WO2024054527A1 - Cellules myéloïdes dérivées de la moelle osseuse génétiquement modifiées pour le traitement de tumeurs du système nerveux central - Google Patents

Cellules myéloïdes dérivées de la moelle osseuse génétiquement modifiées pour le traitement de tumeurs du système nerveux central Download PDF

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WO2024054527A1
WO2024054527A1 PCT/US2023/032123 US2023032123W WO2024054527A1 WO 2024054527 A1 WO2024054527 A1 WO 2024054527A1 US 2023032123 W US2023032123 W US 2023032123W WO 2024054527 A1 WO2024054527 A1 WO 2024054527A1
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cells
gemys
glioma
tumor
cell
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Prajwal RAJAPPA
Alessandro CANELLA
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The Research Institute At Nationwide Children's Hospital
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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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
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    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • LGG represents a heterogeneous group of primary tumors derived from neuroglial cells (Youssef, G. and J.J. Miller, Curr Neurol Neurosci Rep, 2020. 20(7): p. 21), and in LGG AYA patients the most frequent histological types are astrocytoma (pilocytic grade I, diffuse grade II) and oligodendroglioma (grade II). Papageorgiou, JCO Oncol Pract., 2020, 16(4): p. 155-162. In these patients, the five-year progression free survival (PFS) after total resection is above 90%, but it significantly decreases to 55% in those with residual disease. Wisoff, J.H., et al., Neurosurgery, 2011.
  • PFS progression free survival
  • the glioma microenvironment is a pro- tumoral tridimensional architecture mainly constituted by a network of cancer cells, stromal cells (immune cells, fibroblasts, endothelial cells), blood vessels, and extracellular matrix.
  • the stromal components of the TME support homing and proliferation of the cancer cells through the release of pro- or anti-inflammatory chemokines, exosomes, pro-angiogenetic factors, prosurvival chemokines, and growth factors. Guo, S. and C.X.
  • the TME assists in the development of immune escape mechanisms that protect the cancer cells during tumor progression by hindering cancer immune surveillance, led by innate and adaptive immune cells.
  • Cancer cells facilitate the immunosuppressive reprogramming of myeloid cells infiltrating the TME (Bejarano et al., Cancer Discov, 2021. 11(4): p. 933-959) through the release of cytokines (IL4, IL10, and TGFP), restricting the recruitment and activation of cytotoxic effector immune cells, such as cytotoxic T lymphocytes (CD3+CD8+, CTLs), dendritic (DCs) and natural killer (NK) cells.
  • cytotoxic T lymphocytes CD3+CD8+, CTLs
  • DCs dendritic
  • NK natural killer
  • glioma tumor progression is characterized by a significantly increased infiltration of myeloid cells and a reduction of infdtrating cytotoxic T and NK cells in the TME. Grabowski et al., J Neurooncol, 2021. 151(1): p. 3-12. In fact, approximately 50% of the immune cells in the TME are myeloid cells, either resident (microglia) or recruited from the bone marrow (bone marrow-derived myeloid cells - BDMCs).
  • Myeloid cells in the TME can have a range of pro-activating (Ml -like) or immunosuppressive (M2-like) phenotypes (Gabrilovich, D.I. and S. Nagaraj, Nat Rev Immunol, 2009. 9(3): p. 162-74), and the balance between Ml-like and M2-like myeloid cells, together with the infiltration of immunosuppressive regulatory T cells (Tregs) in the tumor niche, are clinically prognostic.
  • Ml -like pro-activating
  • M2-like immunosuppressive phenotypes
  • IL2 The interaction between IL2 and its receptor triggers JAK-STAT and Mtor signaling pathways associated with cellular growth, proliferation, and cell cycle progression to drive the cellular differentiation from the naive to the effector T cell phenotype.
  • JAK-STAT and Mtor signaling pathways associated with cellular growth, proliferation, and cell cycle progression to drive the cellular differentiation from the naive to the effector T cell phenotype.
  • excessive or prolonged IL2 stimulation of T lymphocytes can affect the activation status and lead to T cell exhaustion or inactivation. Liu, Y., et al., Nat Immunol, 2021. 22(3): p. 358-369.
  • Myeloid cells have previously modified to express and release other cytokines, such as inlerleron-y. IL-9, IL-15, IL-18, and IL-21, which are involved in the immune activation and recruitment of myeloid and cytotoxic lymphoid cells, to improve the killing of tumor cells and prevent glioma progression.
  • cytokines such as inlerleron-y.
  • IL-9, IL-15, IL-18, and IL-21 which are involved in the immune activation and recruitment of myeloid and cytotoxic lymphoid cells, to improve the killing of tumor cells and prevent glioma progression.
  • IL-2 cytotoxic lymphoid cells
  • Gliomas are the most prevalent type of brain tumors and one of the leading causes of cancer related death in the adolescent and young adult population (AYA).
  • AYA adolescent and young adult population
  • LGGs low-grade gliomas
  • TME tumor microenvironment
  • Figures 1A-1G provide graphs and images showing the glioma progression from the low- to high-grade affect trafficking and activation of CD3+ T cells in vivo.
  • A)-B) Analysis of the tumor infiltrating immune cells by CyTOF in no tumor (NT), low-grade (LGG), and highgrade glioma (HGG) RCAS/t-va murine model (n 3).
  • TILs tumor infiltrating T lymphocytes
  • C IPA graphical summary of the most important downregulated pathways.
  • D IPA analysis of the TILs trafficking (p ⁇ 0.05).
  • E IPA analysis of the TILs tissue morphology (p ⁇ 0.05).
  • F IPA analysis of the dysregulated pathways associated with T cell activation and proliferation.
  • Figures 2A-2F provide graphs and images showing the generation of bone marrow- derived mature myeloid cells, engineered for the release of IL2 for the treatment of LGGs in vivo, and clone selection.
  • C) Quantification by ELISA of the IL2 secreted in the supernatant of GEMys in culture for 3 days post-LV infection (n 6).
  • Figures 3A-3F provide graphs and images showing the characterization of the myeloid cell composition of GEMys-EV and GEMys-IL2 and the assessment of activation of primary murine CD8+ T cells in vitro.
  • A) CyTOF analysis of the composition of myeloid cells in GEMys-EV and GEMys-IL2 cultivated in selection media (puromycin Ipg/ml, n 3).
  • B) Evaluation of the average of the percent of myeloid cells in GEMys-EV and IL2 (n 6).
  • F Representation of the main goal of the novel cell-based immunotherapy concept in vivo. Unpaired two-tailed student’s t-test calculated statistical significance. *P ⁇ 0.05, **P ⁇ 0.01.
  • FIGS 4A-4I provide graphs and images showing the treatment of RCAS/t-va LGG animals with with a single dose of GEMys-IL2 impact on the transcriptome of tumor infiltrating immune cells.
  • LGG animals were treated systemically by retro-orbital intravenous (IV) injection (80pl ) of GEMys-IL2 or vehicle (PBS).
  • IV intravenous
  • PBS vehicle
  • Plasma samples, brain tissues and tumor infiltrating immune cells were analyzed 3-5 days post-treatment
  • B) ELISA quantitation of circulating IL2 in the peripheral blood at day 5 (n 6).
  • E) RT-qPCR evaluation of LAG3, Tim-3, and FoxP3 in immune cells infiltrating the TME (n 4-6). In D) and E) experiments samples were collected at day 4 posttreatment.
  • G Summary of the gene enrichment analysis by IPA.
  • H Volcano plot of the differential gene expression of tumor infiltrating immune cells. In total 16691 genes. In red are the genes significantly upregulated, and in blue are the genes significantly downregulated. Unpaired two-tailed student’s t-test calculated statistical significance. *P ⁇ 0.05, **P ⁇ 0.01. 1) IPA prediction analysis of the top 25 upregulated (red) and 25 downregulated (blue) upstream regulators. The calculation of p-values with IPA is performed by right-tailed Fisher Exact Test.
  • Figures 5A-5F provide graphs showing the treatment of RCAS/t-va LGG animals with a single dose of GEMys-IL2 modify the composition and activation of tumor infiltrating immune cells and improve the survival.
  • LGG animals treated by retro-orbital intravenous (IV) injection (80 pl) with 8xl0 6 GEMys-IL2 or vehicle (PBS).
  • IV intravenous
  • PBS vehicle
  • IPA enrichment analysis of signaling pathways and biological functions associated with A) Cytotoxic CD8+ T lymphocytes, B) Cytotoxic Natural killers at day 3 post-treatment.
  • A)-B) p-values were calculated by right-tailed Fisher Exact Test.
  • D) Characterization of the tumor infiltrating immune cells CD45+, T, and NK cells (n 3-4) at day 4 post-treatment with GEMys-IL2 by mass cytometry. Unpaired two-tailed student’s t test calculated statistical significance. *P ⁇ 0.05, **P ⁇ 0.01.
  • FIGS 6A-6H provide graphs and images showing analysis of the tumor microenvironment of RCAS/t-va LGG animals treated with a single dose of vehicle or GEMys- IL2.
  • A) IXMC -based molecular imaging analysis of GFP+ cells quantification in brain sections of LGG animals at day 5 post-engraftment with vehicle (PBS) or GEMys-GFP (n 3). Dots represent the GFP intensity colocalized with nuclear staining in 78 (PBS) and 443 (GEMys- GFP) microscopic fields.
  • B) Quantification of TME infiltration by CD45+GFP+ cells by flow cytometry (n 4).
  • D) Characterization of the tumor infiltrating regulatory T lymphocytes (Tregs, CD45+CD25hiFoxP3+, n 4) at day 4 post-treatment with GEMys-IL2 by mass cytometry.
  • E) Cytokine analysis of the total protein extract from the TME of LGG animals treated with vehicle (PBS, upper panel), or GEMys-IL2 (lower panel) (n 4).
  • Figures 7A-7E provide graphs showing the tmpact of the treatment with a single dose of GEMys -IL2 on the transcriptome of tumor infiltrating immune cells in vivo.
  • IP A analysis of differential expressed genes in TME immune cells at day 3 post-treatment with Vehicle, or with 8xlO6GEMys-IL2 (80pl, n 3). Bar graphs represent the gene enrichment analysis related to signaling pathways and biological functions posttreatment of A) Macrophages, B) Granulocytes, C) Monocytes, D) T helper (CD4+) and E) Dendritic cells (DCs), p-values were determined by right-tailed Fisher Exact Test.
  • the present invention provides a method of treating or preventing central nervous system cancer in a subject in need thereof is described.
  • the method includes administering to the subject a therapeutically effective amount of myeloid cells modified to express interleukin- 2 (IL-2).
  • IL-2 interleukin- 2
  • a population of genetically engineered myeloid cells (GEMys) comprising bone marrow derived myeloid cells that have been genetically modified to express IL-2 is also provided.
  • ‘A” or “an” means herein one or more than one; at least one. Where the plural form is used herein, it generally includes the singular.
  • the term “subject” can refer to any warm-blooded organism including, but not limited to, human beings, rats, mice, dogs, goats, sheep, horses, monkeys, apes, pigs, rabbits, cattle, etc.
  • a subject in need thereof When the term is used in the context of a subject needing or requiring compositions of the present application, the term may be referred to as “a subject in need thereof” and include subjects that have been clinically diagnosed (e.g., by a medical professional, e.g., a physician) as being in need of compositions of the present application, subjects that are suspected of being in need of compositions of the present application, subjects at risk for a disease or condition and who may benefit from compositions of the present application, and subjects that are already suffering from a disease or condition and who may benefit from compositions of the present application.
  • a medical professional e.g., a physician
  • the term "pharmaceutically acceptable,” as used herein, refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the term “therapeutically effective amount” is intended to qualify the number or amount of an agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies. A therapeutically effective amount may be administered in one or more doses. Treatments that are therapeutically effective within the meaning of the term as used herein, include treatments that improve a subject's quality of life even if they do not improve the disease outcome per se.
  • an “effective amount” generally means an amount which provides the desired local or systemic effect, e.g., effective to stimulate interleukin release, including achieving the specific desired effects described in this application.
  • an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result.
  • ‘Treat”, “treating”, and “treatment”, etc., as used herein, refer to any action providing a benefit to a subject at risk for or afflicted with a condition or disease such as cancer, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, prevention or delay in the onset of the disease, etc.
  • Preventing refers to any action that decreases the risk that a subject will develop cancer, or that will decrease the risk of progression from a low-grade cancer to a high-grade cancer. Preventing infection can be done in subjects who have an increased risk of developing cancer. Subjects can have an increased risk of developing cancer (e.g., central nervous system cancer) as a result of, for example, radiation exposure, genetic disorder, a family history of CNS tumors, immunodeficiency, stress and a history of previous cancers.
  • cancer e.g., central nervous system cancer
  • cytokine refers to a small protein (-5-20 kDa) that is important in cell signaling, and in particular immunomodulation.
  • cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors.
  • One aspect of the invention provides a method of treating or preventing central nervous system cancer in a subject in need thereof.
  • the method includes administering to the subject a therapeutically effective amount of myeloid cells modified to express interleukin-2 (IL-2).
  • Myeloid cells can be modified to express increased cytokine (e.g., IL-2) levels by various means, such as cell culturing, exposure to infection, exposure to other cytokines, exposure to drugs known to stimulate cytokine release (e.g., various monoclonal antibodies), or by using genetic engineering to increase cytokine (e.g., IL-2) release.
  • the modified myeloid cells are genetically engineered myeloid cells (GEMys) that have been genetically modifed to include an IL-2 gene and in particular an exogenous IL-2 gene.
  • GEMys genetically engineered myeloid cells
  • the myeloid cells are modified to express increased levels of IL-2 relative to their baseline levels of IL-2 release.
  • the modified myeloid cells can be used to treat or prevent central nervous system cancer in a subject.
  • Cancer as defined herein, is a disease based on the development of cells that contain genetic damage resulting in the relatively unrestrained growth of the cells. The genetic damage present in a cancer cell is maintained as a heritable trait in subsequent generations of the cancer cell line. Cancer is generally named based on its tissue of origin. Cancer which has metastasized will typically retain traits associated with its tissue of origin.
  • Central nervous system (CNS) cancer is cancer that occurs in a part of the central nervous system, which is made up of the spinal cord and the brain. Parts of the central nervous system include the cerebrum, the cerebellum, the brain stem, the meninges, and the spinal cord. CNS cancers include medulloblastoma, glioma, astrocytoma, oligodendroglioma, germ cell tumor, and ependymoma. In some embodiments, the CNS cancer is a glioma.
  • a glioma is a type of tumor that starts in the glial cells of the brain or the spine. Symptoms of a glioma vary depending on the specific location. Symptoms of brain gliomas include headaches, vomiting, seizures, and cranial nerve disorders as a result of increased intracranial pressure, whereas a glioma of the optic nerve can cause visual loss while spinal cord gliomas can cause pain, weakness, or numbness in the extremities.
  • the subject has been diagnosed as having CNS cancer.
  • a variety of methods can be used to diagnose a CNS tumor or cancer, including medical history, blood tests, urine tests, medical imaging (e.g., MRI or CT scan), X-ray, and biopsy.
  • Biopsy is the preferred method for diagnosing CNS cancer and can be carried out using sterotactic surgery to avoid risk of damage to the brain.
  • Gliomas are classificed by the type of cell, by grade, and by location. The grade of a glioma is determined by pathological evaluation of the tumor.
  • Grades of gliomas include biologically benign gliomas, low-grade gliomas, and high-grade gliomas (i.e., glioblastomas).
  • Low-grade gliomas are well-differentiated (i.e., not anaplastic) and tend to exhibit benign tendencies and are associated with a better prognosis for the patient.
  • High-grade gliomas are undifferentiated or anaplastic, are malignant, and carry a worse prognosis.
  • the subject has been diagnosed as having a low-grade glioma.
  • treatment prevents progression of the tumor from low-grade to high-grade glioma.
  • the method delays malignant progression from low-grade to high grade glioma. Because low-grade glioma is relatively benign, preventing malignant progression to high-grade glioma can significantly improve the prognosis of a patient having glioma. IL-2 is known to be downregulated in low-grade glioma.
  • Treatment of cancer comprises, but is not limited to, destroying tumor cells, reducing tumor burden, inhibiting tumor growth, reducing the size of the primary tumor, reducing the number of metastatic lesions, increasing survival of the individual, delaying, inhibiting, arresting or preventing the onset or development of metastatic cancer (such as by delaying, inhibiting, arresting or preventing the onset of development of tumor migration and/or tumor invasion of tissues outside of primary cancer and/or other processes associated with metastatic progression of cancer), delaying or arresting primary cancer progression, improving immune responses against the tumor, improving long term memory immune responses against the tumor antigens, and/or improving the general health of the patient with illness.
  • tumor cell death can occur without a substantial decrease in tumor size due to, for instance, the presence of supporting cells, vascularization, fibrous matrices, etc. Accordingly, while reduction in tumor size is preferred, it is not required in the treatment of cancer.
  • CNS cancer Prevention of CNS cancer can be provided for subjects who have an increased risk of developing CNS cancer (e.g., glioma).
  • a subject may have an increased risk of developing CNS cancer if they have one or more risk factors for CNS cancer.
  • risk factors include radiation exposure, a family history of brain tumors, including genetic disorders such as neurofibromatosis types 1 and 2, tuberous clerosis, Von Hippel-Lindau syndrome, Li- Fraumeni syndrome, Turcot syndrome, Gorlin syndrome, and Cowden syndrome, having a weakened immune system, exposure to environmental toxins such as vinyl chloride, or possible high levels of cell phone use.
  • the modified myeloid cells e.g., GEMys cells
  • the effectiveness of cancer treatment may be measured by evaluating a reduction in tumor load or decrease in tumor growth in a subject in response to the administration of the modified myeloid cells.
  • the reduction in tumor load may be represent a direct decrease in mass, or it may be measured in terms of tumor growth delay, which is calculated by subtracting the average time for control tumors to grow over to a certain volume from the time required for treated tumors to grow to the same volume.
  • cancer treatment or prevention using modified myeloid cells is combined with another form of cancer treatment suitable for treating central nervous system cancer.
  • Additional methods of cancer treatment include radiation therapy, chemotherapy, targeted drug therapy (e.g., bevacizumab), radiofrequency ablation, cryoablation, thermal ablation, electroporation, alcohol ablation, high intensity focused ultrasound, photodynamic therapy, hormone-blocking therapy, oncolytic virus treatment, chimeric antigen receptor (CAR) T-Cell therapy, administration of monoclonal antibodies, and administration of immunotoxins.
  • Another aspect of the invention provides a population of genetically engineered myeloid cells (GEMys) comprising bone marrow derived myeloid cells that have been genetically modified to express interleukin-2 (IL-2). More specifically, the myeloid cells have been genetically modifed to include an IL-2 gene and in particular an exogenous IL-2 gene, as well as a suitable expression control sequence.
  • a population of cells refers to a plurality of cells. In some embodiments, the population of cells includes a number of cells sufficient to provide a therapeutically effective dose for treating or preventing central nervous system cancer. In further embodiments, the population of cells includes a plurality of different types of myeloid cells.
  • Granulocytes, monocytes, macrophages, and dendritic cells represent a subgroup of leukocytes, collectively called myeloid cells. Prinz et al., Nat Immunol., 22;18(4):385-392 (2017). Myeloid cells are initially formed in the embryo, but various embodiments of the invention are directed to the use of myloid cells are bone marrow-derived.
  • the myeloid cells comprise macrophages, granulocytes, monocytes, neutrophils, basophils, eosinophils, mast cells, and myloid precursors.
  • the GEMys cell population can also include a small amount of lymphoid cells, such as T lymphocytes, B lymphocytes, and natural killer cells. See Table 1, provided herein.
  • the myeloid cells comprise monocytes, eosinophils, mast cells, and myloid precursor cells. In further embodiments, at least 10%, at least 15%, at least 20%, or at least 25% of these cells (independently for each cell type) are present in the GEMys cell population. The percentages are relative to total cell numbers present in the population.
  • the myeloid cells can be allogenic or autologous cells.
  • the myeloid cell e.g., GEMys cell
  • the myeloid cell is a mammalian myeloid cell.
  • mammals include primates (e.g., human), canines, felines, rodents, porcine, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats and mice.
  • Myeloid cells can be obtained from bone marrow.
  • the myeloid cells can be isolated using magnetic negative selection followed by cultivation in media.
  • Genetic engineering is the modification and manipulation of an organism's genes using technology. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. In the case of the present invention, myeloid cells are genetically modified to express IL-2.
  • a nucleic acid encoding the IL-2 can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcriptionbased amplification system (TAS), the self-sustained sequence replication system (3SR) and the QP replicase amplification system (QB).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcriptionbased amplification system
  • 3SR self-sustained sequence replication system
  • QB QP replicase amplification system
  • a polynucleotide encoding the polypeptide can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule.
  • a wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art.
  • the IL-2 gene can be operatively linked with an expression control sequence that has a regulatory element such as a promoter (constitutive or regulatable) to drive transgene expression and a polyadenylation sequence downstream of the nucleic acid.
  • the expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • Suitable promoters include, but are not limited to, a hVMD2 promoter, an SV40 early promoter, RSV promoter, adenovirus major late promoter, human CMV immediate early I promoter, poxvirus promoter, 30K promoter, 13 promoter, sE/L promoter, 7.5K promoter, 40K promoter, Cl promoter, and EF-la promoter.
  • Enhancer refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
  • a number of polynucleotides comprising promoters also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences.
  • the nucleic acid encoding the polypeptide can be operably linked to a CMV enhancer/chicken -actin promoter (also referred to as a “CAG promoter”).
  • the vector can comprise a reporter to identify the transfection/transduction efficiency of the vector.
  • exemplary reporters include, but are not limited to, EGFR and CD90.1.
  • genetic modification of myeloid cells can be carried out using an appropriate vector (e.g., a viral vector) to insert the interleukin-2 gene into the myeloid cells.
  • suitable vectors include plasmids (e.g., DNA plasmids), bacterial vectors (e.g., a Listeria or Salmonella vector), yeast vectors, and viral vectors.
  • the vector is a viral vector, such as retrovirus, poxvirus (e.g., an orthopox (e.g., vaccinia, modified vaccinia Ankara (MV A), Wyeth, NYVAC, TROYVAC, Dry-Vax, or POXVAC-TC), avipox (e.g., fowlpox, pigeonpox, or canarypox, such as ALVAC), raccoon pox, rabbit pox, capripox (e.g., goat pox or sheep pox), leporipox, or suipox (e.g., swinepox), adenovirus, adeno-associated virus, herpes virus, polio virus, alphavirus, baculorvirus, and Sindbis virus.
  • poxvirus e.g., an orthopox (e.g., vaccinia, modified vaccinia Ankara (MV A), Wyeth
  • Retroviral vectors including lentiviral vectors, are suitable delivery vehicles for the stable introduction of a variety of genes of interest into the genomic DNA of a broad range of target cells. Without being bound by theory, the ability of retroviral vectors to deliver unrearranged, single copy transgenes into cells makes retroviral vectors well suited for transferring genes into cells. Further, retroviruses enter host cells by the binding of retroviral envelope glycoproteins to specific cell surface receptors on the host cells.
  • pseudotyped retroviral vectors in which the encoded native envelope protein is replaced by a heterologous envelope protein that has a different cellular specificity than the native envelope protein (e.g., binds to a different cell-surface receptor as compared to the native envelope protein) also can be used.
  • retroviruses include: murine leukemia virus (MLV), lentivirus such as human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MS V), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
  • MMV murine leukemia virus
  • HMV human immunodeficiency virus
  • EIAV equine infectious anaemia virus
  • MMTV mouse mammary tumor virus
  • RSV Rous sarcoma virus
  • Fujinami sarcoma virus FuSV
  • retroviruses suitable for use include, but are not limited to, Avian Leukosis Virus, Bovine Leukemia Virus, and Mink-Cell Focus-Inducing Virus.
  • the core sequence of the retroviral vectors can be derived from a wide variety of retroviruses, including for example, B, C, and D type retroviruses, as well as spumaviruses and lenti viruses.
  • An example of a retrovirus suitable for use in the compositions and methods disclosed herein includes, but is not limited to, lentivirus.
  • One lentivirus is a human immunodeficiency virus (HIV), for example, type 1 or 2 (i.e., HIV-1 or HIV-2).
  • lentivirus vectors include sheep Visna/maedi virus, feline immunodeficiency virus (FIV), bovine lentivirus, simian immunodeficiency virus (SIV), an equine infectious anemia virus (EIAV), and a caprine arthritis-encephalitis virus (CAEV).
  • Customized vectors are commercially available. See for example VectorBuilder®.
  • Lentivirus vectors suitable for insertion of the IL-2 gene are also commercially available. For example, the Lenti-PacTM lentiviral packaging kit can be used.
  • Interleukin-2 is a cytokine signaling molecule in the immune system.
  • IL-2 has essential roles in key functions of the immune system, tolerance and immunity, primarily via its direct effects on T cells.
  • IL-2 promotes the differentiation of immature T cells, and increases the cell killing activity of both natural killer cells and cytotoxic T cells.
  • a variety of amino acid sequences for IL-2 are known.
  • the human amino acid sequence for interleukin-2 is provided by NCBI Reference Sequence NP_000577.2.
  • the GEMys cells should be administered and dosed in accordance with good medical practice, taking into account the site and method of administration, scheduling of administration, patient age, sex, body weight, the nature and severity of the disorder to be treated or prevented, and other factors known to medical practitioners.
  • the term "administer” refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced.
  • a GEMys cell population, as described herein, can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, and transdermal administration.
  • said dose is about 10 x 10 6 cells/kg of subject weight or lower, is about 9 x 10 6 cells/kg or lower, is about 8 x 10 6 cells/kg or lower, is about 7 x 10 6 cells/kg or lower, is about 6 x 10 6 cells/kg or lower, is about 5 x 10 6 cells/kg or lower.
  • said dose may be between about 0.25 x 10 6 cells/kg to about 5 x 10 6 cells/kg; or more preferably about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg.
  • the dose may be about 0.25 x 10 6 cells/kg, 0.5 x 10 6 cells/kg, 0.6 x 10 6 cells/kg, 0.7 x 10 6 cells/kg; 0.8 x 10 6 cells/kg; 0.9 x 10 6 cells/kg; 1.1 x 10 6 cells/kg; 1.2 x IO 6 cells/kg; 1.3 x IO 6 cells/kg; 1.4 x 10 6 cells/kg; 1.5 x 10 6 cells/kg; 1.6 x 10 6 cells/kg; 1.7 x 10 6 cells/kg; 1.8 x 10 6 cells/kg; 1.9 x 10 6 cells/kg or 2 x 10 6 cells/kg.
  • the dose may, in other embodiments, be between 0.1 and 1 million cells/kg; or between 1 and 2 million cells/kg; or between 2 and 3 million cells/kg; or between 3 and 4 million cells/kg; or between 4 and 5 million cells/kg; or between 5 and 6 million cells/kg; or between 6 and 7 million cells/kg; or between 7 and 8 million cells/kg; or between 8 and 9 million cells/kg; or between 9 and 10 million cells/kg.
  • Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
  • injection includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.
  • the compositions are administered by intravenous infusion or injection.
  • GEMys cells can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration.
  • the composition can be sterile.
  • the formulation should suit the mode of administration.
  • Cell Therapy Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, I. Lister & P. Law, Churchill Livingstone, 2000.
  • Choice of the cellular excipient and any accompanying elements of the composition comprising a population of GEMys cells will be adapted in accordance with the route and device used for administration.
  • the GEMys cells are administered together with a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.
  • Suitable preservatives and buffers can be used in such formulations.
  • such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.
  • HLB hydrophile-lipophile balance
  • the quantity of surfactant in such formulations ranges from about 5% to about 15% by weight.
  • Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.
  • sterile liquid carrier for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • the cells are administered by injection, e.g., intravenously.
  • the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA- LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate.
  • the pharmaceutically acceptable carrier is supplemented with human serum albumen.
  • the inventors evaluated a novel therapeutic concept to deliver bone marrow-derived myeloid cells (GEMys) genetically engineered for the stable expression and release of interleukin-2 (IL2) within the TME to promote the recruitment and activation of CTLs against tumor cells (Fig. 3F). They demonstrated that an intravenous injection of a single dose of GEMys-IL2 in LGG mice was sufficient to rapidly recruit and activate more CTLs and NKs in the TME.
  • the GEMys passed the blood brain barrier (BBB) and released IL2 in the glioma microenvironment, thus promoting inflammation, trafficking and activation of cytotoxic T, NK cells, and reprogramming the transcriptome of the immune cells.
  • BBB blood brain barrier
  • the GEMys-IL2 treatment in vivo delayed tumor progression and significantly improved the overall survival (OS) in LGG bearing animals.
  • glioma cells reprogram immune cells that are infiltrating the tumor microenvironment to trigger drug resistance, immune-evasion mechanisms, and develop an immunosuppressive phenotype, blocking the recruitment and activation of cytotoxic T and NK cells.
  • mass cytometry (CyTOF) analysis in brain tissue of RCAS/t-va animals bearing no tumor, LGG, and HGG showed significantly reduced infiltration of CD3, CD4, and CD8 after malignant progression from low- (LGG) to high-grade glioma (HGG) (Fig. 1A-B).
  • FIG. 1C-F the analysis highlighted the effect of tumor progression on the immunosuppression of the CD3+ cell compartment (T cells). Indeed, we found a strong downregulation of IFNy and a reduction of T cell trafficking (Fig. 1C). Furthermore, the gene enrichment analysis by disease and function showed a strong decrease of genes and pathways involved in activation, migration, and trafficking of T lymphocytes (Fig. ID, red arrows, Fig. IF) resulting in decreased tumor infiltration by T lymphocytes, particularly by CD8+ T cells (Fig. IE, red arrows). In addition, signaling pathways associated with T cell activation and proliferation were significantly downregulated (Fig. IF), such as the T cell receptor (TCR), and the PKC0 signaling pathways. These pathways are critical for effector T cell proliferation, activation and are also associated with naive T cell differentiation into effector T cells.
  • Fig. IF signaling pathways associated with T cell activation and proliferation were significantly downregulated (Fig. IF), such as the T cell receptor (TCR), and
  • GEMys-IL2 demonstrate stable expression of IL2 [0063] Three days post-isolation and infection, the GEMys-IL2 population showed a significant upregulation of IL2 gene expression (Fig. 2B) and IL2 secretion in the culture media (Fig. 2C). Unexpectedly, engineered IL2 secreting myeloid cells consistently expressed and secreted more IFNy than the GEMys-EV cells, and this indirect effect might potentiate the activation of tumor-infiltrating immune cells (Fig. 1A-E). Castro et al., Front Immunol 9, 847 (2016).
  • GEMys media enriched with puromycin Ipg/ml
  • RT-qPCR quantitative real-time PCR
  • GEMys-EV and GEMys-IL2 myeloid composition were characterized by a large immune CyTOF panel, and the percentage of myeloid precursors were assessed by flow cytometry (Table 1). Samples were gated on CD45+ singlets. Nine similar yet distinct clusters were identified via FlowSOM in both the GEMys-EV and GEMys-IL2 and confirmed by manual gating in OMIQ according to phenotypes reported by Becher B. et al. Nat Immunol 15, 1181-1189 (2014). As shown in Fig.
  • Basophils (2.19+0.28%, CD45+ CDllb+ B220- FcRela+), Eosinophils (18.87+4.71%, CD45+ CDllb+ SiglecF+), Mast cells (25.13+6.93%, CD45+ CDllb+/- FceRla+), Neutrophils (4.67+0.90%, CD45+ CDllb+ Ly6G+), Monocytes (19.27+3.25%, CD45+ CDllb+ Ly6G- CD62L+ SiglecF-), Macrophages (1.73+0.26%, CD45+ CDllb+ F4/80+), and Dendritic cells (1.91+2.31%, monocytic: CD45+ CDllb+ CDllc+ MHCII+, conventional: CD45+ CDl lb- CDl l c+ MHCII+, plasmacytoid: CD45+ CDl lb- CDl 1 c+ CD317+), and Myeloid precursor
  • the panel also assessed natural killer cells (0.33+0.39%, CD45+ CDllb- NKp46+), T cells (0.07+0.005%, CD45+ CDllb- CD3+), and B cells (0.002+0.001%, CD45+ CDllb- CD19+), but their numbers were collectively below 0.6% of the composition of both the GEMys-EV and GEMys-IL2 (Table 1).
  • approximately 2.89% of the CD45+ cells were unidentified myeloid cells (CD45+CD1 lb+). These cells expressed CD45 and CD1 lb, but none of the distinctive markers used for myeloid characterization (Fig. 3A-B; Table 1).
  • GEMys-IL2 activate primary murine CD8+ T cells in vitro
  • Cytotoxic T lymphocytes lead the adaptive immune anti-cancer response, and their activation is critical to effective immunotherapies.
  • GEMys-IL2 To test the ability of GEMys-IL2 to activate the target cells in vitro, we co-cultured primary murine CTLs with GEMys for 24h and evaluated the canonical markers associated with cytotoxic T cell activation by RT-qPCR (Fig. 3C).
  • Fig. 3C we also evaluated the expression of Lag3 and Tim-3 among the primary markers associated with T cells inactivation/exhaustion in the same experiment (Fig. 3D).
  • CD25, 4- IBB, and CD69 protein expression measured in CD3+CD8+ T cells co-cultured with GEMys-IL2 were on average respectively 2.0, 1.9 and 4.4-fold significantly higher compared to the levels in T cells treated with control GEMys-EV, thus confirming the results from gene expression (Fig. 3C).
  • GEMys-IL2 ratio 3:1
  • T cell activation markers showed a significant upregulation of IRF4 ( 1.4-fold) in T cells cultured in GEMys-IL2 media compared with T cells in GEMys control media (GEMys-EV).
  • the T cell exhaustion markers Lag3 and Tim-3 were not differentially expressed in CTLs cultivated in media isolated from GEMys-EV or -IL2, suggesting a supportive effect from secreted factors released by GEMys-IL2 on the activation status of CD3+CD8+ T cells.
  • GEMys maintained in culture with the same methods for experiments shown in Figure 3C-D did not show significant differential expression of genes associated with CD8+ T cell activation or exhaustion.
  • GEMys-IL2 are recruited and infiltrate the local glioma microenvironment
  • mice we evaluated the tumor burden at day 25 by measuring the intracranial emission of photons released by the cancer cells using in vivo imaging system (IVIS) to randomize the mice into three treatment groups. Each group was engrafted at day 28 by retro-orbital intravenous injection of PBS (vehicle), 8xl0 6 of GEMys-EV, or 8xl0 6 of GEMys-IL2 cells. Mice were monitored post-treatment daily to evaluate toxicity or stress induced by the treatment and the tumor burden was evaluated by IVIS post-initial GEMys inoculation and at seven days post-treatment. The systemic delivery of syngeneic myeloid cells was well tolerated by the animals, who showed no sign of stress, discomfort, or behavioral issues during the experiments.
  • IVIS in vivo imaging system
  • GEMys-IL2 regulate immune cell transcriptomes in the glioma microenvironment
  • RNAseq total RNA sequencing
  • PBS Vehicle
  • IP A Ingenuity Pathway Analysis software predicted the increased cytotoxicity and proliferation of T and NK cells, as summarized in Figure 4G.
  • IPA software analysis of the top 50 upstream regulators showed that the treatment in vivo of EGG with GEMys-IL2 was associated with the upregulation of IL2 and IFNy (pro-inflammatory and proactivators of cytotoxic T cells), but also the upregulation of STAT1 (Fig. 41, red arrows).
  • STAT1 is critical for the activation and recruitment of CD8+ T cells and for the inhibition of infiltrating suppressive myeloid cells in solid tumors6E
  • the IPA analysis also predicted the downregulation of the master regulators of immunosuppression in M2-like myeloid cells IL10RA, CITED2 and STAT3 (Fig. 41, blue arrows)62,63.
  • SOCS1 was predicted to be one of the top downregulated molecules after treatment ( Figure 4G, I).
  • SOCS1 is a specific inhibitor of the JAK/STAT pathway, and regulatory function is performed by specific binding with phosphorylated JAK molecules, followed by the recruitment of E3 ligases, and proteasome degradation. Sharma et al., Front Pharmacol 10, 324 (2019).
  • the gene enrichment analysis also demonstrated the significant upregulation of a network controlling the trafficking of immune cells and of genes involved in the regulation of the inflammatory response.
  • RNA sequencing signature of the tumor infiltrating immune cells we also assessed the expression of genes in the context of T and myeloid cell activation (Fig. 6C).
  • the analysis by RT-qPCR demonstrated the upregulation of CD25, CD69, STAT1, IRF4, and IL12 in immune cells isolated from the TME of LGG animals engrafted with GEMys-IL2, in comparison to vehicle (PBS).
  • CD25 and CD69 reached statistical significance.
  • the gene enrichment analysis investigated by IPA pointed out that the treatment of LGG animals with GEMys-IL2 triggered the upregulation of signaling pathways and biological functions associated with trafficking and activation of cytotoxicity in CD8+ cells (Fig. 5A). Comparable results were obtained for NK cells (Fig. 5B) and DC cells.
  • the recruitment of GEMys-IL2 at the TME was also associated with the reprogramming of immunosuppressive and M2-like macrophages to become more pro-inflammatory (Fig. 7A).
  • the software also predicted the upregulation of pathways and biological functions correlated with the activation and recruitment of granulocytes, monocytes, and T helper cells (Fig. 7B- 7D).
  • RNAseq signature did not produce any significant data in support of upregulation of pathways or biological functions involved in T cell inactivation, or exhaustion due to the treatment with GEMys-IL2.
  • GEMys-IL2 the transcriptome of immune cells in the tumor microenvironment of LGG mice can be significantly influenced by the recruitment of naive myeloid cells engineered for the release of the pro-inflammatory payload in the TME (GEMys-IL2).
  • GEMys-IL2 potentiate a pro-inflammatory immune cell composition within the glioma microenvironment
  • 5C pointed out the significant upregulation of cytokines in the TME involved in the trafficking of myeloid cells (CCL2, CCL22, CCL3, CXCL2), DCs (CCL2, CCL20, RBP4), NK (CCL2, CCL22, CXCL9, CXCL10) and T cells (CCL2, CCL22, CCL20, CXCL9, CXCL10, IGFBP-5, RBP4).
  • cytokines involved in the activation of myeloid cells CD14, M-CSF, IFNy
  • T cells TIM-1, IL2, IFNy, SAP
  • cytotoxic T cells IL2, CD26, CD160
  • NK cells IL2, SAP, CD160
  • cytotoxic T cells CD45+CD3+CD8+CD25-I-
  • activated NK cells CD45+CD3-CD19-CDl lb-nkp46+
  • Fig. 5F The results concerning the infiltration and activation of regulatory T lymphocytes (Tregs) after treatment with GEMys- IL2 in vivo were controversial. Interleukin-2 promotes proliferation and differentiation of cytotoxic T cells, but also differentiation and homeostasis of regulatory Tregs. Bensinger et al., The Journal of Immunology 172, 5287-5296 (2004). IPA analysis predicted the upregulation post- treatment of genes associated proliferation and trafficking of Tregs (Fig.
  • Longitudinal IVIS monitoring demonstrated that, after an initial and remarkable reduction in tumor progression after seven days in animals treated with GEMys-IL2 compared with control animals (PBS), tumor relapse began two weeks posttreatment (Fig. 5H). Indeed, circulating IL2 levels in serum at the endpoints were similar in animals treated with a single dose of GEMys-IL2 or vehicles (Fig. 6G).
  • these myeloid cells were comprised of neutrophils, monocytes, and macrophages, but it remains to be determined whether the anti-cancer and pro-inflammatory activity in vitro and in vivo was mediated by a defined myeloid cell population or by diverse transduced myeloid cells.
  • the treatment was delivered intravenously and well tolerated in an immunocompetent syngeneic LGG murine model.
  • systemically injected bone marrow cells in glioma animals were able to pass the BBB and be recruited into the TME. Rajappa et al., Clin Cancer Res 23, 3109-3119 (2017).
  • this study demonstrates that GEMys injected intravenously into immunocompetent glioma-bearing mice were recruited to and infiltrated the local tumor microenvironment.
  • rIL2 recombinant interleukin-2
  • mice the intracranial injection of rIL2 caused the disruption of the blood brain barrier (BBB) and the consequent development of cerebral edema.
  • BBB blood brain barrier
  • the intracranial injection of rIL2 was associated with toxicity and development of IL2-induced capillary leak syndrome, edema around the tumor, thrombocytopenia, cardiac arrhythmias, hepatic dysfunction, and fever. Therefore, the development of severe toxicity has been a challenge for the direct delivery of rIL2 into the tumor bed of patients with glioma so far.
  • This study is the first to demonstrate the pre-clinical therapeutic activity of bone marrow-derived naive and pro-inflammatory myeloid cells, engineered to express and secrete IL2 in the TME, which delayed malignant progression of low-grade gliomas and prolong OS.
  • the systemic GEMys treatment approach is a novel concept that leverages tumor homing mechanisms given the crosstalk between tumor and host innate immunity. Due to a well-tolerated treatment course in our pre-clinical models with GEMys and the positive effect on the recruitment of effector T cells, we believe the data suggests that treatment may be more potent if the animals are treated with multiple administrations of GEMys or in combination with checkpoint inhibitors. These approaches may augment or help maintain cytotoxic T cell trafficking and activation and further studies are required given the limitations in first-line immunotherapy regimens observed in clinical care currently.
  • the lentiviral particles were generated by co-transfection in Lenti-XTM 293T cells (Takarabio) of lentiviral packaging kit (Origene) and plasmids for the expression of IL2-GFP+ or the empty vector- GFP+, according to the manufacturer’s protocol (Origene).
  • the lentivirus concentration was quantified by Lenti ELISA Kit (Origene).
  • the cells were scraped, washed, and seeded in fresh media. Three days post-infection, the cells were evaluated for the expression of IL2 by quantitative real-time PCR (RT-qPCR) and for the release of IL2 by ELISA (R&D Systems).
  • Infected myeloid cells were selected by the addition of puromycin Ipg/ml for 2 weeks and both the GFP and IL2 expression were monitored weekly by flow cytometry (LSR Fortessa, BD Biosciences), fluorescent microscopy (EVOS, AMG), RT-qPCR (Applied Biosystems), and ELISA (R&D Systems).
  • NTV-a;Ink4a +/_ ArP /_ ;PTEN +/fl ;LSL-Luc and the RCAS system were adopted to undergo gliomagenesis in vivo.
  • DF-1 chicken fibroblasts transfected with RCAS-PDGFb and DF-1 cells transfected with RCAS-Cre were maintained in DMEM with 10% fetal bovine serum (Gibco).
  • Bone Marrow Femurs and tibias were dissected in sterility from NTV-a; Ink4a +/_ Arf 1 ' / ;PTEN +/fl ;LSL-Luc mice not bearing tumors at 4 weeks of age. Bone marrow was extracted by centrifugation at 10,000 x g for 30 seconds.
  • Spleen Spleens were dissected from NTV-a; Ink4a +/ Arr /_ ;PTEN +/n ;LSL-Luc mice without tumors at 4 weeks of age.
  • mice were anesthetized with an isoflurane vaporizer (Kent Scientific) and cardiac puncture was performed to collect 400pl of peripheral blood. Blood was transferred into microtainer EDTA blood collection tubes (BD), and animals were immediately euthanized. Blood samples were centrifuged at 2,000 x g for 10 minutes at +4°C, serum was collected and immediately analyzed by ELISA or stored at -80°C.
  • Brain Whole brains were dissected from mice at endpoint and fixed in 10% buffered formalin phosphate for 24 hours for histology. The tumor area was also extracted from the whole right hemisphere with a scalpel and digested to investigate the inflammatory status (by cytokine array). Alternatively, the tumor area was dissociated to isolate immune cells of the glioma microenvironment to assess cellular composition (by CyTOF and flow cytometry) and perform analysis of the transcriptome (by RT-qPCR and Nanostring).
  • the stratification with 25% Percoll was performed to deplete the myelin layer at 500g for 20 minutes at 18°C in absence of break.
  • the pellet was washed in DPBS+10% FBS and stained for flow cytometry or processed for RNA isolation and analysis of the transcriptome.
  • Tumor or normal tissue was excised from the brain’ s right hemisphere at the injection site. The tissue was then dissected into smaller pieces, digested with 2 mg of Papain (Brainbits) for 20 minutes at 37°C, and filtered through a 70 pm cell strainer. CD45+ cells were isolated by magnetic positive selection (Miltenyi). Sequencing was performed by the Genomic core facility of the Nationalwide Children’s Hospital of Columbus, Ohio. Samples were run on a Chromium controller (10X Genomics), using Next GEM Single cell 3’ Reagent kit 3.1. Results were mapped to the mmlO mouse genome reference using CellRangerv3.0.2 (lOXGenomics).
  • Seurat v.4 and ShinyCell packages were utilized to evaluate gene expression analysis in R., and scRNAseq data were deposited to GEO (GSE221440).
  • GEO GEO
  • the database generated for the Ingenuity Pathway Analysis (IPA, Qiagen) is publicly available.
  • RNA was extracted from the immune cells infiltrating the glioma microenvironment after three days post treatment of LGG RCAS/t-va mice with vehicle (PBS) or 8xl0 6 GEMys-IL2 (n 3), using RNeasy micro kit (Qiagen). Quality was assessed using Agilent Bioanalyzer RNA chips, and quantification via Qubit RNA High Sensitivity assay. Libraries were generated with NEBNext Ultra II Directional RNA prep (Biolabs) and sequenced with Illumina NovaSeq6000 (Illumina).
  • IPA Ingenuity Pathway Analysis
  • RT-qPCR Quantitative Real Time PCR
  • RNA from primary isolated murine cytotoxic T cells and myeloid cells was extracted using RNeasy mini kit (Qiagen), and total RNA from primary immune cells isolated in the glioma microenvironment was extracted using RNeasy micro kit (Qiagen).
  • RNA quantity and quality were assessed by Nanodrop (Thermo Fisher), and cDNA was synthetized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in accordance with the manufacturer’s protocol.
  • SYBR Green RT-qPCR experiments were executed following the manufacturer’s protocols (Applied Biosystems) using PrimeTime qPCR primer assays (IDT), and in a StepOnePlus system (Applied Biosystems).
  • GEMys Composition To determine the composition of GEMys in vitro, a large twenty-six parameter mass cytometry (CyTOF) panel consisting of both innate and adaptive immune cell markers was applied to both GEMys-EV and GEMys-IL2 in triplicate to ensure consistency in the generation of GEMys.
  • CyTOF Maxpar Cell Staining buffer
  • isolated immune cells were washed twice with Maxpar Cell Staining buffer (Fluidigm), incubated in Fc-blocking solution for 10 minutes, stained with 100 pL of a cocktail of metal-conjugated surface antibodies and incubated for 30 minutes at 4°C with vortexing.
  • CD8+ T cells co-culture with GEMys. Spleens were harvested from 4-week-old NTV-a/Ink4a +/_ Arf t/ 7PTEN fl /LSL-Luc mice without tumors and immediately mechanically dissociated on a 70pm cell strainer (Thermo Scientific).
  • T cells were washed in PBS and processed for CD8+ selection (StemCell Technologies).
  • T cells were co-cultured in 24 well plates with GEMys-EV or GEMys-IL2 with a T-cell : GEMys ratio of 1:3 for 1 to 4 days in RPMT media supplemented with 10% FBS (Gibco).
  • CD8+ cells were co-cultured with GEMys-EV or GEMys-IL2 for 24hrs, followed by CD8+ T cells magnetic negative selection (StemCell Technologies), total RNA extraction, and real-time PCR. Mass cytometry and flow cytometry were used to evaluate the protein expression.
  • CTL cytotoxic T lymphocytes
  • CTLs were stimulated for 24 and 48hr with anti-CD3/28 Dynabeads (Thermofischer 11456D) as described by Patel et al. Stained cells were acquired on a LSR Fortessa (BD Biosciences) and analyzed using FlowJo software.
  • Cytokine array Immune cells infiltrating the glioma microenvironment in the RCAS murine model were collected from the tumoral mass in the right hemisphere of brains from animals at week 3 (low-grade glioma), or at week 7 (high-grade glioma). Tumor progression was validated by histological evaluation of 5pm paraffin-embedded brain tissue sections stained by H&E. Total protein lysates were generated with R1PA buffer supplemented with HALT proteases inhibitor cocktail (Thermo Scientific), and the total protein concentration was quantified by BCA (Thermo Scientific), per manufacturer’s protocol recommendations. The cytokine profile in Figure 1G was investigated by Mouse Cytokine 44-plex Discovery Assay (Discovery Assay), and in Figure 5C by mouse XL cytokine array in accordance with the manufacturer’s instruction (R&D Systems).
  • Enzyme-Linked immunosorbent Assay To perform the quantification of the circulating murine IL2 in the peripheral blood, or of the released IL2 in the culture media, we performed ELISA as described by the manufacturer (R&D Systems). All incubations were conducted at room temperature unless otherwise noted. Briefly, the plate was coated overnight with lOOpL of primary antibody and blocked in 1% BSA for Ih. lOOpL samples or standards were seeded in triplicate and incubated for 2hrs. The plate was washed, and biotinylated anti- IL2 was added to each well and incubated for 2hrs. lOOpL Streptavidin-HRP was added and allowed to stand for 20 min.
  • ELISA Enzyme-Linked immunosorbent Assay

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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode de traitement ou de prévention du cancer du système nerveux central chez un sujet en ayant besoin. La méthode consiste à administrer au sujet une quantité thérapeutiquement efficace de cellules myéloïdes modifiées afin d'exprimer l'interleukine-2. L'invention concerne également une population de cellules myéloïdes génétiquement modifiées (GEMy) comprenant des cellules myéloïdes dérivées de la moelle osseuse qui ont été génétiquement modifiées afin d'exprimer l'interleukine-2.
PCT/US2023/032123 2022-09-06 2023-09-06 Cellules myéloïdes dérivées de la moelle osseuse génétiquement modifiées pour le traitement de tumeurs du système nerveux central WO2024054527A1 (fr)

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