is esophageal cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is gastric cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is pancreatic cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is liver cancer or its metastatic tumor. In some embodiments, the CD70 positive tumor is mesothelioma or its metastatic tumor.
  • IL-15 is a cytokine that plays a crucial role in the activation and proliferation of nature NK cells, T cells, as well as NKT cells.
  • IL-15 has garnered significant attention for its potential to enhance anti-tumor immunity.
  • Preclinical and clinical studies have demonstrated that IL-15 can boost the cytotoxic activity of NK cells and CD8+T cells, leading to improved tumor surveillance and destruction.
  • various forms ofIL-15 including recombinant IL-15 and IL-15 superagonists, are being investigated for their efficacy in treating a range of cancers, either as monotherapies or in combination with other immunotherapeutic agents.
  • IL-15 has a short half-life in the bloodstream, which limits its effectiveness. High doses required for therapeutic efficacy can lead to severe toxicity and adverse immune response. Rapid clearance from blood necessitates frequent dosing, making treatment burdensome. Thus, improving IL-15 therapy with better bioavailability represents an urgent unmet need.
  • IL-15 peptides comprising a signal peptide (SP) that is different from the SP of wildtype IL-15, which results in improved secretion and bioavailability ofIL-15.
  • SP signal peptide
  • This optimization can lead to higher concentrations ofIL-15 in the tumor microenvironment, thereby potentiating its ability to activate and expand the immune effector cells.
  • Enhanced secretion of IL-15 can improve its pharmacokinetics, reduce the required dosage, and minimize potential side effects, making the therapy more effective and safer for patients.
  • IL-15 peptide comprises a signal peptide (SP) .
  • SP can be located at the N-terminus.
  • the IL-15 peptide comprises, from N terminus to C terminus, a SP, and the mature IL-15, or a functional variant thereof (e.g., N72D mutant of mature IL-15) .
  • the IL-15 peptide comprises an SP that is the wild-type SP of IL-4, or a variant thereof.
  • the IL-15 peptide comprises an SP that is the wild-type SP of human IL-4, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 50.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 50.
  • the IL-15 peptide comprises an SP that is the wild-type SP of mouse IL-4, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 74.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 74.
  • the IL-15 peptide comprises an SP that is the wild-type SP of monkey IL-4, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 75.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 53.
  • the IL-15 peptide comprises an SP that is the wild-type SP of IgK, or a variant thereof. In some embodiments, the IL-15 peptide comprises an SP that is the wild-type SP of human IgK, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 76. In some embodiments, the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 76.
  • the IL-15 peptide comprises an SP that is the wild-type SP of mouse IgK, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 51.
  • the IL-15 peptide comprises an SP that is the wild-type SP of mouse IgK, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 51.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 54.
  • the IL-15 peptide comprises an SP that is the wild-type SP of human GM-CSF, or a variant thereof.
  • the SP can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 52.
  • the SP has up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to SEQ ID NO: 52.
  • the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 55.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of IL-2, or a variant thereof.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of human IL-2 (SEQ ID NO: 77) , or a variant thereof.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of immunoglobulin (e.g., IgE) , or a variant thereof.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of human IgE (SEQ ID NO: 80) , or a variant thereof.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of CD33, or a variant thereof.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of human CD33 (SEQ ID NO: 79) , or a variant thereof.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of tissue plasminogen activator (tPA) , or a variant thereof.
  • the IL-15 peptide can further comprise SP that is the wild-type SP of human tPA (SEQ ID NO: 78) , or a variant thereof.
  • the IL-15 peptide can further comprise SP having an amino acid sequence of SEQ ID NOs: 18 and 81-85.
  • the SP has the amino acid sequence of SEQ ID NO: 18.
  • the SP has the amino acid sequence of SEQ ID NO: 81.
  • the SP has the amino acid sequence of SEQ ID NO: 82.
  • the SP has the amino acid sequence of SEQ ID NO: 83.
  • the SP has the amino acid sequence of SEQ ID NO: 84.
  • the SP has the amino acid sequence of SEQ ID NO: 85.
  • the IL-15 peptide comprises the SP ofIL-4, IgK, GM-CSF, IL-2, IgE, tPA, CD33, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15) .
  • the IL-15 peptide comprises wildtype IL-15 (e.g., SEQ ID NO: 28) .
  • the IL-15 peptide comprises a variant of the wildtype IL-15 with enhanced activity and binding affinity.
  • the IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and a variant of the mature IL-15 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • the IL-15 peptide comprises a variant of mature IL-15, which has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to wildtype IL-15 (SEQ ID NO: 28) . It is also understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the polypeptide half-life.
  • the IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and an IL-15 agonist or superagonist.
  • an IL-15 agonist or superagonist One skilled in the art can simply identify an IL-15-agonist or-superagonist. A list of known IL-15-agonist or-superagonist can be found in the US11273204B2, or Cai et al., Frontiers in Pharmacology 14 (2023) .
  • the IL-15 agonist or superagonist contains a substitution at L45, S51, or N72 in reference to wildtype IL-15 (SEQ ID NO: 28) .
  • the IL-15 agonist or superagonist contains a substitution selected from the group consisting of L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y and N72P, in reference to wildtype IL-15 (SEQ ID NO: 28) .
  • the IL-15 agonist or superagonist contains N72D substitution and has the amino acid sequence of SEQ ID NO: 71.
  • the IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and IL-15 with a N72D substitution (SEQ ID NO: 71) .
  • the IL-15 peptide disclosed herein that comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof can be further linked to an IL-15R ⁇ peptide, and/or an immunoglobulin fragment to form more stable and long-lasting complexes.
  • the IL-15 peptide disclosed herein is linked to wildtype human IL-15R ⁇ , or a function variant thereof.
  • the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • the IL-15R ⁇ peptide has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to wildtype IL-15 (SEQ ID NO: 72) . It is also understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the polypeptide half-life.
  • the IL-15 peptide disclosed herein comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof is linked to an IL-15R ⁇ peptide that is a function fragment of the full-length IL-15R ⁇ .
  • the IL-15R ⁇ peptide can be the sushi domain of a full-length IL-15R ⁇ or a function variant thereof.
  • the sushi domain of IL-15R ⁇ refers to a domain beginning at the first cysteine residue (C1) after the signal peptide of IL-15R ⁇ and ending at the fourth cysteine residue (C4) after said signal peptide.
  • the sushi domain corresponding to a portion of the extracellular region of IL-15R ⁇ is necessary for its binding to IL-15 (Wei et al., J. Immunol., vol. 167 (1) , p: 277-282, 2001) .
  • the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • the IL-15R ⁇ peptide has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution in reference to wildtype IL-15 sushi domain (SEQ ID NO: 73) . It is also understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the polypeptide half-life.
  • the IL-15 peptide disclosed herein comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof can be linked to an Fc domain.
  • the IL-15 peptide is linked to an IL-15R ⁇ peptide and also an Fc domain.
  • the Fc domain can be human IgG1 Fc domain.
  • the IL-15 peptide and IL-15R ⁇ peptide can be linked non-covalently such as in the complex disclosed in U.S. Pat. No. 8,124,084 B2. Said complex can be simply obtained by expressing both peptides in the same cell.
  • the IL-15 peptide and IL-15R ⁇ peptide can also be covalently linked in a fusion protein.
  • the IL-15 peptide can be in a C-terminal or in an N-terminal position relative to IL-15R ⁇ peptide.
  • the IL-15 peptide and IL-15R ⁇ peptide (e.g., a sushi domain) can be linked by a peptide linker.
  • the peptide linker can be of a length sufficient to ensure that the fusion protein forms proper secondary and tertiary structures.
  • the length of the linker can vary without significantly affecting the biological activity of the fusion protein.
  • the linker comprises 2-30 amino acids.
  • the linker comprises 10-30 amino acids.
  • the linker comprises 15-30 amino acids.
  • the linker comprises 15-25 amino acids.
  • the linker comprises 18-22 amino acids.
  • the linkers allow the fusion to adopt a proper conformation (i.e., aconformation allowing a proper signal transducing activity through the IL-15Rbeta/gamma signaling pathway) .
  • the linkers (1) adopt a flexible extended conformation, (2) do not exhibit a propensity for developing ordered secondary structure which could interact with the functional domains of fusion proteins, and (3) have minimal hydrophobic or charged character which could promote interaction with the functional protein domains.
  • linker sequences are described in U.S. Pat. Nos. 5,073,627; 5,108,910; and 11,273,204.
  • peptides comprising, from N-terminus to C terminus, the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, mature IL-15 or a functional variant thereof (e.g., SEQ ID NO: 28 or 71) , and IL-15R ⁇ (e.g., SEQ ID NO: 72) or a function fragment (e.g., SEQ ID NO: 74) thereof.
  • peptides comprising, from N-terminus to C terminus, the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, IL-15R ⁇ (e.g., SEQ ID NO: 72) or a function fragment (e.g., SEQ ID NO: 74) thereof, and mature IL-15 or a functional variant thereof (e.g., SEQ ID NO: 28 or 71) .
  • IL-15R ⁇ e.g., SEQ ID NO: 72
  • a function fragment e.g., SEQ ID NO: 74
  • mature IL-15 or a functional variant thereof e.g., SEQ ID NO: 28 or 71
  • nucleic acids that encode the IL-15 peptides disclosed herein, wherein the IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15a) .
  • nucleic acids that encode peptides comprising, from N-terminus to C terminus, the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, mature IL-15 or a functional variant thereof (e.g., SEQ ID NO: 28 or 71) , and IL-15R ⁇ (e.g., SEQ ID NO: 72) or a function fragment (e.g., SEQ ID NO: 74) thereof.
  • nucleic acids that encode peptides comprising, from N-terminus to C terminus, the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, IL-15R ⁇ (e.g., SEQ ID NO: 72) or a function fragment (e.g., SEQ ID NO: 74) thereof, and mature IL-15 or a functional variant thereof (e.g., SEQ ID NO: 28 or 71) .
  • IL-15R ⁇ e.g., SEQ ID NO: 72
  • a function fragment e.g., SEQ ID NO: 74
  • mature IL-15 or a functional variant thereof e.g., SEQ ID NO: 28 or 71
  • the vector can be an expression vector.
  • the vector can be a viral vector.
  • the vector is a retroviral vector, for example, a gamma retroviral vector, which is employed for the introduction of the nucleic acids described herein into a target cell.
  • the vector can be a lentiviral vector.
  • the vector can be an adenoviral vector.
  • the vector can be an adeno-associated viral vector.
  • the vectors and constructs can optionally be designed to include a reporter.
  • compositions comprising the nucleic acid disclosed herein.
  • Pharmaceutical compositions comprising nucleic acids as therapeutics can be formulated to ensure stability, efficacy, and safety. These formulations include a variety of components, such as buffers, salts, stabilizers, surfactants, lipids, cryoprotectants, chelating agents, or preservatives, or combinations thereof.
  • Buffers are used to maintain the pH of the solution, which is crucial for the stability of the nucleic acid, preventing degradation.
  • Common buffers include phosphate-buffered saline (PBS) , Tris (tris (hydroxymethyl) aminomethane) , and citrate buffer.
  • PBS phosphate-buffered saline
  • Tris tris (hydroxymethyl) aminomethane)
  • citrate buffer for example, Onpattro (Patisiran) contains a citrate buffer to maintain an acidic pH, which is essential for the stability of its lipid nanoparticles.
  • Salts are also included in the pharmaceutical compositions to provide ionic strength, which helps maintain the stability and solubility of the nucleic acid.
  • Typical salts used in these compositions include sodium chloride, potassium chloride, and magnesium chloride. These salts mimic physiological conditions and improve the osmolarity of the solution.
  • Stabilizers can protect nucleic acids from degradation and extend the shelf life of the product.
  • Sugars such as sucrose and trehalose, polyols like mannitol, and amino acids such as glycine and arginine are common stabilizers.
  • Surfactants can be included to enhance the solubility and stability of nucleic acids in the formulation and to prevent aggregation.
  • Polysorbate 80, poloxamers, and sodium dodecyl sulfate (SDS) are commonly used surfactants.
  • Onpattro includes Polysorbate 80 to stabilize its lipid nanoparticle formulation and prevent aggregation.
  • Lipid nanoparticles or liposomes that encapsulate and protect the nucleic acid can be used for facilitating its delivery to target cells.
  • Common lipids used include DSPC (1, 2-distearoyl-sn-glycero-3-phosphocholine) , cholesterol, and PEGylated lipids (PEG2000-C-DMG) .
  • Onpattro uses a combination of these lipids to create stable lipid nanoparticles that encapsulate the siRNA.
  • Cryoprotectants can be used to protect the nucleic acid and other components during freeze-drying (lyophilization) processes.
  • Sugars like sucrose and trehalose, and polyols like mannitol, are common cryoprotectants. These agents are crucial in maintaining the integrity of lipid nanoparticles during lyophilization.
  • Chelating agents such as EDTA (ethylenediaminetetraacetic acid) and citric acid, can be included to bind metal ions that could catalyze the degradation of nucleic acids. EDTA is frequently used to prevent metal ion-induced degradation.
  • Preservatives can be added to prevent microbial growth in multi-dose formulations or during storage.
  • Common preservatives include benzyl alcohol, phenol, and methylparaben.
  • Multi-dose formulations might include preservatives like benzyl alcohol to ensure sterility over time.
  • the general formulation strategy for nucleic acid therapeutics involves ensuring sterility, of ten achieved through filtration or aseptic processing, and adjusting the formulation to be isotonic with body fluids to prevent irritation upon administration, using agents like sodium chloride or mannitol. Buffers are used to maintain an optimal pH for stability and activity, and delivery vehicles such as lipids or polymers are employed to encapsulate the nucleic acid, protecting it from degradation and enhancing delivery to target cells.
  • excipients are carefully selected based on their compatibility with the nucleic acid and their ability to maintain the integrity and efficacy of the therapeutic product.
  • the precise composition varies depending on the specific requirements of the nucleic acid therapeutic and its intended route of administration.
  • engineered cells expressing the IL-15 peptides disclosed herein that comprise the SP of human IL-4 (e.g., SEQ ID NO: 50) , human IgK (e.g., SEQ ID NO: 51) , or human GM-CSF (e.g., SEQ ID NO: 52) , or a variant thereof, and mature IL-15 or a functional variant thereof (e.g., SEQ ID NO: 28 or 71) .
  • the engineered cells disclosed herein further express a synthetic receptor.
  • the term “synthetic receptor” refers to an engineered cell surface protein or protein complex comprising (1) a target-binding domain that can specifically bind a target molecule, and (2) a functional domain that can activate a signaling pathway in the engineered cell.
  • the target-binding domain comprises an extracellular domain.
  • the functional domain comprises an intracellular domain.
  • the synthetic receptor further includes a transmembrane sequence.
  • the synthetic receptor can be a protein complex that comprises proteins expressed from exogenous nucleic acids.
  • the synthetic receptor can also be a protein complex that comprises at least one protein that is exogenously expressed, and at least one protein that is endogenously expressed.
  • the engineered cell can be an immune cell, such as a T cell, a NK cell, a NKT cell, a B cell, a macrophage, etc., and the functional domain can activate the immune cell, either directly or indirectly.
  • the synthetic receptor can be selected from the group consisting of: a chimeric antigen receptor ( “CAR” ) , a T cell receptor ( “TCR” ) , a chimeric TCR, a TCR receptor fusion construct ( “TRuC” ) , a synthetic T cell Receptor and Antigen Receptor ( “STAR” ) , a T cell antigen coupler ( “TAC” ) , an antibody TCR receptor ( “AbTCR” ) , and a chimeric CD3 ⁇ receptor.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TRuC TCR receptor fusion construct
  • TRuC synthetic T cell Receptor and Antigen Receptor
  • TAC T cell antigen coupler
  • the synthetic receptor is a CAR. In some embodiments, the synthetic receptor is a TCR. In some embodiments, the synthetic receptor is a chimeric TCR. In some embodiments, the synthetic receptor is a TRuC. In some embodiments, the synthetic receptor is a STAR. In some embodiments, the synthetic receptor is a TAC. In some embodiments, the synthetic receptor is an AbTCR. In some embodiments, the synthetic receptor is a chimeric CD3 ⁇ receptor.
  • the IL-15 peptides disclosed herein and the synthetic receptor are connected with a self-cleaving peptide, such as a 2A peptide.
  • the engineered cell comprising a nucleic acid containing an expression cassette encoding the IL-15 peptide and also an expression cassette encoding the synthetic receptor.
  • the expression cassettes are connected by an IRES sequence.
  • the synthetic receptor can be a CAR, a TCR, a chimeric TCR, a TRuC, a STAR, a TAC, an AbTCR, or a chimeric CD3 ⁇ receptor.
  • the engineered cells disclosed herein further express a CAR.
  • the engineered cells disclosed herein further express a TCR. In some embodiments, the engineered cells disclosed herein further express a chimeric TCR. In some embodiments, the engineered cells disclosed herein further express a STAR. In some embodiments, the engineered cells disclosed herein further express a TRuC. In some embodiments, the engineered cells disclosed herein further express a TAC. In some embodiments, the engineered cells disclosed herein further express an AbTCR. In some embodiments, the engineered cells disclosed herein further express a chimeric CD3 ⁇ receptor.
  • the genetically engineered cell provided herein can be of any type.
  • the cell is suitable for transplantation.
  • the cell is suitable for allogeneic transplantation.
  • the cell is selected from the group consisting of a stem cell, a pluripotent cell, a progenitor cells, a hematopoietic stem and/or progenitor cell, aCD34+mobilized peripheral blood cell, a CD34+cord blood cell, a CD34+bone marrow cell, ahepatocyte, a somatic cell, an immune cell, and a non-transformed cell.
  • the cell is a stem cell.
  • the cell is a pluripotent cell.
  • the cell is a progenitor cell. In some embodiments, the cell is a hematopoietic stem and/or progenitor cell. In some embodiments, the cell is a CD34+mobilized peripheral blood cell. In some embodiments, the cell is a CD34+cord blood cell. In some embodiments, the cell is a CD34+bone marrow cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a non-transformed cell.
  • the cell provided herein is a stem cell.
  • the cell can be a somatic cell.
  • the cell can be a non-pluripotent cell.
  • the cell can be an incompletely or partially pluripotent stem cell.
  • the cell can be a multipotent cell.
  • the cell can be an oligopotent cell,
  • the cell can be a unipotent cell.
  • the cell can be a terminally differentiated cell.
  • Pluripotent cells suitable for use in particular embodiments include, but are not limited to, naturally-occurring stem cells, embryonic stem cells, or induced pluripotent cells (iPSCs) .
  • iPSCs induced pluripotent cells
  • Provided herein are also a mixed population of cells combining any of the cells mentioned above.
  • a population of cells provided herein can comprise cells undergoing reprogramming, which comprise pluripotent cells, partially pluripotent cells, and non-pluripotent cells, such as fully differentiated cells.
  • the cell provided herein is an adult stem/progenitor cell. In some embodiments, the cell provided herein is a neonatal stem/progenitor cell. In some embodiments, the cell is selected from the group consisting of: a mesodermal stem/progenitor cell, an endodermal stem/progenitor cell, and an ectodermal stem/progenitor cell. In some embodiments, the cell is a mesodermal stem/progenitor cell. In some embodiments, the cell is an endodermal stem/progenitor cell. In some embodiments, the cell is an ectodermal stem/progenitor cell.
  • mesodermal stem/progenitor cells include, but are not limited to: mesodermal stem/progenitor cells, endothelial stem/progenitor cells, bone marrow stem/progenitor cells, umbilical cord stem/progenitor cells, adipose tissue derived stem/progenitor cells, hematopoietic stem/progenitor cells (HSCs) , mesenchymal stem/progenitor cells, muscle stem/progenitor cells, kidney stem/progenitor cells, osteoblast stem/progenitor cells, chondrocyte stem/progenitor cells, and the like.
  • the cell is a mesodermal stem/progenitor cell.
  • the cell is an endothelial stem/progenitor cell. In some embodiments, the cell is a bone marrow stem/progenitor cell. In some embodiments, the cell is an umbilical cord stem/progenitor cell. In some embodiments, the cell is an adipose tissue derived stem/progenitor cell. In some embodiments, the cell is a hematopoietic stem/progenitor cell (HSC) . In some embodiments, the cell is a mesenchymal stem/progenitor cell. In some embodiments, the cell is a muscle stem/progenitor cell. In some embodiments, the cell is a kidney stem/progenitor cell. In some embodiments, the cell is an osteoblast stem/progenitor cell. In some embodiments, the cell is a chondrocyte stem/progenitor cell.
  • HSC hematopoietic stem/progenitor cell
  • ectodermal stem/progenitor cells include, but are not limited to, neural stem/progenitor cells, retinal stem/progenitor cells, skin stem/progenitor cells, and the like.
  • the cell is a neural stem/progenitor cell.
  • the cell is a retinal stem/progenitor cell.
  • the cell is a skin stem/progenitor cell.
  • endodermal stem/progenitor cells include, but are not limited to, liver stem/progenitor cells, pancreatic stem/progenitor cells, epithelial stem/progenitor cells, and the like.
  • the cell is an endodermal stem/progenitor cell.
  • the cell is a liver stem/progenitor cell.
  • the cell is a pancreatic stem/progenitor cell.
  • the cell is an epithelial stem/progenitor cell.
  • the cell provided herein is selected from the group consisting of: pancreatic islet cells, CNS cells, PNS cells, cardiac muscle cells, skeletal muscle cells, smooth muscle cells, hematopoietic cells, bone cells, liver cells, an adipose cells, renal cells, lung cells, chondrocyte, skin cells, follicular cells, vascular cells, epithelial cells, immune cells, endothelial cells, and the like.
  • the cell is a pancreatic islet cell.
  • the cell is a CNS cell.
  • the cell is a PNS cell.
  • the cell is a cardiac muscle cell.
  • the cell is a skeletal muscle cell.
  • the cell is a smooth muscle cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a bone cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is an adipose cell. In some embodiments, the cell is a renal cell. In some embodiments, the cell is a lung cell. In some embodiments, the cell is a chondrocyte. In some embodiments, the cell is a skin cell. In some embodiments, the cell is a follicular cell. In some embodiments, the cell is a vascular cell. In some embodiments, the cell is an epithelial cell. In some embodiments, the cell is an endothelial cell.
  • the cell provided herein is an immune cell.
  • the cell can be a leukocyte.
  • a leukocyte is a karyocyte developed from a hematopoietic stem cell and plays important roles in the hematopoietic system and the lymphatic system.
  • Leukocytes includes myeloid cells, lymphoid cells, granulocytes (such as neutrophils, eosinophils, basophils) , lymphocytes (such as T cells, B cells, NK cells, NKT cells) , plasma cells, mast cells, dendritic cells, monocytes and cells differentiated therefrom such as a macrophage.
  • the cell provided herein is a leukocyte.
  • the cell provided herein is a myeloid cell. In some embodiments, the cell provided herein is a lymphoid cell. In some embodiments, the cell provided herein is a granulocyte. In some embodiments, the cell provided herein is a neutrophil. In some embodiments, the cell provided herein is an eosinophil. In some embodiments, the cell provided herein is a basophil. In some embodiments, the cell provided herein is a lymphocyte. In some embodiments, the cell provided herein is a T cell. In some embodiments, the cell provided herein is a B cell. In some embodiments, the cell provided herein is a plasma cell. In some embodiments, the cell provided herein is an NK cell.
  • the cell provided herein is an NKT cell. In some embodiments, the cell provided herein is a dendritic cell. In some embodiments, the cell provided herein is a monocyte. In some embodiments, the cell provided herein is a macrophage. In some embodiments, the cell provided herein is a mast cell. In some embodiments, the cell provided herein is a PBMC.
  • the cell provided herein is a tumor-infiltrating lymphocyte (TIL) .
  • TIL tumor-infiltrating lymphocyte
  • a population of the cells disclosed herein can be a homogenous population of cells.
  • the population of cells can be a heterogeneous population of cells.
  • the population of cells can be a heterogeneous population of cells comprising any combination of the cells disclosed herein.
  • the population of cells consists essentially of the engineered cells disclosed herein that express an IL-15 peptide and a synthetic receptor.
  • T cells provided herein can be used in allogeneic transplantation.
  • some CAR-T cells disclosed herein can be universal CARTs that can be used in allogeneic transplantation for cancer treatment.
  • provided herein are genetically engineered T cells that express the IL-15 peptide disclosed herein, which comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15) .
  • IL-15 peptide disclosed herein, which comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15.
  • the CAR and IL-15 peptide are linked by a self-cleaving linker, such as a 2A linker.
  • the 2A linker is T2A, P2A, E2A, or F2A.
  • the genetically engineered cells express the IL-15 peptide connected to CAR as a fusion protein through a 2A peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26.
  • the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 23.
  • the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24. In some embodiments, the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 25.
  • the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26.
  • the synthetic receptors provided herein include a target-binding domain.
  • the target-binding domain is an extracellular domain that can bind a target molecule.
  • the target molecule is an antigen on a target tissue.
  • the target molecule is a viral antigen.
  • the target molecule is a cancer antigen.
  • the target-binding domain of a synthetic receptor provided herein can also be an epitope that can be recognized by an antibody (e.g., a bispecific or multispecific antibody) that can bind a target molecule on a target tissue, such as a cancer antigen.
  • Such an antigen binding domain is generally derived from an antibody.
  • the target-binding domain can be an antibody fragment, derivative or mimetic thereof, where these fragments, derivatives and mimetics have the requisite binding affinity for the target molecule.
  • Such antibody fragments or derivatives generally include at least the VH and VL domains of the subject antibodies, so as to retain the binding characteristics of the subject antibodies.
  • An antibody fragment as used herein refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally an antigen-binding site.
  • antibody fragments include, but are not limited to, Fab, Fab', F (ab’ ) 2, Fv, single chain antibody molecules (e.g., scFv) , disulfide-linked scFv (dsscFv) , diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD) , single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies) , single variable domain of heavy chain antibodies (VHH) , nanobodies, and multispecific antibodies formed from antibody fragments.
  • the target-binding domain is a Fab.
  • the target-binding domain is a scFv.
  • the target-binding domain comprises a single variable domain antibody.
  • the target-binding domain comprises an antibody mimetic.
  • An antibody mimetic can be molecules that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies.
  • the antibody mimetics are usually artificial peptides within a molar mass of about 2 to 20 kDa. Nucleic acids and small molecules are sometimes considered antibody mimetics as well.
  • Antibody mimetics known in the art include adnectins, affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, avimers, bicyclic peptides, Centyrin (J&J) , DARPins, Fynomers, Knottins, Kunitz domain peptides, monobodies, and nanoCLAMPs.
  • the target-binding domain comprises antibody-like scaffolds (e.g., Owens, Nature Biotechnology 35: 602–603 (2017) ; Simeon and Chen, Protein Cell 9 (1) : 3-14 (2018) ) .
  • a target-binding domain of a synthetic receptor can comprise a ligand or extracellular ligand binding domain of a receptor (see Sadelain et al., Cancer Discov. 3: 388-398 (2013) ; Sharpe et al., Dis. Model Mech. 8: 337-350 (2015) ) .
  • the ligand or extracellular ligand binding domain of a receptor provides to the synthetic receptor the ability to target the cell expressing the synthetic receptor to the corresponding receptor or ligand.
  • the ligand or extracellular ligand binding domain is selected such that the cell expressing the synthetic receptor is targeted to a cancer cell or tumor.
  • the ligand or extracellular ligand binding domain is selected to bind to a cancer antigen that is the corresponding receptor or ligand.
  • the synthetic receptor comprises an antigen-binding domain that specifically binds a viral antigen.
  • the viral antigen is EBV.
  • the viral antigen is HPV. It is understood that these or other viral antigens can be utilized for targeting by a synthetic receptor disclosed herein.
  • the antigen binding domain of the synthetic receptor is selected to bind to an antigen expressed on a cancer cell.
  • a cancer antigen can be uniquely expressed on a cancer cell, or the cancer antigen can be overexpressed in a cancer cell relative to noncancerous cells or tissues.
  • the cancer antigen to be bound by the synthetic receptor is chosen to provide targeting of the cell expressing the synthetic receptor over noncancerous cells or tissues.
  • the cancer antigen can be a tumor antigen. Any suitable cancer antigen can be chosen based on the type of cancer exhibited by a subject (cancer patient) to be treated. It is understood that the selected cancer antigen is expressed in a manner such that the cancer antigen is accessible for binding by the synthetic receptor.
  • the cancer antigen to be targeted by a cell expressing a synthetic receptor is expressed on the cell surface of a cancer cell.
  • any cancer antigen that is accessible for binding to a synthetic receptor is suitable for targeting the synthetic receptor expressing cell to the cancer cell.
  • Suitable antigens include, but are not limited to, B-cell maturation antigen (BCMA) , mesothelin (MSLN) , prostate specific membrane antigen (PSMA) , prostate stem cell antigen (PCSA) , carbonic anhydrase IX (CAIX) , carcinoembryonic antigen (CEA) , CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD123, CD133, CD138, CD33, IL3Ra2, CS1, C-Met, epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM) , folate-binding protein (FBP) , fetal acetylcholine receptor (AChR) , folate receptor- ⁇ and ⁇ (FR ⁇ and ⁇ ) , Gan
  • the engineered cells provided herein express a synthetic receptor (e.g., CAR) targeting a tumor antigen selected from the group consisting of CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3/HER3, ITER-2, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Fltl, KDR, Flt4, Flt3, CEA, CA125, CTLA-4
  • CAR
  • the engineered cells provided herein express a synthetic receptor (e.g., CAR) targeting a tumor antigen selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, Her3, GD2, MAGE, GPC3, Mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1.
  • the engineered cells provided herein express a synthetic receptor (e.g., CAR) targeting CD70.
  • the engineered cells provided herein express a synthetic receptor (e.g., CAR) targeting GPC3.
  • the engineered cells provided herein express a synthetic receptor (e.g., CAR) targeting FOLR1.
  • the synthetic receptor is a CAR
  • the IL-15 peptide provided herein can be co-expressed with a CAR in the engineered cell.
  • the CAR comprises an antigen-binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and/or intracellular signaling domain.
  • the transmembrane domain can be derived from one or more proteins selected from the following: CD8, CD28, CD27, CD7, TRAC, TRBC, CD3 ⁇ , CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, and BTLA.
  • the CAR provided herein includes a CD28 transmembrane domain.
  • the CAR provided herein includes a CD8 transmembrane domain.
  • the CD8 transmembrane domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 20.
  • the co-stimulatory domain can be derived from one or more proteins selected from the following: CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, CD2, NKG2C, LIGHT, and DAP12.
  • the CAR provided herein includes a CD28 co-stimulatory domain.
  • the CAR provided herein includes a 4-1BB co-stimulatory domain.
  • the 4-1BB co-stimulatory domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 21.
  • the intracellular signaling domain can include one or more of the following: CD3 ⁇ activation domain, CD3 ⁇ activation domain, CD3 ⁇ activation domain, Fc ⁇ RI ⁇ activation domain, Fc ⁇ RI ⁇ activation domain, immunoglobulin ⁇ activation domain, immunoglobulin ⁇ activation domain, bovine leukemia virus gp30 activation domain, EB virus LMP2A activation domain, simian immunodeficiency virus PBj14Nef activation domain, Kaposi's sarcoma herpesvirus activation domain, DAP-12 activation domain, and immune receptor tyrosine activation motif (ITAM) .
  • the CAR provided herein includes a CD3 ⁇ activation domain.
  • the CD3 ⁇ signaling domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 22.
  • the hinge domain can be derived from one or more proteins selected from the following: CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, and PD-1.
  • the CAR provided herein includes a CD8 hinge.
  • the CD8 hinge has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 19.
  • IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15a, and wherein the CAR includes an antigen-binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain; wherein the hinge domain is derived from one or more proteins selected from the following: CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, and PD-1; the transmembrane domain is derived from one or more proteins selected from the following: CD8, CD28, CD27, CD7, TRAC, TRBC, CD3 ⁇ , CD4, 4-1BB, OX40, ICOS,
  • engineered NKT cells expressing an IL-15 peptide and a CAR wherein the IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15a)
  • the CAR includes an antigen-binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain; wherein the hinge domain is derived from one or more proteins selected from the following: CD8, CD28, IgG1, and IgG4; the transmembrane domain is derived from one or more proteins selected from the following: CD8 ⁇ , CD28, CD3 ⁇ , and CD4; the co-stimulatory domain is derived from 4-1BB, CD28, or both; and the intracellular signaling
  • engineered NKT cells expressing an IL-15 peptide and a CAR wherein the IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15a)
  • the CAR includes an antigen-binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain; wherein the hinge domain is the CD8 hinge domain; the transmembrane domain is the CD8 transmembrane domain; the co-stimulatory domain is the 4-1BB co-stimulatory domain; and the intracellular signaling domain is the CD3 ⁇ activation domain.
  • the amino acid sequence of the exemplary CD8 hinge domain is shown as SEQ ID NO: 19 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 19.
  • the amino acid sequence of the exemplary CD8 transmembrane domain is shown as SEQ ID NO: 20 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 20.
  • the amino acid sequence of the exemplary 4-1BB co-stimulatory domain is shown as SEQ ID NO: 21 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 21.
  • the amino acid sequence of the exemplary CD3 ⁇ activation domain is shown as SEQ ID NO: 22 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ ID NO: 22.
  • engineered NKT cells expressing an IL-15 peptide and a CAR wherein the CAR and the IL-15 peptide are separated by a self-cleaving peptide such as T2A, P2A, E2A, or F2A.
  • the CAR targets CD70.
  • the CAR targets FOLR1.
  • the CAR targets GPC3.
  • the CAR targets FOLR1.
  • the FOLR1 targeting CAR has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 69.
  • the engineered NKT cells expressing a peptide comprising an IL-15 peptide and a FOLR1-targeting CAR linked by a 2A peptide, wherein the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-65.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 63. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 64. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 65.
  • the CAR targets GPC3.
  • the GPC3 targeting CAR has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 70.
  • the engineered NKT cells expressing a peptide comprising an IL-15 peptide and a GPC3-targeting CAR linked by a 2A peptide, wherein the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-68.
  • the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 66. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 67. In some embodiments, the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 68.
  • compositions comprising the cells disclosed herein.
  • the pharmaceutical composition comprises an effective amount of a cell disclosed herein and a pharmaceutically acceptable carrier.
  • the cells disclosed herein and compositions comprising the cells can be conveniently provided in sterile liquid preparations, for example, typically isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, or the like, which are typically buffered to a selected pH.
  • the compositions can comprise carriers, for example, water, saline, phosphate buffered saline, and the like, suitable for the integrity and viability of the cells, and for administration of a cell composition.
  • Sterile injectable solutions can be prepared by incorporating cells disclosed herein in a suitable amount of the appropriate solvent with various amounts of the other ingredients, as desired.
  • Such compositions can include a pharmaceutically acceptable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like, that are suitable for use with a cell composition and for administration to a subject such as a human.
  • Suitable buffers for providing a cell composition are well known in the art. Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells disclosed herein.
  • compositions will generally be isotonic, that is, they have the same osmotic pressure as blood and lacrimal fluid.
  • the desired isotonicity of the cell compositions provided herein can be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, or other inorganic or organic solutes.
  • Sodium chloride is preferred particularly for buffers containing sodium ions.
  • One particularly useful buffer is saline, for example, normal saline.
  • the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the cells disclosed herein and will be compatible for administration to a subject, such as a human.
  • the skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions to be administered in methods of the invention.
  • the cells disclosed herein can be administered in any physiologically acceptable vehicle. Suitable doses for administration are described herein.
  • a cell population comprising cells disclosed herein can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of cells in a cell population using various well-known methods, as described herein.
  • the ranges of purity in cell populations comprising genetically modified cells provided herein can be from about 20%to about 25%, from about 25%to about 30%, from about 30%to about 35%, from about 35%to about 40%, from about 40%to about 45%, from about 45%to about 50%, from about 55%to about 60%, from about 65%to about 70%, from about 70%to about 75%, from about 75%to about 80%, from about 80%to about 85%; from about 85%to about 90%, from about 90%to about 95%, or from about 95 to about 100%.
  • the ranges of purity in cell populations comprising genetically modified cells provided herein can be from about 20%to about 30%, from about 20%to about 50%, from about 20%to about 80%, from about 20%to about 100%, from about 50%to about 80%, or from about 50%to about 100%. Dosages can be readily adjusted by those skilled in the art; for example, adecrease in purity may require an increase in dosage.
  • kits for preparation of cells disclosed herein comprises one or more vectors for generating a genetically engineered cell, such as a T cell, that expresses a fusion protein disclosed herein for allogeneic transplant.
  • the kits can be used to generate genetically engineered cells from non-autologous cells to be administered to a compatible subject.
  • the kits can comprise cells disclosed herein, for example, non-autologous cells, for administration to a subject.
  • the kits comprise the cells disclosed herein in one or more containers.
  • methods disclosed herein comprise transducing the cell with a nucleic acid disclosed herein. In some embodiments, methods disclosed herein comprise transducing the cell with a vector comprising a nucleic acid disclosed herein. In some embodiments, the nucleic acid encodes a disclosed herein.
  • the nucleic acids encode the IL-15 peptides disclosed herein, wherein the IL-15 peptide comprises the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, and the mature IL-15 (e.g., SEQ ID NO: 28) , or a functional variant thereof (e.g., N72D mutant of mature IL-15a) .
  • the nucleic acids encode peptides comprising, from N-terminus to C terminus, the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, mature IL-15 or a functional variant thereof (e.g., SEQ ID NO: 28 or 71) , and IL-15R ⁇ (e.g., SEQ ID NO: 72) or a function fragment (e.g., SEQ ID NO: 74) thereof.
  • the nucleic acids encode peptides comprising, from N-terminus to C terminus, the SP of human IL-4, human IgK, or human GM-CSF, or a variant thereof, IL-15R ⁇ (e.g., SEQ ID NO: 72) or a function fragment (e.g., SEQ ID NO: 74) thereof, and mature IL-15 or a functional variant thereof (e.g., SEQ ID NO: 28 or 71) .
  • the nucleic acids further encode a synthetic receptor (e.g., a CAR) wherein the CAR and the IL-15 peptide are connected with a self-cleaving peptide (e.g., a 2A peptide) .
  • the cells disclosed herein can be subjected to conditions that favor maintenance or expansion of cells as well known in the art.
  • stem cells or immune cells can be subjected to conditions that favor maintenance or expansion of cells as well known in the art.
  • the cells disclosed herein e.g. stem cells or immune cells
  • Thermo Fisher Scientific Waltham, MA
  • Thermo Fisher Scientific Waltham, MA
  • the cells disclosed herein can optionally be expanded prior to or after ex vivo genetic engineering. Expansion of the cells is particularly useful to increase the number of cells for administration to a subject. Such methods for expansion of cells are well known in the art (see e.g.
  • the cells can optionally be cryopreserved after isolation and/or genetic engineering, and/or expansion of genetically engineered cells (see Kaiser et al., supra, 2015) ) .
  • Methods for cyropreserving cells are well known in the art (see, for example, Freshney, Culture of Animal Cells: A Manual of Basic Techniques, 4th ed., Wiley-Liss, New York (2000) ; Harrison and Rae, General Techniques of Cell Culture, Cambridge University Press (1997) ) .
  • one or more nucleic acids encoding the IL-15 peptide and the CAR are introduced into the target cell using a suitable expression vector.
  • the target cells e.g., stem cells or immune cells
  • the target cells are transduced with one or more nucleic acids encoding an IL-15 peptide, or a synthetic receptor and an IL-15 peptide.
  • the synthetic receptor and an IL-15 peptide encoding nucleic acids can be on separate vectors or on the same vector, as desired.
  • a nucleic acid encoding a synthetic receptor or an IL-15 peptide disclosed herein can be cloned into a suitable vector, such as a retroviral vector, and introduced into the target cell using well known molecular biology techniques (see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999) ) .
  • a suitable vector such as a retroviral vector
  • Any vector suitable for expression in a cell of the invention particularly a human immune cell or a stem cell, can be used.
  • the vectors contain suitable expression elements such as promoters that provide for expression of the encoded nucleic acids in the target cell.
  • cells can optionally be activated to increase transduction efficiency (see Parente-Pereira et al., J. Biol. Methods 1 (2) e7 (doi 10. 14440/jbm. 2014.30) (2014) ; Movassagh et al., Hum. Gene Ther. 11: 1189-1200 (2000) ; Rettig et al., Mol. Ther. 8: 29-41 (2003) ; Agarwal et al., J. Virol. 72: 3720-3728 (1998) ; Pollok et al., Hum. Gene Ther. 10: 2221-2236 (1998) ; Quinn et al., Hum. Gene Ther. 9: 1457-1467 (1998) ; see also commercially available methods such as Dynabeads TM human T cell activator products, Thermo Fisher Scientific, Waltham, MA) .
  • the vector is a retroviral vector, for example, a gamma retroviral or lentiviral vector, which is employed for the introduction of a fusion protein and/or synthetic receptor into the target cell.
  • a retroviral vector is generally employed for transduction.
  • any suitable viral vector or non-viral delivery system can be used.
  • Combinations of a retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells.
  • Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., Mol. Cell. Biol.
  • Non-amphotropic particles are suitable too, for example, particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art (Relander et al., Mol. Therap. 11: 452-459 (2005) ) .
  • Possible methods of transduction also include direct co-culture of the cells with producer cells (for example, Bregni et al., Blood 80: 1418-1422 (1992) ) , or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (see, for example, Xu et al., Exp. Hemat. 22: 223-230 (1994) ; Hughes, et al. J. Clin. Invest. 89: 1817-1824 (1992)) .
  • the chosen vector exhibits high efficiency of infection and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8: 423-430 (1997) ; Kido et al., Current Eye Research 15: 833-844 (1996) ; Bloomer et al., J. Virol. 71: 6641-6649 (1997) ; Naldini et al., Science 272: 263267 (1996) ; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319-10323 (1997) ) .
  • viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus derived vector, or a herpes virus, such as Epstein-Barr Virus (see, for example, Miller, Hum. Gene Ther. 1 (1) : 5-14 (1990) ; Friedman, Science 244: 1275-1281 (1989) ; Eglitis et al., BioTechniques 6: 608-614 (1988) ; Tolstoshev et al., Current Opin. Biotechnol.
  • Epstein-Barr Virus see, for example, Miller, Hum. Gene Ther. 1 (1) : 5-14 (1990) ; Friedman, Science 244: 1275-1281 (1989) ; Eglitis et al., BioTechniques 6: 608-614 (1988) ; Tolstoshev et al., Current Opin. Biotechno
  • Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med. 323: 370 (1990) ; Anderson et al., U.S. Pat. No. 5,399,346.
  • the vectors used herein employ suitable promoters for expression in a particular host cell.
  • the promoter can be an inducible promoter or a constitutive promoter.
  • the promoter of an expression vector provides expression in a stem cell, such as a hematopoietic stem cell.
  • the promoter of an expression vector provides expression in an immune cell, such as a T cell.
  • Non-viral vectors can be used as well, so long as the vector contains suitable expression elements for expression in the target cell.
  • Some vectors, such as retroviral vectors can integrate into the host genome.
  • targeted integration can be implemented using technologies such as a nuclease, transcription activator-like effector nucleases (TALENs) , Zinc-finger nucleases (ZFNs) , and/or clustered regularly interspaced short palindromic repeats (CRISPRs) , homologous recombination, non-homologous end joining, microhomology-mediated endjoining, homology-mediated endjoining and the like (Gersbach et al., Nucl. Acids Res. 39: 7868-7878 (2011) ;ánva, et al. Cell Death Dis. 6: e1831. (Jul 23 2015) ; Sontheimer, Hum. Gene Ther. 26 (7) : 413-424 (2015) ; Yao et al. Cell Research volume 27, pages 801–814 (2017) .
  • technologies such as a nuclease, transcription activator-like effector nucleases (TALENs) , Zinc-finger nucleases
  • nucleic acids encoding the IL-15 peptides disclosed herein, engineered cells expressing the IL-15 peptides disclosed herein can be administered to a subject to illicit or enhance an immune response against cancer tissue or viral infection.
  • the nucleic acids or engineered cells provided herein can be administered as a single therapy.
  • the nucleic acids or engineered cells provided herein can be administered in combination with a second therapy to enhance efficacy of the therapy.
  • provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the nucleic acids disclosed herein.
  • methods of treating the nucleic acids disclosed herein in treatment of tumor or cancer comprising administering to the subject a therapeutically effective amount of the nucleic acids disclosed herein.
  • uses of the nucleic acids disclosed herein in treatment of tumor or cancer comprising uses of the nucleic acids provided herein for the preparation of a medicament for the treatment of tumor or cancer.
  • methods of treating tumor or cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the engineered cells disclosed herein.
  • provided herein are uses of the engineered cells disclosed herein in treatment of tumor or cancer.
  • provided herein are uses of the engineered cells provided herein for the preparation of a medicament for the treatment of tumor or cancer.
  • apopulation of cells comprising the genetically engineered cells is used in the treatment.
  • the population of cells can be homogenous.
  • the population of cells can be heterogenous.
  • provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein.
  • provided herein are uses of the pharmaceutical composition disclosed herein in treatment of tumor or cancer.
  • provided herein are uses of the pharmaceutical composition provided herein for the preparation of a medicament for the treatment of tumor or cancer.
  • the methods and uses provided herein include administering cancer antigen-specific immune cells to a subject in need thereof, wherein the cells recombinantly express a synthetic receptor (e.g., CAR) comprising an antigen binding domain that specifically binds the cancer antigen.
  • a synthetic receptor e.g., CAR
  • the cancer antigen-specific immune cell also expresses an IL-15 peptide provided herein.
  • the cancer antigen can be any cancer antigen disclosed herein or otherwise known in the art.
  • the cancer antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, FOLR1, GPC3, Mesothelin, PSMA, ROR1, EGFR, MUC1, or NY-ESO-1.
  • the present disclosure also provides methods of using the nucleic acids, genetically engineered cells or pharmaceutical compositions disclosed herein in treating viral infection.
  • methods of treating viral infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the nucleic acids, the genetically engineered cells or the pharmaceutical compositions disclosed herein.
  • methods of the nucleic acids, the genetically engineered cells, or the pharmaceutical compositions disclosed herein in treatment of viral infection comprising uses of the nucleic acids, the genetically engineered cells, or the pharmaceutical compositions disclosed herein in treatment of viral infection.
  • provided herein are uses of the nucleic acids, the genetically engineered cells, or the pharmaceutical compositions provided herein for the preparation of a medicament for the treatment of viral infection.
  • nucleic acids or genetically engineered immune cells provided herein in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the cells provided herein can be administered as a dose based on cells per kilogram (cells/kg) of body weight of the subject to which the cells are administered.
  • the cell doses are in the range of about 10 4 to about 10 10 cells/kg of body weight, for example, about 10 5 to about 10 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 , or about 10 5 to 10 6 , depending on the mode and location of administration.
  • a higher dose is used than in regional administration, where the immune cells are administered in the region of a tumor.
  • Exemplary dose ranges include, but are not limited to, 1x10 4 to 1x10 8 , 2x10 4 to 1x10 8 , 3x10 4 to 1x10 8 , 4x10 4 to 1x10 8 , 5x10 4 to 1x10 8 , 6x10 4 , to 1x10 8 , 7x10 4 to 1x10 8 , 8x10 4 to 1x10 8 , 9x10 4 to 1x10 8 , 1x10 5 to 1x10 8 , for example, 1x10 5 to 9x10 7 , 1x10 5 to 8x10 7 , 1x10 5 to 7x10 7 , 1x10 5 to 6x10 7 , 1x10 5 to 5x10 7 , 1x10 5 to 4x10 7 , 1x10 5 to 3x10 7 , 1x10 5 to 2x10 7 , 1x10 5 to 1x10 7 , 1x10 5 to 9x10 6 , 1x10 5 to 8x10 6 , 1x10 5 to
  • cells are provided in a dose of 1x10 5 to 1x10 8 , for example 1x10 5 to 1x10 7 , 1x10 5 to 1x10 6 , 1x10 6 to 1x10 8 , 1x10 6 to 1x10 7 , 1x10 7 to 1x10 8 , 1x10 5 to 5x10 6 , in particular 1x10 5 to 3x10 6 or 3x10 5 to 3x10 6 cells/kg for regional administration, for example, intrapleural administration.
  • 1x10 5 to 1x10 8 for example 1x10 5 to 1x10 7 , 1x10 5 to 1x10 6 , 1x10 6 to 1x10 7 , 1x10 7 to 1x10 8 , 1x10 5 to 5x10 6 , in particular 1x10 5 to 3x10 6 or 3x10 5 to 3x10 6 cells/kg for regional administration, for example, intrapleural administration.
  • Exemplary dose ranges also can include, but are not limited to, 5x10 5 to 1x10 8 , for example, 6x10 5 to 1x10 8 , 7x10 5 to 1x10 8 , 8x10 5 to 1x10 8 , 9x10 5 to 1x10 8 , 1x10 6 to 1x10 8 , 1x10 6 to 9x10 7 , 1x10 6 to 8x10 7 , 1x10 6 to 7x10 7 , 1x10 6 to 6x10 7 , 1x10 6 to 5x10 7 , 1x10 6 to 4x10 7 , 1x10 6 to 3x10 7 cells/kg, and the like. Such does can be particularly useful for systemic administration.
  • cells are provided in a dose of 1x10 6 to 3x10 7 cells/kg for systemic administration.
  • Exemplary cell doses include, but are not limited to, a dose of 1x10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 4 ,
  • the dose can also be adjusted to account for whether a single dose is being administered or whether multiple doses are being administered.
  • the precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject, as described above. Dosages can be readily determined by those skilled in the art based on the disclosure herein and knowledge in the art.
  • nucleic acids, engineered cells, and pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
  • subcutaneous administration is adopted.
  • intravenous administration is adopted.
  • oral administration is adopted.
  • the nucleic acids or cells provided herein can be delivered regionally to a tumor using well known methods, including but not limited to, hepatic or aortic pump; limb, lung or liver perfusion; in the portal vein; through a venous shunt; in a cavity or in a vein that is nearby a tumor, and the like.
  • the nucleic acids or cells provided herein can be administered systemically.
  • the nucleic acids or cells are administered regionally at the site of a tumor.
  • the nucleic acids or cells can also be administered intratumorally, for example, by direct injection of the nucleic acids or cells at the site of a tumor and/or into the tumor vasculature.
  • administration is preferably by intrapleural administration (see Adusumilli et al., Science Translational Medicine 6 (261) : 261ra151 (2014) ) .
  • One skilled in the art can select a suitable mode of administration based on the type of cancer and/or location of a tumor to be treated.
  • the nucleic acids or cells can be introduced by injection or catheter.
  • the nucleic acids or cells are pleurally administered to the subject in need, for example, using an intrapleural catheter.
  • expansion and/or differentiation agents can be administered to the subject prior to, during or after administration of cells to increase production of the cells provided herein in vivo.
  • Proliferation of the cells provided herein is generally done ex vivo, prior to administration to a subject, and can be desirable in vivo after administration to a subject (see Kaiser et al., Cancer Gene Therapy 22: 72-78 (2015) ) .
  • Cell proliferation should be accompanied by cell survival to permit cell expansion and persistence, such as with T cells.
  • cancers or tumors that can be treated with the nucleic acids, cells or pharmaceutical compositions disclosed herein are solid tumors. Cancers or tumors to be treated using the nucleic acids, cells or pharmaceutical compositions provided herein comprise cancers typically responsive to immunotherapy.
  • the cancer or tumor can be carcinomas, sarcoma, melanoma (e.g., cutaneous or intraocular malignant melanoma) , glioma, glioblastoma, brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors, carcinoid tumors, gastric cancer, esophageal cancer, liver cancer, lung cancer (e.g., small cell lung cancer, or non-small cell lung cancer) , head and neck cancer, skin cancer, nasopharyngeal cancer, kidney cancer, colorectal cancer, breast cancer, pancreatic cancer, testicular cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer (for example, hormone refractory prostate adenocarcinoma) , bladder cancer, colon cancer, endocrine cancer, basal cell cancer, squamous cell cancer, dermatofibrosarcoma protuberans, mesothelioma, Merkel cell carcinoma, bone cancer
  • cancers or tumors that can be treated with the nucleic acids, cells, or pharmaceutical compositions disclosed herein are hematological cancers.
  • hematological cancer can be lymphoma, leukemia, multiple myeloma (MM) , or myelodysplastic syndrome (MDS) .
  • the hematological cancer can be polycythemia vera, acute leukemia, acute myeloid leukemia (AML) , acute lymphocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia (CML) , chronic myelocytic leukemia, chronic lymphocytic leukemia, chronic myelomonocytic leukemia (CMML) , natural killer cell leukemia (NK leukemia) , Hodgkin’s disease, non-Hodgkin’s disease, Waldenstrom’s macroglobulinemia, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, natural killer cell lymphoma (NK lymphoma) , cutaneous T-Cell lymphoma (CTCT
  • an anti-tumor effect can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An anti-tumor effect can also be manifested by the ability of the nucleic acids, cells or pharmaceutical compositions provided herein in prevention of the occurrence of tumor in the first place.
  • an “anti-tumor effect” can be manifested by the reduction in cancer-induced immunosuppression.
  • Clinical improvement comprises decreased risk or rate of progression or reduction in pathological consequences of the cancer or tumor.
  • a method of treating cancer can include any effect that ameliorates a sign or symptom associated with cancer.
  • signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting growth of a tumor, slowing the growth rate of a tumor, reducing the size of a tumor, reducing the number of tumors, eliminating a tumor, all of which can be measured using routine tumor imaging techniques well known in the art.
  • Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.
  • the methods or uses provided herein can reduce tumor burden.
  • administration of the nucleic acids, cells or pharmaceutical compositions disclosed herein can reduce the number of tumor cells, reduce tumor size, and/or eradicate the tumor in the subject.
  • Methods for monitoring patient response to administration of a pharmaceutical composition disclosed herein are known in the art and can be employed in accordance with methods disclosed herein.
  • methods known in the art can be employed to monitor the patient for response to administration of therapeutic methods disclosed herein.
  • methods known in the art can be used to monitor size of lesions, and/or size of lymph nodes.
  • contrast-enhanced CT scans can detect and/or monitor lesions and/or lymph nodes in a patient.
  • administration of a pharmaceutical composition disclosed herein can reduce the size of lesions detected by CT scans in a patient. In some embodiments, administration of a pharmaceutical composition disclosed herein can cause shrinkage of abnormal lymph nodes. In some embodiments, the methods or uses provided herein can provide for increased or lengthened survival of a subject having cancer. In some embodiments, the methods or uses provided herein can provide for an increased immune response in the subject against the cancer.
  • a therapeutically effective amount of the nucleic acids, cells or pharmaceutical compositions disclosed herein is administered to a subject in need of cancer treatment.
  • the subject can be a mammal.
  • the subject is a human.
  • Another group of suitable subjects can be a subject who has a history of cancer, but has been responsive to another mode of therapy.
  • the prior therapy can have included, but is not restricted to, surgical resection, radiotherapy, and chemotherapy.
  • these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group can be further subdivided into high-risk and low-risk individuals.
  • the subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts and are suitably defined for different types of cancers. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.
  • the subject can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects.
  • the subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective can be to decrease or delay the risk of recurrence.
  • refractory or recurrent malignancies can be treated using the nucleic acids, genetically engineered cells or pharmaceutical compositions disclosed herein.
  • Combination therapy using agents with different mechanisms of action can result in additive or synergetic effects.
  • Combination therapy can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent disclosed herein.
  • Combination therapy can decrease the likelihood that resistant cancer cells will develop.
  • the additional therapy results in an increase in the therapeutic index of the nucleic acids, cells or pharmaceutical compositions described herein.
  • the additional therapy results in a decrease in the toxicity and/or side effects of the nucleic acids, cells or pharmaceutical compositions described herein.
  • the nucleic acids, cells, or pharmaceutical compositions described herein can be administered in combination with an additional therapy.
  • the additional therapy can be surgical resection, radiotherapy, or chemotherapy.
  • the additional therapy can be administered prior to, concurrently with, or subsequent to administration of the nucleic acids, cells, or pharmaceutical compositions described herein.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • a person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.
  • Embodiment 1 An engineered natural killer T (NKT) cell expressing a chimeric antigen receptor (CAR) targeting CD70, wherein endogenous CD70 expression on cell surface of the NKT cell is reduced or eliminated.
  • NKT natural killer T
  • CAR chimeric antigen receptor
  • Embodiment 2 The engineered NKT cell of Embodiment 1, wherein the endogenous CD70 expression of the NKT cell is reduced or eliminated by gene editing.
  • Embodiment 3 The engineered NKT cell of Embodiment 1 or 2, wherein the engineered NKT cell is CD70 negative.
  • Embodiment 4 The engineered NKT cell of any one of Embodiments 1 to 3, wherein the CAR targeting CD70 comprises a CD70-binding domain selected from the group consisting of an anti-CD70 scFv, an anti-CD70 VHH, a full-length or truncated CD27 peptide, and any combinations thereof.
  • the CAR targeting CD70 comprises a CD70-binding domain selected from the group consisting of an anti-CD70 scFv, an anti-CD70 VHH, a full-length or truncated CD27 peptide, and any combinations thereof.
  • Embodiment 5 The engineered NKT cell of Embodiment 4, wherein the CD70-binding domain is an anti-CD70 scFv or an anti-CD70 VHH.
  • Embodiment 6 The engineered NKT cell of Embodiment 5, wherein the anti-CD70 scFv or anti-CD70 VHH is humanized.
  • Embodiment 7 The engineered NKT cell of any one of Embodiments 4 to 6, wherein the anti-CD70 scFv comprises a light chain variable region (VL) and a heavy chain variable region (VH) , wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, and the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 2.
  • VL light chain variable region
  • VH heavy chain variable region
  • Embodiment 8 The engineered NKT cell of any one of Embodiments 4 to 6, wherein the anti-CD70 VHH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15.
  • Embodiment 9 The engineered NKT cell of any one of Embodiments 1 to 8, wherein the NKT cell also expresses an exogenous IL-15 peptide.
  • Embodiment 10 The engineered NKT cell of Embodiment 9, wherein the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • Embodiment 11 The peptide of Embodiment 10, wherein the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28.
  • Embodiment 12 The peptide of Embodiment 10, wherein the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28, with a N72D substitution.
  • Embodiment 13 The peptide of any one of Embodiments 9 to 12, wherein the IL-15 peptide further comprises an IL-15R ⁇ peptide or a functional fragment thereof.
  • Embodiment 14 The peptide of Embodiment 13, wherein the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • Embodiment 15 The peptide of any one of Embodiments 9 to 12, wherein the IL-15 peptide further comprises a sushi domain of IL-15R ⁇ .
  • Embodiment 16 The peptide of Embodiment 15, wherein the sushi domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • Embodiment 17 The engineered NKT cell of any one of Embodiments 9 to 16, wherein the IL-15 peptide is connected to the CAR through a self-cleaving peptide.
  • Embodiment 18 The engineered NKT cell of Embodiment 17, wherein the self-cleaving peptide is a 2A peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26.
  • Embodiment 19 The engineered NKT cell of any one of Embodiments 9 to 18, wherein the IL-15 peptide comprises a signal peptide at its N-terminus.
  • Embodiment 20 The engineered NKT cell of Embodiment 19, wherein the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 27 and 50-52.
  • Embodiment 21 The engineered NKT cell of any one of Embodiments 1 to 20, wherein the CAR targeting CD70 further comprises (1) a hinge domain; (2) a transmembrane domain; (3) a co-stimulatory domain; and/or (4) an intracellular signaling domain.
  • Embodiment 22 The engineered NKT cell of Embodiment 21, comprising (1) the hinge domain of CD8, CD28, IgG1, or IgG4; (2) the transmembrane domain of CD8 ⁇ , CD28, CD3 ⁇ , or CD4; (3) the co-stimulatory domain of CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, FceRI ⁇ , BTLA, GITR, HVEM, CD2, NKG2C, LIGHT, or DAP12; and/or (4) the activation domain of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FceRI ⁇ , FceRI ⁇ , immunoglobulin ⁇ , immunoglobulin ⁇ , bovine leukemia virus gp30, EB virus LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus,
  • Embodiment 23 The engineered NKT cell of Embodiment 21, wherein (1) the hinge domain is a CD8 hinge domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 19; (2) the transmembrane domain is a CD8 transmembrane domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 20; (3) the co-stimulatory domain is a 4-1BB co-stimulatory domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 21; and/or (4)
  • Embodiment 24 The engineered NKT cell of any one of Embodiments 1 to 23, wherein the CAR targeting CD70 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-33.
  • Embodiment 25 An engineered NKT cell expressing a peptide having a CAR targeting CD70 connected to an IL-15 peptide through a self-cleaving peptide, wherein endogenous CD70 expression on cell surface of the NKT cell is reduced or eliminated, wherein the peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-49 and 56-62.
  • Embodiment 26 A population of cells consisting essentially of NKT cells and comprising a plurality of the engineered NKT cells of any one of Embodiments 1 to 25.
  • Embodiment 27 The population of cells of Embodiment 26, wherein the engineered NKT cells constitute at least 30%, at least 50%, or at least 70%of the cell population.
  • Embodiment 28 The population of cells of Embodiment 26 or 27, wherein at least 90%, at least 70%, at least 50%, at least 30%, or at least 10%of NKT cells of the cell population have reduced CD70 expression.
  • Embodiment 29 The population of cells of Embodiment 28, wherein at least 90%of the NKT cells of the cell population have reduced CD70 expression.
  • Embodiment 30 The population of cells of any one of Embodiments 26 to 29, wherein the engineered NKT cells are activated.
  • Embodiment 31 The population of cells of any one of Embodiments 26 to 30, wherein the cell population can persist in vivo for at least 7 days, at least 14 days, at least 21 days, at least 28 days, or at least 35 days.
  • Embodiment 32 The population of cells of Embodiment 31, wherein the cell population can persist in vivo for at least 35 days.
  • Embodiment 33 A method for preparing the engineered NKT cell of any one of Embodiments 1 to 32, comprising sequentially performing the following steps: (i) reducing or eliminating the expression of endogenous CD70 of a NKT cell; and (ii) introducing into the NKT cell an expression construct encoding a CAR targeting CD70.
  • Embodiment 34 The method of Embodiment 33, wherein the engineered NKT cell has enhanced proliferation efficiency.
  • Embodiment 35 The method of Embodiment 33 or 34, wherein the interval between step (i) and step (ii) is at least 48 hours, or at least 72 hours.
  • Embodiment 36 The method of Embodiment 35, wherein the interval between step (i) and step (ii) ranges between about 48 to about 72 hours.
  • Embodiment 37 The method of any one of Embodiments 33 to 36, wherein step (i) is achieved based on the CRISPR-Cas based system, Base Editor, Prime Editor, CRISPRi, ZFN, zinc finger transcriptional repressor, TALEN, TALE repressor, meganuclease, mega-TAL, RNA-guided endonuclease, RNA editing system, ADAR, RNA interference, antisense oligonucleotides, antisense RNA, microRNA, siRNA, shRNA, or protein expression blocker (PEBL) .
  • CRISPR-Cas based system Base Editor, Prime Editor, CRISPRi, ZFN, zinc finger transcriptional repressor, TALEN, TALE repressor, meganuclease, mega-TAL, RNA-guided endonuclease, RNA editing system, ADAR, RNA interference, antisense oligonucleotides, antisense RNA, microRNA
  • Embodiment 38 A pharmaceutical composition comprising the engineered NKT cell of any one of Embodiments 1 to 25 or the population of cells of any one of Embodiments 26 to 32, and a pharmaceutically acceptable carrier.
  • Embodiment 39 A method of promoting macrophage polarization towards the M1 type, comprising contacting a macrophage with the engineered NKT cell of any one of Embodiments 1 to 25, the population of cells of any one of Embodiments 26 to 32, or the pharmaceutical composition of Embodiment 38.
  • Embodiment 40 A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the engineered NKT cell of any one of Embodiments 1 to 25, the population of cells of any one of Embodiments 26 to 32, or the pharmaceutical composition of Embodiment 38.
  • Embodiment 41 The method of Embodiment 40, further comprising administering an additional therapy to the subject.
  • Embodiment 42 The method of Embodiment 40 or 41, wherein the subject is a human.
  • Embodiment 43 Use of the engineered NKT cell of any one of Embodiments 1 to 25 or the population of cells of any one of Embodiments 26 to 32 in cancer treatment.
  • Embodiment 44 Use of the engineered NKT cell of any one of Embodiments 1 to 25 or the population of cells of any one of Embodiments 26 to 32 in the preparation of a medicament for treating cancer.
  • Embodiment 45 The method or use of any one of Embodiments 40 to 44, wherein the cancer is renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung cancer, cervical cancer, breast cancer, ovarian cancer, colorectal cancer, endometrial cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, or mesothelioma, or metastatic cancers thereof.
  • the cancer is renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung cancer, cervical cancer, breast cancer, ovarian cancer, colorectal cancer, endometrial cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, or mesothelioma, or metastatic cancers thereof.
  • Embodiment 46 The method or use of Embodiment 45, wherein the cancer is renal cell carcinoma or metastatic renal cell carcinoma, or lung cancer or metastatic lung cancer.
  • Embodiment 47 The method or use of Embodiment 46, wherein the renal cell carcinoma is refractory metastatic clear cell renal cell carcinoma.
  • Embodiment 48 The method or use of any one of Embodiments 40 to 47, wherein the cancer is CD70 positive cancer.
  • Embodiment 49 An engineered NKT cell expressing a CAR targeting CD70, wherein the CAR targeting CD70 comprises (1) an anti-CD70 scFv comprising a VL and a VH, wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, and the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 2; or (2) an anti-CD70 VHH having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs
  • Embodiment 50 A non-naturally occurring peptide comprising, from N terminus to C terminus, a signal peptide (SP) and an IL-15 peptide.
  • SP signal peptide
  • Embodiment 51 The peptide of Embodiment 50, wherein the SP is an IL-4 SP, an IgK SP,or a GM-CSF SP.
  • Embodiment 52 The peptide of Embodiment 51, wherein the SP is an IL-4 SP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 50.
  • Embodiment 53 The peptide of Embodiment 51, wherein the SP is an IgK SP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 51.
  • Embodiment 54 The peptide of Embodiment 51, wherein the SP is a GM-CSF SP having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 52.
  • Embodiment 55 The peptide of any one of Embodiments 50 to 54, wherein the IL-15 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • Embodiment 56 The peptide of Embodiment 55, wherein the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28.
  • Embodiment 57 The peptide of Embodiment 55, wherein the IL-15 peptide has the amino acid sequence of SEQ ID NO: 28, with a N72D substitution.
  • Embodiment 58 The peptide of any one of Embodiments 50 to 57, wherein the IL-15 peptide further comprises an IL-15R ⁇ peptide or a functional fragment thereof.
  • Embodiment 59 The peptide of Embodiment 58, wherein the IL-15R ⁇ peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • Embodiment 60 The peptide of any one of Embodiments 50 to 57, wherein the IL-15 peptide further comprises a sushi domain of IL-15R ⁇ .
  • Embodiment 61 The peptide of Embodiment 60, wherein the sushi domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • Embodiment 62 The peptide of any one of Embodiments 50 to 61, wherein the IL-15 peptide further comprises an Fc domain.
  • Embodiment 63 The peptide of Embodiment 50, having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-55.
  • Embodiment 64 A nucleic acid, encoding the peptide of any one of Embodiments 50 to 63.
  • Embodiment 65 A vector, comprising the nucleic acid of Embodiment 64.
  • Embodiment 66 The vector of Embodiment 65, wherein the vector is a viral vector.
  • Embodiment 67 The vector of Embodiment 66, wherein the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.
  • Embodiment 68 A genetically engineered cell expressing the peptide of any one of Embodiments 50 to 63.
  • Embodiment 69 A genetically engineered cell comprising the nucleic acid of Embodiment 64, or the vector of any one of Embodiments 65 to 67.
  • Embodiment 70 The genetically engineered cell of Embodiment 68 or 69, further expressing a synthetic receptor.
  • Embodiment 71 The genetically engineered cell of Embodiment 70, wherein the synthetic receptor is selected from the group consisting of a CAR, a TCR, a chimeric TCR, aTRuC, a TAC, an AbTCR, a STAR, and a chimeric CD3 ⁇ receptor.
  • the synthetic receptor is selected from the group consisting of a CAR, a TCR, a chimeric TCR, aTRuC, a TAC, an AbTCR, a STAR, and a chimeric CD3 ⁇ receptor.
  • Embodiment 72 The genetically engineered cell of Embodiment 70, wherein the synthetic receptor is a CAR.
  • Embodiment 73 The genetically engineered cell of any one of Embodiments 70 to 72, wherein the synthetic receptor comprises an antigen-binding domain targeting a tumor antigen.
  • Embodiment 74 The genetically engineered cell of Embodiment 73, wherein the tumor antigen is CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, Her3, GD2, MAGE, FOLR1, GPC3, Mesothelin, PSMA, ROR1, EGFR, MUC1, or NY-ESO-1.
  • the tumor antigen is CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, Her3, GD2, MAGE, FOLR1, GPC3, Mesothelin, PSMA, ROR1, EGFR, MUC1, or NY-ESO-1.
  • Embodiment 75 The genetically engineered cell of Embodiment 70, wherein the synthetic receptor is a CAR targeting CD70 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-33, a CAR targeting FOLR1 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 69, or a CAR targeting GPC3 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70.
  • the synthetic receptor is a CAR targeting CD70 having at least 80%, at least 85%, at least 90%
  • Embodiment 76 The genetically engineered cell of any one of Embodiments 68 to 75, wherein the cell is an immune cell.
  • Embodiment 77 The genetically engineered cell of Embodiment 76, wherein the immune cell is a leukocyte selected from the group consisting of a T cell, a NK cell, a NKT cell, a B cell, a plasma cell, a dendritic cell, a neutrophil, a monocyte, a macrophage, a granulocyte, a mast cell, a lymphocyte, a leukocyte, and a peripheral blood mononuclear cell.
  • the immune cell is a leukocyte selected from the group consisting of a T cell, a NK cell, a NKT cell, a B cell, a plasma cell, a dendritic cell, a neutrophil, a monocyte, a macrophage, a granulocyte, a mast cell, a lymphocyte, a leukocyte, and a peripheral blood mononuclear cell.
  • Embodiment 78 The genetically engineered cell of Embodiment 77, wherein the immune cell is a T cell, a NK cell, or a NKT cell.
  • Embodiment 79 A pharmaceutical composition comprising the cell of any one of Embodiments 68 to 78, or the nucleic acid of Embodiment 64, and a pharmaceutically acceptable carrier.
  • Embodiment 80 A method of increasing IL-15 level in a subject in need thereof comprising administering a therapeutically effective amount of the cell of any one of Embodiments 68 to 78, or the nucleic acid of Embodiment 64, or the pharmaceutical composition of Embodiment 79 to the subject.
  • Embodiment 81 A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the cell of any one of Embodiments 68 to 78, or the nucleic acid of Embodiment 64, or the pharmaceutical composition of Embodiment 79 to the subject.
  • Embodiment 82 The method of 80 or 81, wherein the subject is a human.
  • Embodiment 83 Use of the cell of any one of Embodiments 68 to 78 or the nucleic acid of Embodiment 64 in cancer treatment.
  • Embodiment 84 Use of the cell of any one of Embodiments 68 to 78 or the nucleic acid of Embodiment 64 for the preparation of a medicament for the treatment of cancer.
  • Embodiment 85 The method or use of any one of Embodiments 81 to 84, wherein the cancer is a hematological cancer or a solid tumor.
  • CAR-NKT NKT cells that express a CAR
  • CD70 CAR CAR that specifically targets CD70
  • CD70 CAR-NKT CAR that specifically targets CD70
  • CD70 CAR-NKT NKT cells that express CD70 CAR
  • CAR-IL-15 fusion of CAR and IL-15, connected by 2A peptide, or construct that includes the encoding sequences for the fusion
  • FOLR1 CAR CAR that specifically targets FOLR1
  • GPC3 CAR CAR that specifically targets GPC3
  • FOLR1 CAR-NKT NKT cells that express FOLR1 CAR
  • GPC3 CAR NKT cells that express GPC3 CAR
  • Armored CAR-NKT cells CD70 knockout NKT cells expressing CD70 CAR-IL-15.
  • CAR-NKT production was performed mostly following methods described in Heczey et al., Nature Medicine 2020, 26: 1686-90.
  • Genetically modified NKT cells expressing a CAR targeting human CD70 and a secretory IL-15, and further, having endogenous CD70 gene knockout (FIG. 1) were prepared as described in detail below.
  • CAR-2A-IL-15 molecules (FIG. 2) that included an CD70 targeting CAR (including an anti-CD70 antibody, CD8 hinge, CD8 transmembrane domain, 4-1BB co-stimulatory domain, and CD3 ⁇ signal domain) and an IL-15 molecule connected via 2A peptide (e.g., SEQ ID NOs: 34-49) were constructed.
  • CD70 targeting CAR including an anti-CD70 antibody, CD8 hinge, CD8 transmembrane domain, 4-1BB co-stimulatory domain, and CD3 ⁇ signal domain
  • 2A peptide e.g., SEQ ID NOs: 34-49
  • NKT Cells On the starting day, PBMCs were isolated from single blood collection using Ficoll separation fluid. NKT cells were enriched through positive selection using magnetic beads (Miltenyi Cat#130-094-842) and antibodies targeting NKT-specific constant TCR.
  • NKT cells were mixed with processed autologous PBMCs and stimulated by ⁇ -Galcer, which specifically promoted the proliferation of NKT cells.
  • NKT cells Under days 4 to 9 of cell culture, NKT cells underwent CRISPR/Cas9-mediated gene editing to knockout the CD70 gene.
  • SpCas9 protein and chemically synthesized sgRNA sgRNA target sequence: 5’ -GCGCTGGATGCACACCACG-3’ , SEQ ID NO: 16, purchased from GenScript
  • sgRNA target sequence: 5’ -GCGCTGGATGCACACCACG-3’ SEQ ID NO: 16, purchased from GenScript
  • NKT cells were collected into 15ml or 50ml centrifuge tubes. A small number of cells were counted (centrifuging at 350g for 5 mins, discarding the supernatant) .
  • sgRNA and Cas9 protein were taken out from the-20°Cfreezer.
  • Opti-MEM Opti-MEM
  • Cas9 protein and sgRNA were mixed and incubated at room temperature for 10 mins to form RNP complexes.
  • NKT complete medium (Stemcell product number 10981 medium, plus 200 IU/ml IL-2, 10 ng/ml IL-21) was added to appropriate wells of a plate and pre-warmed in a CO 2 incubator.
  • the cell suspension and RNP were mixed at 6 ⁇ l: 6 ⁇ l, and the mixture was electroporated using a Neon Electroporation Device (Invitrogen product number MPK5000) at 1500V for 10ms for 3 times. After electroporation, cells were cultured in the pre-warmed medium and transferred to a CO 2 incubator. Data from over ten donors showed an average knockout efficiency of 88%-95%.
  • a Neon Electroporation Device Invitrogen product number MPK5000
  • CD70 CAR sequences or the CAR-IL-15 sequences were transduced using ⁇ -retrovirus (transduction method as in Xu et al., Clin Cancer Res 2019, 25: 7126-38) .
  • the data showed that the transduction efficiency ranged between 30%-80%.
  • CAR-NKT cells were cultured, with media replenishment or exchange as necessary, to reach the required cell number. Quality control mid-assessment was performed after around two weeks.
  • the antibodies used for detection include: Biotin-labeled Human CD70 protein (Acrobiosystems, CDL-H82D7) , APC streptavidin (Biolegend, 405207) , BV421 Mouse Anti-Human CD3 (BD, 562426) , BV510 Mouse Anti-Human CD4 (BD, 562970) , FITC Mouse Anti-Human CD8 (BD, 555366) , PE anti-human TCR V ⁇ 24-J ⁇ 18 (6B11) iNKT Antibody (Biolegend, 342904) , CD3-APC-CY7 (Biolegend, 300318) , iNKT-FITC (Biolegend, 342906) , anti-CD70-PE (Biolegend, 355104) .
  • Biotin-labeled CD70 protein and APC streptavidin were used to detect the CAR targeting CD70.
  • Anti-CD3 and anti-human TCR V ⁇ 24-J ⁇ 18 antibodies were used for staining samples, with double-positive cells identified as iNKT cells.
  • Anti-CD70 antibody staining was used to assess the efficiency of CD70 knockout.
  • Cells were prepared using the method described in Example 1, including gene knockout and viral transduction, to obtain Armored CAR-NKT cells expressing the following CAR-IL-15: SEQ ID NOs: 34-37.
  • flow cytometry was employed for characterization of the product, assessing purity, CAR expression, CD4/CD8 ratio, and gene knockout, among others.
  • IL-15 was linked to the CAR molecule through different 2A peptides, including E2A (SEQ ID NO: 23) , P2A (SEQ ID NO: 24) , T2A (SEQ ID NO: 25) , and T2A_R (SEQ ID NO: 26) .
  • NKT cells were prepared as described in Example 1 without CAR transduction or CD70 knockout. The cells were stained with both a CD3 antibody and the monoclonal antibody TCR V alpha 24 J alpha 18 (6B11) , which specifically detected NKT. As shown in FIG. 5, only 6.41%NKT cells stained positive for CD70 before stimulation by ⁇ -Galcer, which lowered to 82.48%on the 7th day post-stimulation.
  • NKT cells Three groups of NKT cells were prepared using methods described in Example1: (1) Group iNKT: without CD70 knockout or CAR transductions, (2) Group CAR70: transduced with CD70 CAR (SEQ ID NO: 30) and without CD70 knockout, and (3) Group CJP: transduced with CD19 CAR (SEQ ID NO: 12 in Chinese patent application CN106220739A) and without CD70 knockout. Cell counts were taken at various time points. As shown in FIG. 6, the NKT cells in both the iNKT group and the CJP group significantly increased over time during culture, whereas the cell number of the CAR70 group barely increased. Generally, NKT cells account for less than 1%in human peripheral blood PBMCs, necessitating effective expansion to meet clinical needs. This result indicated that effective expansion of CAR70-NKT required knockout of endogenous CD70.
  • CD70 knockout in the preparation of CD70 CAR-NKT, cells from three healthy donors (02W-NKT, 23W-NKT, and 19W-NKT) were divided into two groups for each donor: one underwent CRISPR/Cas9-mediated gene editing knockout (KO) of CD70, and the other did not.
  • FIG. 7A most NKT cells expressed high levels of CD70 upon stimulation.
  • FIG. 7B the knockout (KO) group showed a significant reduction in CD70 expression compared to the group without knockout.
  • the expression of CD70 in cells from the three donors decreased from 59.24%to 3.43%, from 91.15%to 9.75%, and from 81.09%to 16.03%, respectively.
  • CAR-IL-15 molecule was transduced to the NKT cells from healthy donors with CD70 knockout (the “KO-CAR” group) or without CD70 knockout (the “NKT-CAR” group) .
  • Three experiments were done in parallel using cells from three donors. The fold expansion of the cell numbers between the 3rd and 5th days post-viral transduction was measured. As shown in FIG. 8A, the fold expansion of the KO-CAR group was higher than that of the NKT-CAR group in all three experiments.
  • the CAR positivity rates in the NKT-CAR group dropped to 0%by the 5th day post-transduction, hence zero growth, demonstrating a significant reduction in CAR-NKT cells prepared without CD70 gene knockout.
  • CD70 knockout during the preparation of CAR-NKT was also evaluated.
  • electroporation for CD70 knockout was performed on day 4, 5 or 6 (D4, D5, or D6 in FIG. 8B) after ⁇ -Galcer stimulation and the gene editing efficiency was assessed at different time points post electroporation (K1, K2, K3, K4, K5, K6, and K7 in FIB. 8B representing day 1, 2, 3, 4, 5, 6 and 7 days post electroporation, respectively) using flow cytometry with anti-CD70 antibody.
  • K1, K2, K3, K4, K5, K6, and K7 in FIB. 8B representing day 1, 2, 3, 4, 5, 6 and 7 days post electroporation, respectively.
  • the percentage decrease in CD70 positive cells post-knockout compared to pre-knocked out cells reflected gene editing efficiency. As shown in FIG.
  • the cytotoxic activity of the Armored CAR-NKT cells was measured.
  • the 786-O wild-type cell line with no endogenous CD1d expression was transduced with lentivirus carrying Luciferase and GFP reporter genes to construct the 786-O-luc-GFP cell line.
  • endogenous CD70 was knockout from the 786-O-luc-GFP cell line, resulting in the CD70-CD1d-target cell strain “786-O--. ”
  • CD1d gene was transduced into 786-O-luc-GFP cell line using lentivirus, resulting in the CD70+CD1d+target cell strain “786-O++.
  • the 786-O++cells or 786-O--cells were mixed with Armored CAR-NKT or control NKT cells at gradient effector-to-target ratios and cultured overnight. On the next day, luciferase substrate was added to measure the chemiluminescence signal value, and a reduction in target cells based on the decrease in signal value to calculate the in vitro killing rate.
  • Armored CAR-NKT cells were mixed with the two types of target cells, 786-O++and 786-O--cells respectively, in a 96-well plate at a fixed 1: 1 effector-to-target ratio and cultured overnight.
  • the control group (NC) included only the effector cells.
  • cytokine concentrations were measured using flow CBA or ELISA methods.
  • Armored CAR-NKT cells secreted both IFN- ⁇ and IL-15 (FIGs. 9C-9D) .
  • IL-15 was constitutively secreted by Armored CAR-NKT with or without the presence of target cells.
  • IL-15 was not expressed in NKT cells without exogenous IL-15 gene.
  • IFN- ⁇ secretion was dependent on the interaction between the CAR and the CD70 positive target, with CAR-NKT cells secreting significant amounts of IFN- ⁇ only when stimulated by positive target cells.
  • Armored CAR-NKT cells (CAR-IL-15 of SEQ ID NO: 34) were used in this study.
  • the renal cancer cell line 786-O++overexpressing CD1d and expressing CD70 was subcutaneously inoculated into NOG mice with severe immunodeficiency, with each mouse receiving 5x10 ⁇ 6 cells, to establish a renal cancer CDX subcutaneous transplant tumor model. Once the tumor volume exceeded 50 mm ⁇ 3, the mice were randomly divided into groups and received intravenous injections of Armored CAR-NKT cells, control NKT cells, or PBS.
  • 3E5, 1E6, and 5E6 respectively represent 3x10 ⁇ 5, 1x10 ⁇ 6, and 5x10 ⁇ 6 cells/mouse.
  • Antibodies used in this study included: PerCP-Cy 5.5 Mouse Anti-Human CD45 (BD, 564105) , APC/Cyanine7 anti-human CD3 Antibody (BioLegend, 300318) , BV510 Mouse Anti-Human CD4 (BD, 562970) , PE-Cy 7 Mouse Anti-Human CD8 (BD, 557746) , APC Streptavidin (BioLegend, 405207) , Biotinylated CD70 Protein (Acrobiosystems, CDL-H82D7-200ug) , and PE anti-human TCR V ⁇ 24-J ⁇ 18 (iNKT cell) Antibody (BioLegend, 342904) .
  • tumor volume was measured twice a week.
  • high-dose Armored CAR-NKT (5x10 ⁇ 6 cells/mouse) exhibited a significant tumor suppression effect. Compared to the control groups, the tumor suppression effect became evident from day 15.
  • FIG. 10B at the end of the experiment on day 54, the average tumor volume was 789.3 mm ⁇ 3 in the PBS control group and was 794.0 mm ⁇ 3 in the NKT control group, while in the high-dose Armored CAR-NKT treated group, the average tumor volume was only 23.3 mm ⁇ 3, showing even a significant reduction of tumor size compared to 54.0 mm ⁇ 3 on day 0.
  • the concentration of IFN- ⁇ in mouse serum was analyzed at different time points during the study. As shown in FIG. 11, consistent with the efficacy results, in the high-dose Armored CAR-NKT group, the concentration of IFN- ⁇ in serum exceeded 100 pg/ml, indicating that an increase in serum IFN- ⁇ concentration in the early phase of administration correlated positively with anti-tumor activity.
  • mice blood samples were collected on Days 7, 14, 21, 28, and 35, and the proportion of CAR-NKT cells in mouse peripheral blood was detected using flow cytometry.
  • CAR-NKT cells were detected in the blood samples of mice treated with Armored CAR-NKT, with significant expansion observed in the high-dose group. Additionally, the CAR-NKT cells persisted in the mouse body for a long time (more than 35 days) .
  • mice During another in vivo safety study to study the safety, the daily health status of the mice was observed and recorded, especially for potential side effects that might arise in preclinical studies of cell therapy, including abdominal swelling, narrowed eyes, prostration, paralysis, tremors, arched back, piloerection, reduced activity, hypothermia, etc.
  • CAR-NKT Compared to CAR-T therapy, CAR-NKT exhibited very good safety in vivo, with no significant toxic side effects observed in the CAR-NKT administration group in the animal model.
  • mouse body weight remained stable during the study, with no abnormal clinical manifestations observed within 54 days post-administration, indicating good safety of Armored CAR-NKT.
  • CD70 CAR-NKT without exogenous IL-15 was also produced using the method described in Example 1, except that in the CAR delivery viral transduction step, retroviruses containing the CAR expression cassette but not the IL-15 expression sequence (i.e., CAR sequences without IL-15 expression sequence as shown in SEQ ID NOs: 30-33) were used.
  • the CD70 targeting CAR-NKT cells without exogenous IL-15 still showed effective tumor killing activities, specifically targeting CD70 positive tumor cells, and exhibited tumor-suppressing effects in mice.
  • the CD70 CAR can feature a variety of antigen-binding domains.
  • Armored CAR-NKT cells were prepared as described in Example 1, and different CD70 CAR sequences (SEQ ID NOs: 31-33 and 38-49) were used, which contained anti-CD70 VHHs (SEQ ID NOs: 3 to 15) .
  • CAR-NKTs containing these VHH also demonstrated potent tumor cell killing ability and showed effective in vivo tumor suppression in mice.
  • CAR2a NKT cells having CAR-IL-15 of SEQ ID NO: 34 with an anti-CD70 scFv
  • CAR3d NKT cells having CAR-IL-15 of SEQ ID NO: 41, with an anti-CD70 VHH
  • CAR5d NKT cells having CAR-IL-15 of SEQ ID NO: 49, with an anti-CD70 VHH.
  • CD70+lung cancer cell line NCI-H460 and the ovarian cancer cell line SK-OV-3, for in vitro efficacy study following the same methods described above.
  • CD70 CAR-NKT exhibited strong cytotoxic effects.
  • FIGs. 15A and 16A CAR2a, CAR3d, and CAR5d all showed CAR-mediated specific killing of CD70+cancer cell lines, with E: T ratio-dependent cytotoxicity.
  • E T ratio-dependent cytotoxicity
  • mice When the tumor volume reached 84 mm 3 , the mice were randomly divided into groups and Armored CAR-NKT cells administered via tail vein injection in a single dose. Each group contained 9 mice. Following cell injection, tumor volumes were measured twice weekly, and blood samples were collected from the mouse orbital sinus once a week to detect the amplification and persistence of Armored CAR-NKT in peripheral blood. Additionally, the health status of the mice was monitored weekly, and abnormalities were recorded.
  • both the low-dose groups (5x10 ⁇ 6 cells/mouse) and high-dose groups (1x10 ⁇ 7 cells/mouse) of CAR2a, CAR3d, and CAR5d exhibited robust tumor suppression.
  • CAR3d and CAR5d demonstrated the most pronounced tumor suppression effects.
  • the tumor inhibition effects began to manifest from as early as day 6.
  • the average tumor volume was 526 mm 3 in the PBS control group and 573 mm 3 in the NKT control group, indicating no tumor suppression in the NKT group.
  • the tumor volume dropped to zero by day 22 post-administration.
  • mice blood samples were collected on Day 7, Day 14, Day 20, and Day 27, and human immune cells in the peripheral blood of mice were measured by flow cytometry to reflect the amplification and persistence of Armored CAR-NKT in vivo.
  • the antibody used included: PerCP-Cy 5.5 Mouse Anti-Human CD45 (BD, 564105) , Brilliant Violet 421 anti-human CD3 Antibody (BD, 562426) , APC-Cy7 Mouse Anti-Human CD4 (BD, 557871) , Alexa Fluor 700 Mouse Anti-Human CD8 (Biolegend, 301028) , APC Streptavidin (BioLegend, 405207) , Biotinylated CD70 Protein (Acrobiosystems, CDL-H82D7-200 ⁇ g) , PE anti-human TCR V ⁇ 24-J ⁇ 18 (iNKT cell) Antibody (BioLegend, 342904) , FITC anti-human CD27 (Biolegend, 356404) , BV510 anti-human CD45
  • Routine Observation Records a) daily observation records; b) daily clinical observation: once per day; c) daily food intake records: twice per week; 2) sampling at the endpoint of the experiment, analyzed using the following indicators: a) gross anatomy records; b) organ and tissue weighing; c)complete blood count; d) blood biochemistry; e) histopathological analysis of tissue sections.
  • mice in each group were euthanized and sampled for analysis of the specified indicators. During the necropsy, no macroscopic pathological abnormalities were found. The mass of organs such as the heart, liver, spleen, and kidneys were measured (Table 3) . As shown, notably, the spleen mass in the CAR2a 1E7 group significantly increased compared to the NKT control group, without causing apparent damage to the mice, and the overall health status of the mice remained good. The organ mass in the other groups showed no significant difference compared to the NKT control group.
  • CBC complete blood count
  • the test indicators included red blood cells (RBC) , hemoglobin (HGB) , hematocrit (HCT) , mean corpuscular volume (MCV) , mean corpuscular hemoglobin (MCH) , mean corpuscular hemoglobin concentration (MCHC) , white blood cells (WBC) , lymphocytes (LYMPH) , monocytes (MONO) , neutrophils (NEUT) , eosinophils (EOS) , basophils (BASO) , platelets (PLT) , and mean platelet volume (MPV) .
  • RBC red blood cells
  • HGB hemoglobin
  • HCT hematocrit
  • MCV mean corpuscular volume
  • MHCT mean corpuscular hemoglobin
  • MHC mean corpuscular hemoglobin concentration
  • WBC white blood cells
  • LYMPH lymphocytes
  • MONO monocytes
  • NEUT neutrophils
  • EOS eosinophil
  • mice plasma was collected for blood biochemistry tests to evaluate liver and kidney functions.
  • the test indicators included alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , gamma-glutamyl transferase (GGT) , total bilirubin (TB) , creatine kinase (CK) , lactate dehydrogenase (LDH) , blood urea nitrogen (BUN) , glucose (GLU) , and total cholesterol (TC) , triglycerides (TG) , creatinine (CRE) .
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • GTT gamma-glutamyl transferase
  • TB total bilirubin
  • CK creatine kinase
  • LDH lactate dehydrogenase
  • BUN blood urea nitrogen
  • BUN glucose
  • TC total cholesterol
  • TG
  • Armored CAR-NKTs having the CAR-IL-15 of SEQ ID NO: 34 were prepared according to methods described in Example 1 using cells from a healthy donor, with viral transduction performed at different time points after CD70 gene knockout. In the study groups D1, D2, and D3, the viral transduction was performed 1 day, 2 days, and 3 days post-knockout, respectively. Results showed no significant differences in NKT purity and viability across the different study groups (data not shown) .
  • Table 7 Percentage of CAR on Day 12 of Product Preparation After CAR Transduction at Different Time Points Following CD70 Knockout
  • NKT cells obtained from healthy donors were used to prepare CD70 CAR-NKT cells without IL-15 expression ( “CAR-NKT wo IL-15” ) using a virus containing CD70 targeting CAR expression sequence (SEQ ID NO: 30) as detailed in Example 6.
  • Armored CAR-NKT cells were prepared using a virus containing the CAR-IL-15 expression sequence (SEQ ID NO: 34) , referred to as “CAR-NKT-IL-15” in this Example.
  • CAR-NKT wo IL-15, CAR-NKT-IL-15, and negative control NKT cells were cultured for 6 days in RPMI 1640 medium (Gibco, Cat#C11875500CP) supplemented with 10%FBS (Thermo Fisher, Cat#A5669701) without any cytokines.
  • Positive controls were established by treating CAR-NKT wo IL-15 with external recombinant IL-15 protein (Miltenyi Biotec., Cat#130-095-762) supplementation at concentrations of0.5 ng/ml and 5 ng/ml.
  • IL-15 The secretion of IL-15 in the culture supernatant of CAR-NKT-IL-15 was quantifiable by ELISA, with a mean concentration of 60.1 pg/ml ( ⁇ 8.6 pg/ml) .
  • IL-15 was undetectable in the supernatants from cultures of CAR-NKT wo IL-15 and NKT cells alone (data not shown) .
  • FIG. 23A As shown in FIG. 23A, on day 6, cell viability of NKT and CAR-NKT wo IL-15 decreased compared to day 0, whereas both metrics increased for CAR-NKT-IL-15 and the positive control groups treated with recombinant IL-15 protein, indicating that IL-15 enhanced survival of the CAR-NKT cells. Consistent results were obtained with total cell counts (data not shown) .
  • CAR-NKT wo IL-15 and CAR-NKT-IL-15 along with CAR-T cells prepared from healthy donor T cells with CD70 targeting CAR alone (SEQ ID NO: 30) , were separately co-cultured with CD70-expressing Raji cells at a 1: 1 ratio. Raji cells were added on day 0 and day 6, and the expansion of CAR-NKT or CAR-T cells in response to stimulation by Raji target cells was measured.
  • CAR-NKT-IL-15 demonstrated superior proliferative capacity compared to CAR-NKT wo IL-15 and to CART cells.
  • mice were injected with PBS, NKT, 5x10 ⁇ 6 cell/mouse Armored CAR-NKT, or 25x10 ⁇ 6 cell/mouse Armored CAR-NKT.
  • Each group consisted of 6 mice for measuring tumor volume twice weekly post-cell injection, and blood samples were taken weekly to detect the persistence of CAR-NKT in mouse peripheral blood.
  • 27 more mice were included in the PBS, NKT, and 5x10 ⁇ 6 cell/mouse CAR-NKT groups, with 3 euthanized weekly for 7 weeks for sampling tissue organs to detect the tissue distribution of CAR-NKT.
  • Antibodies used in this study included: PerCP-Cy 5.5 Mouse Anti-Human CD45 (BD, 564105) , APC/Cyanine7 anti-human CD3 Antibody (BioLegend, 300318) , BV510 Mouse Anti-Human CD4 (BD, 562970) , PE-Cy 7 Mouse Anti-Human CD8 (BD, 557746) , APC Streptavidin (BioLegend, 405207) , Biotinylated CD70 Protein (Acrobiosystems, CDL-H82D7-200ug) , PE anti-human TCR V ⁇ 24-J ⁇ 18 (iNKT cell) Antibody (BioLegend, 342904) .
  • both dosage groups of Armored CAR-NKT showed significant tumor suppression effects, with tumors completely disappearing in 4 out of 6 mice in the high-dose group post-treatment, and noticeable suppression of tumor growth in the low-dose group.
  • the survival period was extended in both dosage groups injected with Armored CAR-NKT compared to the control groups of PBS and NKT.
  • CAR-NKTPeripheral Blood Expansion Blood samples were collected at different time points post infusion, and flow cytometry was used to detect the proportion of CAR-NKT cells in mouse peripheral blood, which reflected the expansion and persistence of CAR-NKT in the mouse body. As shown in FIG. 24C, CAR-NKT cells were detected in blood samples of mice of both dosage groups receiving the Armored CAR-NKT treatment, demonstrating long persistence and significant expansion of the CAR-NKT cells in the mouse body. In the high-dose group, CAR-NKT cells exceeding 10 ⁇ 6 cells/ml (dashed line position) were detected in peripheral blood on day 92. These results were consistent with the efficacy outcomes, indicating that the effective expansion of CAR-NKT cells in the body directly correlated with their anti-tumor efficacy.
  • Tissue Copy Number Proportion Tissue Copy Number/Total Copy Number x 100% (FIG. 24E) .
  • CAR-NKT mainly concentrated in tumors, with the proportion of CAR copy numbers in tumors reaching over 80%of the total by day 35.
  • Armored CAR-NKT cells (SEQ ID NO: 34) were prepared using NKT cells derived from 5 different donors (0101, 0102, 0103, 0105, 0106) .
  • NKT cells sourced from 4 different donors were used to prepare Armored CAR-NKT (SEQ ID NO: 34) .
  • Monocytes were cultured for 6 days at 5 ⁇ 10 ⁇ 5 cells per well in RPMI 1640 medium (Gibco, Cat#C11875500CP) supplemented with 20 ng/ml GM-CSF (Invitrogen, Cat#RGMCSF20) and 10%FBS (Thermo Fisher, Cat#A5669701) . At the end of the 6-day monocyte culture, the majority of the cells (>60%) were macrophages.
  • macrophages (CD45+CD11b+) were gated into M1 (CD80+CD86+) , and M2 (CD163+CD206+) ; each data point in the figure represented a different donor.
  • M1 accounted for only 1.6 ⁇ 1.0%
  • M1 accounted for 29.3 ⁇ 13.6%
  • M1 accounted for 70.4 ⁇ 2.8%and 85.2 ⁇ 6.8%, respectively.
  • CCR7 and CD45RA were used to differentiate the T cell subtypes at various stages of differentiation: Tn (CCR7+CD45RA+) , Tcm (CCR7+CD45RA-) , Tem (CCR7-CD45RA-) , and Temra (CCR7-CD45RA+) .
  • the antibodies used in the detection included: FITC anti-human CD27 (Biolegend 356404) , BV510 anti-human CD28 (BD 563075) , PE-Cy7 anti-human CCR7 (Biolegend 353226) , APC anti-human CD45RA (BD 550855) , PerCP-Cy5.5 anti-human CD62L (Biolegend 304824) , PE Streptavidin (BD 554061) , Biotinylated CD70 Protein (Acrobiosystems, CDL-H82D7-200ug) , and BV605 anti-human TCR V ⁇ 24-J ⁇ 18 (iNKT cell) Antibody (BioLegend, 342930) .
  • CD27 positive CAR-NKT cells As shown in FIG. 27A, after co-culturing with CD70-positive 786-O target cells, there was a substantial up-regulation of CD27 positive CAR-NKT cells, and the percentages of CD27/CD28 double-positive cells were up-regulated in CAR-NKT cells.
  • FIG. 27B in the CD27 positive CAR-NKT cells, CCR7 positive cells were up-regulated (left) , indicating an increase in naive and central memory T cells, Tn and Tcm, whereas CD27-negative CAR-NKT cells expressed very low levels of CCR7.
  • This example describes interim findings from an investigator-initiated trial (IIT) of Armored CAR-NKT, an autologous anti-CD70 CAR-NKT product, in patients with refractory metastatic clear cell renal cell carcinoma (ccRCC) .
  • IIT investigator-initiated trial
  • ccRCC metastatic clear cell renal cell carcinoma
  • This is a single-arm, open-label IIT (NCT06182735) using 3+3 design to evaluate three dose levels of Armored CAR-NKT: 5 ⁇ 10 ⁇ 6/m 2 (DL1) , 1.5 ⁇ 10 ⁇ 7/m 2 (DL2) , and 4.5 ⁇ 10 ⁇ 7/m 2 cells (DL3) .
  • Armored CAR-NKT cells were manufactured as described in examples above, harvested and cryopreserved in injectable freezing medium. Functional assays were performed as described in Example 4 and all cell products showed cytotoxicity against CD70-positive 786-O cells, constitutive secretion of IL-15, and target-cell-
  • the overall response rate (ORR) was 50% (2/4) in all evaluable patients, and 66.7% (2/3) in CD70-positive patients.
  • 0101 and 0103 patients had deep tumor reduction of-64.8%at Week 36 and-80.4%at Week 20, respectively.
  • all CD70-positive patients had low CD70 expression (H-score ⁇ 100) in ccRCC, demonstrating efficacy of Armored CAR-NKT even in CD70-low patients.
  • Table 10 Tumor Response in IIT* *: CT imaging was performed and results were assessed using RECIST 1.1; **: d, day; w, week.
  • CD70-positive T cells in the peripheral blood decreased significantly after Armored CAR-NKT infusion and remained at a low level up to Week 20, evidencing a durable activity of Armored CAR-NKT.
  • CD27 positive CAR-NKT cells were measured by flow cytometry in drug products (DP) and in patients’ peripheral blood following infusion. It was also found that, after infusion into patients, CD27 was substantially up-regulated in CAR-NKT cells and positive CD27 positive percentage increased over time (FIG. 30A) . While CD27 was detected in a moderate percentage ( ⁇ 40%) of cells from different patients; >90%CAR-NKT cells in peripheral blood at the time of CAR peak expansion expressed CD27, much higher than CD27+%in bulk T cells in patients’ blood.
  • CD27+CAR-NKT cells were analyzed using anti-CCR7 and anti-CD45RA to differentiate the T cell subtypes at various stages of differentiation: Tn or naive T cells (CCR7+CD45RA+) , Tcm or central-memory T cells (CCR7+CD45RA-) , Tem or effector-memory T cells (CCR7-CD45RA-) , and Temra or terminally-differentiated effector-memory T cells (CCR7-CD45RA+) . As shown in FIG.
  • CD27+CAR-NKT cells contained 1.83%Tn, 23.85%Tcm, and 74.31%Tem; CD27-CAR-NKT cells only contained Tem and did not have Tn or Tcm, indicating that CD27+CAR-NKT had greater stem-memory phenotype compared to CD27-CAR-NKT.
  • This example describes in vitro data showing that using different signal peptides (SP) for IL-15 in the expression cassettes affected the levels of IL-15 secreted by NKT or regular T cells, which affected efficacy of the cell therapy products.
  • IL-4 signal peptide SEQ ID NO: 50
  • IgK signal peptide SEQ ID NO: 51
  • GM-CSF signal peptide SEQ ID NO: 52
  • SEQ ID NO: 27 wild-type IL-15 signal peptide
  • CD70 CAR-IL-15 NKT Armored CAR-NKT with different signalpeptidesfor IL-15:
  • Armored CAR-NKT cells were manufactured from two healthy donors labeled 1172W and 2041W. During manufacturing, cell culture supernatants from NKT and Armored CAR-NKT with different signal peptides for IL-15 were collected, and IL-15 concentrations were measured using ELISA. As shown in FIG. 31, the Armored CAR-NKTs had enhanced IL-15 secretion when IL-15 SP was replaced with IL-4 SP or IgK SP IL-15.
  • Co-culture of Armored CAR-NKTs with tumor target cells were performed by plating 1E4 target cells with 1E4 Armored CAR-NKT cells in 200 ⁇ l in a 96-well plate, and incubating for 48 hours. Supernatants were collected and IL-15 levels were measured using ELISA method. As shown in FIG. 32, when these Armored CAR-NKTs were co-cultured with CD70-positive target tumor cells 786-O++and Raji, similar trends of IL-15 secretion were observed. Both IL-4 SP and IgK SP resulted in increased levels of IL-15 compared to wild-type SP. These data showed that IL-15 secretion level could be enhanced by using different single peptides.
  • Additional Armored CAR-NKTs containing other CD70 binder e.g., anti-CD70 VHH
  • CD70 binder e.g., anti-CD70 VHH
  • results consistent with those described above obtained the Armored CAR-NKTs having the anti-CD70 scFv 41D12) were obtained (data not shown) .
  • FOLR1 CAR-NKT cells were prepared similarly as described in Example 1, except without CD70 knock-out step.
  • Viruses containing FOLR1 CAR-IL-15 sequences (SEQ ID NO: 63 for wt SP, SEQ ID NO: 64 for IL-4 SP, SEQ ID NO: 65 for IgK SP) were used.
  • CAR-T cells were manufactured by stimulating PBMCs with CD3/28 beads (Thermo, Cat#40203D) , and cells were transduced by lentiviral vectors containing GPC3 CAR-IL-15 sequences with different signal peptides (SEQ ID NO: 66 for wt SP, SEQ ID NO: 67 for IL-4 SP, SEQ ID NO: 68 for IgK SP) .
  • This example describes in vivo data showing that using different signal peptides (SP) for IL-15 in the expression cassettes affected the levels of IL-15 secretion, which resulted in different efficacy or properties of the cell therapy products. Sequences used in the viral vectors were the same as in the example above (Example 13) .
  • CD70 CAR-IL-15 NKT Armored CAR-NKT
  • Signal Peptidesfor IL-15 :
  • the Armored CAR-NKT cells with xSP-IL-15 sequences were evaluated in the same animal model as in Example 5.
  • PBS approximately 5x10 ⁇ 6/mouse CAR-NKTs or control NKTs were injected intravenously; each treatment group contained 6 mice.
  • Mouse samples were analyzed using the same methods as described in Example 10.
  • mice were sacrificed, and the tumors were processed for DNA extraction and qPCR to detect CAR copy numbers.
  • Table 12 displays CAR copy number per microgram of DNA from tumor in different groups.
  • IL-4 SP and IgK SP groups had very high CAR copy numbers which were 2 orders of magnitude higher than that in wt SP.
  • the efficacy correlated with IL-15 secretion when using these different SPs (Example 13) , indicating that greater IL-15 secretion achieved by changing SP could lead to higher efficacy and/or tumor infiltration of cell therapy products in vivo.
  • NKTs were co-transduced with a FOLR1 CAR-IL-15 with varying xSP-IL-15, and GFP-luciferase (GFP-luc) using retroviral vectors.
  • Female NOG mice were divided into two groups, tumor-free and tumor-bearing, respectively. Both groups were injected via the tail vein with 5E6/mouse GFP-luc labeled control NKT cells or CAR-IL-15 NKT cells (wt SP-IL-15, IL-4 SP-IL-15, IgK SP-IL-15) , and the tumor-bearing group were also injected via the tail vein with 2E6 SK-OV-3 tumor cells 7 days before NKT/CAR-NKT injection. Each group contained 9 mice.
  • mice On the 1st, 5th, 8th, 12th, 15th, and 19th days after the injection of GFP-luc labeled NKT cells and CAR-NKT cells, the mice were injected intraperitoneally with the luciferase substrate D-Luciferin potassium salt, and bioluminescent imaging was performed using the PekingElmer IVIS LuminaIII to assess the proliferation and persistence of CAR-NKT cells in the mice. Plasma IL-15 concentrations were determined using ELISA from blood samples collected on Day 19 from the tumor-bearing group.
  • FIGs. 38A-38B bioluminescence measured at each time point showed the levels of NKT or CAR-NKT cells persisted in mice.
  • IL-4 SP enhanced the ability of NKTs to expand and persist compared to wt SP; this effect was more prominent in tumor-bearing mice.
  • FIG. 38C plasma IL-15 levels were also elevated in the IL-4 SP group compared to other CAR-NKT groups.

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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

La présente invention porte sur des lymphocytes T tueurs naturels (NKT) modifiés exprimant des récepteurs antigéniques chimériques (CAR) ciblant CD70, dans lesquels l'expression endogène de CD70 est réduite ou éliminée. Ces lymphocytes CAR-NKT peuvent proliférer in vivo et persister pendant une longue période, exerçant ainsi d'importants effets suppresseurs de tumeurs. La présente invention concerne également des peptides IL-15 à biodisponibilité améliorée, des cellules immunitaires modifiées exprimant ces peptides IL-15. La présente invention concerne également les procédés de fabrication des cellules immunitaires NKT ou IL-15 ciblées sur le CD70, ainsi que l'utilisation de ces cellules, par exemple, dans le traitement du cancer.
PCT/CN2024/098303 2023-06-09 2024-06-09 Cellules immunitaires modifiées et leurs utilisations Pending WO2024251290A1 (fr)

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CN119912588B (zh) * 2025-04-01 2025-08-12 浙江大学 嵌合抗原受体、人诱导多能干细胞、髓系前体细胞样巨噬细胞及其制备方法

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