EP4731776A2 - Cellules progénitrices myéloïdes modifiées - Google Patents

Cellules progénitrices myéloïdes modifiées

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Publication number
EP4731776A2
EP4731776A2 EP24832807.2A EP24832807A EP4731776A2 EP 4731776 A2 EP4731776 A2 EP 4731776A2 EP 24832807 A EP24832807 A EP 24832807A EP 4731776 A2 EP4731776 A2 EP 4731776A2
Authority
EP
European Patent Office
Prior art keywords
cells
population
engineered
cell
myeloid progenitor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24832807.2A
Other languages
German (de)
English (en)
Inventor
Jessica M. HAVERKAMP
Wilhelmus Theodorus HENDRIKS
Diego Rodrigues COELHO
Gregory MOTZ
Chew-Li SOH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BlueRock Therapeutics LP
Original Assignee
BlueRock Therapeutics LP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BlueRock Therapeutics LP filed Critical BlueRock Therapeutics LP
Publication of EP4731776A2 publication Critical patent/EP4731776A2/fr
Pending legal-status Critical Current

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
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    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates generally to the field of biotechnology and immunotherapy. More specifically, this disclosure pertains to genetically engineered immune cells for treating tumors.
  • Cancer is a major public health concern worldwide, causing significant morbidity and mortality.
  • Traditional cancer treatments such as surgery, radiation therapy, and chemotherapy, are often associated with serious side effects and may not be effective in all cases.
  • immunotherapy has emerged as a promising alternative or adjunct to conventional cancer therapies. Immunotherapy harnesses the power of the patient’s immune system to recognize and eliminate cancer cells, offering a more targeted and potentially less toxic approach to cancer treatment.
  • This disclosure relates generally to populations of engineered cells, including engineered immune cells or engineered myeloid progenitor cells, that can influence a tumor microenvironment to treat certain tumors, including glioblastoma.
  • the disclosed populations of engineered cells possess the remarkable ability to remodel the tumor microenvironment (TME) into a supportive environment that facilitates cytotoxic immune function and enables effective immunotherapy treatment in brain and other solid tumors.
  • TEE tumor microenvironment
  • This remodeling effect is achieved through the stable secretion of one or more cytokines, e.g., IL-12.
  • the engineered immune cells are designed to secrete IL-12 at levels that effectively suppress tumor growth while ensuring the safety profile associated with a safe treatment for the subject.
  • the populations of engineered immune cells are genetically modified by the integration of a heterologous nucleic acid encoding the one or more cytokines downstream of a promoter of an endogenous gene.
  • the endogenous gene includes a sustained transgene expression locus (STEL), for example, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
  • this disclosure provides a population of engineered immune cells that secrete interleukin- 12 (IL-12) at a concentration of between 1.0 and 1,000 ng/ml, as measured by homogeneous time resolved fluorescence (HTRF), when said cells are cultured at a density of 1 million cells per milliliter for 24 hours.
  • IL-12 interleukin- 12
  • HTRF homogeneous time resolved fluorescence
  • the population of engineered immune cells secretes IL-12 at a concentration of 500 ng/ml when said cells are cultured at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells are genetically modified to comprise a heterologous nucleic acid encoding IL-12.
  • expression of the heterologous nucleic acid is driven by a promoter sequence of an endogenous gene.
  • the endogenous gene comprises a sustained transgene expression locus (STEL).
  • the endogenous gene comprises Glyceraldehyde 3- phosphate dehydrogenase (GAPDH).
  • GPDH Glyceraldehyde 3- phosphate dehydrogenase
  • the population of immune cells comprises one or more of hematopoietic stem cells, myeloid progenitor cells, microglia precursor cells, microglial cells, dendritic cells, T cells, B cells, natural killer cells, or macrophages.
  • the population of immune cells is a population of myeloid progenitor cells.
  • the myeloid progenitor cells are derived from pluripotent stem cells, for example, induced pluripotent stem cells (iPSCs).
  • the population of engineered immune cells express CD1 lb, CD14, and CD45. In some embodiments, at least 80 percent of the engineered immune cells express CDl lb, CD14, and CD45. In some embodiments, the population of engineered immune cells are positive for one or more of CD1 lb, CD14, and CD45. In some embodiments, the engineered immune cells are positive for each of CD1 lb, CD14, and CD45.
  • the population of engineered immune cells express HLADR, CD86, CD80, and CD40.
  • the population of engineered immune cells do not express CD206, CD 163, or MerTK. In some embodiments, the population of engineered immune cells do not express CD206, CD163, and MerTK.
  • the population of immune cells are able to present tumor antigens to other immune cells capable of recognizing antigens.
  • each cell within the population of the immune cells comprises a major histocompatibility complex (MHC) molecule on the cell surface, and wherein said MHC molecule binds to and presents the tumor antigens to the other immune cells.
  • MHC major histocompatibility complex
  • antigens may comprise peptide antigens that bind to MHC class I molecules.
  • antigens may comprise lipid antigens presented by non-classical molecules.
  • the MHC molecule comprises an MHC class I molecule or an MHC class II molecule.
  • each cell of the population of immune cells comprises a co-stimulatory molecule on the cell surface, and wherein said co-stimulatory molecule enhances an immune response by interacting with immune cells.
  • the immune response is a CD8+ T cell-mediated immune response.
  • the co-stimulatory molecule comprises CD80, CD86, or CD40.
  • each of the immune cells comprises one or more genetic alterations that results in increased expression of MHC molecule on the cell surface, thereby increasing the capability of the immune cells to present the tumor antigens to T cells.
  • each of the immune cells comprises a targeting moiety on the cell surface.
  • the targeting moiety comprises an exogenously expressed chimeric or naturally occurring protein capable of binding to an antigen to elicit activation of an immune cell.
  • the targeting moiety comprises a chimeric antigen receptor.
  • the targeting moiety comprises a T cell receptor.
  • this disclosure provides a method for generating engineered immune cells, including introducing a heterologous nucleic acid encoding IL- 12 into a stem cell using a CRISPR/Cas system; and deriving the immune cells from the stem cell, thereby producing the population of engineered immune cells.
  • the stem cell comprises an induced pluripotent stem cell.
  • the heterologous nucleic acid encoding IL-12 is integrated into a sustained transgene expression locus (STEL).
  • the STEL loci comprises a GAPDH gene.
  • the heterologous nucleic acid encoding IL- 12 is integrated into a sustained transcriptionally active payload region (STAPLR).
  • STAPLR sustained transcriptionally active payload region
  • the heterologous nucleic acid encoding IL-12 is integrated into the STAPLR, the heterologous nucleic acid further including an exogenous gene promoter.
  • this disclosure provides a pharmaceutical composition for treating a tumor, the composition comprising: a population of engineered immune cells as described herein; and further including a pharmaceutical acceptable carrier, carrier, or dilutant.
  • this disclosure provides a method of stimulating an immune response against a tumor, the method comprising: administering the population of engineered immune cells as described herein; and allowing the population of engineered immune cells to secrete IL- 12, thereby stimulating a CD8+ T cell-mediated response against the tumor.
  • the tumor is a glioblastoma.
  • the administering comprises direct injection of the population of cells into the site of the tumor.
  • the population of cells stimulates an immune response against the tumor in the subject.
  • the immune response is a CD8+ T cell-mediated response.
  • the immune response comprises secretion of pro-inflammatory cytokines by T regs.
  • the pro-inflammatory cytokines comprise IFNy, TNFa, or both IFNy and TNFa.
  • the method further comprises administering an additional anti-cancer therapy to the subject.
  • additional anti-cancer therapy comprises a CD28 agonist.
  • the additional anti-cancer therapy does not comprise a CD28 agonist.
  • the CD28 agonist is an anti-CD28 antibody.
  • the subject is a human.
  • this disclosure provides a kit comprising a dosage form suitable for administration to a subject comprising the engineered immune cells described herein, and instructional material for the use of said dosage form.
  • this disclosure provides a kit comprising a dosage form suitable for administration to a subject comprising a pharmaceutical composition described herein, and instructional material for the use of said dosage form.
  • this disclosure provides a device for administering a therapy to a subject comprising a pharmaceutical composition or population of engineered immune cells as described here.
  • this disclosure provides an engineered cell comprising a heterologous nucleic acid encoding a cytokine, or a fragment thereof, wherein expression of the heterologous nucleic acid is driven by a promoter of an endogenous gene.
  • the endogenous gene comprises a STEL.
  • the endogenous gene comprises GAPDH.
  • the cytokine or fragment thereof comprises IL-12.
  • the engineered cell comprises a stem cell or an immune cell.
  • the immune cell comprises a myeloid progenitor cell.
  • this disclosure further provides a population of cells derived from an engineered cell described herein, wherein the population of cells express the cytokine at a concentration of between 1.0 and 1,000 ng/ml, as measured by homogeneous time resolved fluorescence (HTRF), when said cells are cultured at a density of 1 million cells per milliliter for 24 hours.
  • HTRF homogeneous time resolved fluorescence
  • the population of cells secrete the cytokine at a concentration below 500 ng/ml, as measured by HTRF, when said cells are cultured at a density of 1 million cells per milliliter for 24 hours.
  • FIG. 1 illustrates an exemplary cell differentiation scheme employed to generate myeloid progenitor cells from induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the progenitor cells underwent a subsequent maturation process, after which they were subjected to cry opreservation to maintain viability during storage.
  • FIG. 2 is an illustration of the cloning strategy used to integrate IL- 12 transgenes into the GAPDH STEL site.
  • the IL- 12 transgenes were integrated into the GAPDH STEL site by using a 2 A sequence replacing the GAPDH stop codon to integrate the IL- 12 transgene in-frame to the endogenous GAPDH sequence before the STOP codon at the 3’UTR.
  • the IL-12 transgenes were integrated using CRISPR-based gene editing systems as described below.
  • FIGS. 3A-3D show exemplary FACS data collected to characterize iPSC-derived myeloid progenitor cells genetically engineered to secrete IL-12.
  • FIGS. 3A and 3B show genetically engineered cells express well-established myeloid progenitor cell markers, CD45+, CD1 lb+, and CD14+.
  • FIG. 3C shows an exemplary FACs profile that confirm the myeloid progenitor cells do not express neutrophil marker CD66b.
  • FIG. 3D is an exemplary FACS profile showing the cells do not express proliferation markers Ki67 and pHH3.
  • FIG. 4 shows exemplary experimental results confirming iPSC-derived myeloid progenitor cells genetically engineered to secrete IL-12 upregulate Ml markers, z.e., HLADR, and CD86.
  • the results are bar graphs based on FACS data collected from myeloid progenitor cell lines that were co-cultured with T cells. These data show that engineered myeloid progenitor cells (CD19t-IL-12, IL-12 biallelic, and IL-12 monoallelic - as indicated on x- axis), but not “control cells” (i.e., non-edited myeloid progenitor cells), upregulated Ml markers when co-cultured in the presence of T cells.
  • control cells i.e., non-edited myeloid progenitor cells
  • FIG. 5 shows exemplary experimental results demonstrating M2 markers (CD206, CD 163, and MerTK) were downregulated by the genetically engineered myeloid progenitor cells after co-culture with T cells.
  • the results are bar graphs based on FACS data collected from myeloid progenitor cell populations co-cultured with T cells. These data show that the engineered myeloid progenitor cells (CD19t-IL-12, IL-12 biallelic, and IL-12 monoallelic - as indicated on x-axis), but not “control cells” (i.e., non-edited myeloid progenitor cells), downregulated genes that are typically present in tumor-associated macrophages and M2 macrophages.
  • control cells i.e., non-edited myeloid progenitor cells
  • FIG. 6 shows exemplary experimental results of cytokine profiles for iPSC derived myeloid progenitor cells genetically engineered to secrete IL-12 and control cells (z.e., non-edited myeloid progenitor cells).
  • the results are bar graphs based on flowcytometry experiments performed to detect cytokines of interest. These data show a side-by- side comparison of cytokine secretion profiles for two genetically engineered myeloid progenitor cell lines (CD19t-IL-12, and IL-12 monoallelic) and control cells (z.e., non-edited myeloid progenitor cells).
  • the cytokines assayed included IL-4, IL-2, IP-10, IL-1 beta, TNF- alpha, MCP-1, IL- 17 A, IL-6, IL- 10, IFN-gamma, IL- 12, IL-8, and TGF-beta 1.
  • FIG. 7 provides exemplary experimental results showing IL-12 output of iPSC- derived myeloid progenitor cells genetically engineered to secrete IL-12.
  • these exemplary data show rates of IL-12 production of control cells (non-edited myeloid progenitor cells) and myeloid progenitor cells generated from iPSCs genetically engineered to secrete IL- 12 according to three different engineering strategies.
  • the rate of IL- 12 produced was measured using homogeneous time resolved fluorescence (HTRF).
  • the amount of IL-12 produced is identified on the y-axis in nanograms per milliliter per one million cells per twenty-four hours (IL-12 ng/mL/lM/24hr).
  • the control and genetically engineered myeloid progenitor cell lines are identified on the x-axis.
  • FIG. 8 shows exemplary experimental results demonstrating iPSC-derived myeloid progenitor cells genetically engineered to secrete IL-12 effectively kill tumor cells and inhibit tumor cell growth in vitro.
  • FIG. 8 is exemplary data taken from cell killing assays that compare varying quantities of two genetically engineered myeloid progenitor cell lines — one line with CD19t-IL-12 integration, the other with monoallelic, IL- 12 integration — and control cells (non-edited myeloid progenitors).
  • the y-axis shows the number of apoptotic U251 cells.
  • the x-axis identifies the cells (and cell populations) present in the corresponding wells.
  • FIGS. 9A and 9B show exemplary experimental results demonstrating the ability of genetically engineered myeloid progenitor cells to kill tumor cells, thus leading to increased tumor cell death.
  • FIGS. 9A and 9B show bar graphs quantifying the growth of U251 tumor cells in co-culture with CD8 T cells and varying quantities of control cells (FIG. 9A), as compared with genetically engineered myeloid progenitor cell lines: monoallelic IL-12 engineered myeloid progenitor cells (FIG. 9B).
  • the x-axis represents cell numbers for each cell type, while the y-axis corresponds to U251 tumor cell growth. Measurements were taken after a 5-day co-culture period.
  • FIG. 10 illustrates an exemplary workflow that was used to evaluate the impact of M2 polarization on CD8 T cell activity mediated by genetically modified myeloid progenitor cells.
  • Myeloid progenitor cells derived from iPSCs genetically engineered to secrete IL-12 and control cells (myeloid progenitor cells derived from non-edited iPSCs) were cultured, separately, under conditions that simulate the tumor microenvironment. This stimulation involved culturing cell populations in the presence of TGF-beta and IL-10 to induce M2 skewing.
  • CD8 T cells were separated from donor PBMCs using a commercially available CD8 T cell isolation kit.
  • FIGS. 11A and 11B are exemplary experimental results that show myeloid progenitor cells rescue IFN-gamma secretion from CD8 T cells despite M2 polarization. Specifically, FIG.
  • FIG. 11A shows the amounts of IFN-gamma (y-axis) that was detected by flow cytometry following the co-culture of T cells with varying quantities of M2 polarized myeloid progenitor cells (indicated on x-axis). This data demonstrate that the secretion of IFN-gamma by T cells is rapidly elevated by the addition of genetically engineered myeloid progenitor cells despite M2 polarization.
  • FIG. 11B is exemplary data showing the amounts of IL- 12 (y axis) that was detected by varying quantities of M2 polarized myeloid progenitor cells (identified along x-axis).
  • FIGS. 12A-12G are exemplary experimental results showing myeloid progenitor cells genetically engineered to secrete IL- 12 activate CD8 T cell cytolytic activity despite M2 polarization. More particularly, FIG. 12A shows the amount of IL-12 (indicated on y-axis) that was detected from the cell populations indicated on the x-axis. FIGS. 12B-12G show the amounts of effector CD8 T cell cytokines and cytolytic enzymes (TNF-alpha, IL-2, Perforin, Granzyme A, Granzyme B, and Granulysin - as indicated) that was detected from corresponding cell populations.
  • CD8 T cell cytokines and cytolytic enzymes TNF-alpha, IL-2, Perforin, Granzyme A, Granzyme B, and Granulysin - as indicated
  • FIG. 13 provides exemplary data showing the significant tumor suppressive capabilities of genetically engineered myeloid progenitor cells, in vivo. Specifically, FIG. 13 shows line graphs of tumor volumes (indicated on y-axis) that were detected over time (indicated on x-axis) from mice across different treatment groups. The upper panel shows all mice in the study and the lower panels shows individual mice per treatment group. These exemplary data demonstrate the anti-tumor efficacy of genetically engineered myeloid progenitor cells.
  • FIGS. 14A and 14B show exemplary experimental results further demonstrating the in vivo therapeutic efficacy of genetically engineered myeloid progenitor cells.
  • FIG. 14A shows exemplary data that was obtained by collecting tumor measurements from pre-clinical animal models of glioblastoma (GBM) that had been subjected to the indicated treatments (shown on the x-axis). Surprisingly, these exemplary data clearly demonstrate a significantly lower tumor growth inhibition ratio (y-axis) when using myeloid progenitor cells genetically engineered to secrete IL-12 than control non-engineered cells or anti-PDl monoclonal antibody benchmark.
  • FIG 14B shows representative images of the tumors which were harvested from the pre-clinical mouse models at humane end points. The tumors were cleared of surrounding fat and muscle tissue to ensure accurate size measurements.
  • FIG. 15 shows IL-12 levels detected in the serum of pre-clinical animal models over a 20-day period. Serum samples were collected from the animal models at specific time points as indicated, and IL-12 secretion was analyzed using bead-based Luminex approaches.
  • FIGS 16A-16C provide exemplary results showing serum cytokine levels associated with cytokine release syndrome (CRS), as compared to treatment with an anti-PD- 1 antibody.
  • CRS cytokine release syndrome
  • the evaluation was performed on the intratumoral (IT) treatment with genetically engineered myeloid progenitor cells (CD19t-IL-12) in comparison to the benchmark monoclonal antibody (mAb) treatment anti-human PD-1 (aPDl) administered intraperitoneally (IP).
  • mAb monoclonal antibody
  • aPDl monoclonal antibody
  • IP monoclonal antibody
  • the data were obtained from humanized mice, where human flank tumors were established, followed by the transfer of human peripheral blood mononuclear cells (huPBMCs). Subsequently, the mice received either the myeloid progenitor cell treatment or aPDl treatment.
  • FIG. 16A shows levels of IL-6 (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL-12), and aPDl.
  • FIG. 16B shows levels of IL- 8 (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL-12), and aPDl.
  • FIG. 16A shows levels of IL-6 (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL-12), and aPDl.
  • 16C shows levels of IL 1 -beta (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL-12), and aPDl.
  • FIG. 17A-17D show exemplary experimental results that demonstrate the remodeling of the tumor microenvironment (TME) towards an anti-tumor cytolytic T lymphocyte (CTL) response following the administration of myeloid progenitor cells.
  • TME tumor microenvironment
  • CTL anti-tumor cytolytic T lymphocyte
  • FIG. 17A experiment data showing the ratios of anti-tumor cytolytic CD8 T cells to protumor immunosuppressive FoxP3+ T cells (Tregs) in the tumor after treatment with myeloid progenitor cells genetically engineered to secrete IL-12 or control cells (non-edited myeloid progenitor cells).
  • Tregs protumor immunosuppressive FoxP3+ T cells
  • FIG. 17B demonstrates an infiltration of T cells (CD3+) within the TME with an overall decreased exhaustion phenotype (PD1/TIM3 negative), indicating a shift from immunosuppressive to pro-inflammatory phenotypes.
  • FIG. 17B demonstrates an increased frequency of total T cells within the TME
  • FIGS. 17C and 17D show a decrease in exhaustion markers (PD1/TIM3) within CD4 and CD8 subsets in the TME. This observation suggests that the shift in T cell frequency is accompanied by a functional change in T cell activation in the TME by in vivo intratumoral (IT) treatment with genetically engineered myeloid progenitor cells.
  • IT in vivo intratumoral
  • FIGS. 18A and 18B show exemplary experimental results demonstrating that engineered M2 polarized myeloid progenitor cells (indicated as MPCs) can restore both CD8 IFNy (FIG. 18A) and TNFa (FIG. 18B) to a level of pro-inflammatory cytokine that is comparable between CD8 T cells stimulated with or without anti-CD28 monoclonal antibody (mAb).
  • FIGS. 18A and 18B are bar graphs of exemplary results showing levels of IFNy (pg/mL) and TNFa (pg/mL), respectively, secreted from CD8 T cells under conditions identified along the X-axis.
  • FIG. 18A compares levels of IFNy secreted from CD8 T cells when the cells are cultured with or without M2 myeloid progenitor cells, and with and without the addition of an anti-CD28 mAb.
  • FIG. 18B compares levels of TNFa secreted from CD8 T cells when the cells are cultured with or without M2 myeloid progenitor cells, and with and without the addition of an anti-CD28 mAb.
  • FIGS. 19A and 19B show exemplary results demonstrating the impact of myeloid progenitor cells (and control cells) on tumor spheroid size (x axis) when cultured with anti- CD3 mAb alone (FIG. 19A) or with a combination of anti-CD3 and anti-CD28 mABs (FIG. 19B)
  • the dark lines indicate observations from myeloid progenitor cells.
  • the gray lines indicate observations from control cells.
  • FIGS. 20A and 20B show exemplary results demonstrating myeloid progenitor cells (indicated as MPCs) promote pro-inflammatory cytokine production from Tregs.
  • FIG. 20A shows levels of ZFNy secreted from Tregs co-cultured with a certain number of myeloid progenitor cells indicated along the X axis. The Y axis indicates the fold change in IFNy (as measured in pg/mL) from myeloid progenitor cells in combination with Tregs as compared with Tregs only.
  • FIG. 20B shows levels of TNFa secreted from Tregs co-cultured with a certain number of engineered myeloid progenitor cells indicated along the X axis. The Y axis indicates the fold change in TNFa (as measured in pg/mL) from engineered myeloid progenitor cells in combination with Tregs as compared with Tregs only.
  • aspects of the present disclosure relate to methods, populations of engineered cells, pharmaceutical compositions, kits, and devices for stimulating an immune response against a tumor in a subject.
  • the methods, populations of engineered cells, pharmaceutical compositions, kits, and devices for stimulating an immune response against a tumor in a subject include a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that secrete IL- 12.
  • the population of engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • the population of engineered cells can elicit or enhance an immune response (e.g., an anti -tumor immune response) in a subject.
  • the immune response has an important role in cancer, including for the identification and elimination of tumors.
  • transformed cells of tumors can express antigens (e.g., tumor antigens) that are not found on normal cells.
  • antigens e.g., tumor antigens
  • these antigens appear foreign, and their presence causes immune cells to attack the transformed tumor cells.
  • the main response of the immune system to tumors is to destroy the abnormal cells using killer T cells, sometimes assisted by helper T cells.
  • Tumor antigens are presented on MHC molecules, which allows cytotoxic T cells and NK cells to recognize the tumor cell as abnormal, as well as the generation of antibodies against the tumor cells allowing for their destruction by the complement system.
  • populations of engineered cells for treatment of a subject having a tumor (e.g., glioblastoma).
  • the population of engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • TME tumor microenvironment
  • effective immunotherapy treatment e.g., CAR-T therapy
  • Interleukin- 12 is a heterodimeric cytokine with multiple biological effects on the immune system. It includes two subunits, p35 and p40, both of which are required for the secretion of the active form of IL-12, p70. Interleukin- 12 acts on dendritic cells (DC), leading to increased maturation and antigen presentation, which can allow for the initiation of a T cell response to tumor specific antigens. It also drives the secretion of IL-12 by dendritic cells, creating a positive feedback mechanism to amplify the response.
  • DC dendritic cells
  • IL- 12 plays a fundamental role in directing the immune system in activating a CD4+ T cell- mediated and/or a CD8+ T cell response. Furthermore, IL-12 can promote the activation of innate immune cells such as macrophages and eosinophils through its induction of other pro- inflammatory cytokines. This activation then leads to IL- 12 secretion by these cells and further amplification of both innate and adaptive immune responses.
  • the embodiments described herein use novel populations of engineered cells (e.g., population of engineered immune cells or populations of engineered myeloid progenitor cells) a broadly applicable therapy that will elicit or enhance an anti-tumor response in a subject.
  • Such novel populations are based, at least in part, on the surprising and unexpected observation that secretion of specific amounts of IL-12 by engineered cells, such as engineered immune cells or engineered myeloid progenitor cells, activates cytotoxic T cells and orchestrates remodeling of the tumor microenvironment to promote tumor elimination through the enhancement of anti-tumor immune responses.
  • the population of engineered cells described herein present an improvement in the treatment of solid tumors, such as glioblastoma.
  • this disclosure provides populations of engineered cells (e.g., myeloid progenitor cells) that secrete IL-12 at a therapeutic rate that ensures antitumoral efficacy while minimizing the risk of toxicity.
  • IL-12 is a potent cytokine with immunomodulatory properties that plays a crucial role in anti-tumor immune responses.
  • excessive IL- 12 production may lead to adverse effects, including systemic toxicity.
  • the disclosed populations of engineered cells are genetically modified to achieve a rate of IL-12 secretion that activates an immune response against tumors without reaching toxic concentrations.
  • the disclosure provides strategies for generating populations of engineered cells that secrete an optimal level of IL-12 secretion.
  • this disclosure describes engineering strategies that result in the production of engineered cells with IL-12 secretion profiles that maximize therapeutic efficacy while minimizing toxicities associated with IL-12 overexpression.
  • the disclosed engineered cells offer an improved approach for cancer immunotherapy, which harnesses the benefits of IL- 12 while addressing the challenges associated with toxicity.
  • the controlled secretion of IL-12 by these cells ensures a therapeutic effect within a safe range, providing a new avenue for enhancing anti-tumor immune responses without compromising patient safety.
  • this disclosure provides an engineered cell (e.g., myeloid progenitor cell) that includes a heterologous nucleic acid encoding IL- 12 downstream of a promoter of an endogenous gene associated with a sustained transgene expression locus (STEL), such as, GAPDH.
  • STL sustained transgene expression locus
  • IL- 12 By placing IL- 12 behind the GAPDH promoter — an endogenous gene whose consistent expression is important for cellular function — the engineered cells can exploit endogenous expression mechanisms to control IL- 12 production.
  • This arrangement ensures that IL-12 secretion, driven by the stable expression of the GAPDH gene, remains within non-toxic levels, yet retains the potency required for antitumoral efficacy.
  • these cells secrete IL-12 at concentrations that strike an optimal balance between therapeutic impact and safety.
  • the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “administration,” “administering” and variants thereof refer to the introduction of a composition or therapeutic agent (e.g., a population of cells) into a subject. Administration includes concurrent and sequential introduction of the composition or therapeutic agent. Administration of the composition or therapeutic agent (e.g., a population of cells) into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. A suitable route of administration allows the composition or the agent to perform its intended function. Administration also includes self-administration and the administration by another. The administration can also be performed systemic, or it can be local. For instance, a composition or therapeutic agent (e.g., a population of cells) can be administered locally, e.g., by local injection into a tissue.
  • the term “and/or” should be understood to mean either one, or both of, or any combination of the alternatives.
  • the term “antibody” refers to an immunoglobulin molecule which specifically binds with an antigen (e.g., a cell surface antigen).
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
  • anti-cancer therapy refers to any therapy used for treating a cancer in a subject.
  • exemplary anti -cancer therapies include, but are not limited to, hormone blocking therapy, chemotherapy, small molecule drugs, kinase inhibitor, therapeutic antibodies or binding fragments thereof, cell therapies, radiation, and surgery.
  • the term “antigen” refers to a molecule that provokes an immune response. This immune response can involve either antibody production, or the activation of specific immunologically-competent cells (e.g., T cells), or both.
  • Antigens can be any type of molecule including, for example, peptides, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins.
  • the term antigen can refer to a substance that induces an immune response.
  • antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
  • an APC refers to a cell that can process and display foreign antigens (e.g., tumor antigens) in association with major histocompatibility complex (MHC) molecules on its surface.
  • MHC major histocompatibility complex
  • an APC can display antigen-class I and II major histocompatibility complex (MHC) on the membrane together with co-stimulatory signals to activate antigen-specific T cells (e.g., tumor antigen-specific T cells), which can lead to an adaptive immune response.
  • MHC major histocompatibility complex
  • Cas protein As used herein, the terms “Cas protein,” “Cas nuclease” and “Cas molecule” are interchangeable and refer to the enzyme responsible for cutting DNA in the CRISPR/Cas system. It can include enzymes from type I, II, and III CRISPR/Cas systems (e.g., Cas3, Cas9, CaslO, Casl2, etc).
  • Cas9 protein generally refers to the enzyme from the bacterial type II CRISPR/Cas system responsible for cutting DNA. Cas9 can include wild-type proteins and functional mutants thereof.
  • Cas 12 protein generally refers to the enzyme from the bacterial type II CRISPR/Cas system responsible for cutting DNA. Cas 12 can include wild-type proteins and functional mutants thereof. For instance, in some instances, the Cas 12 is a Casl2a protein, also known as Cpfl.
  • chimeric antigen receptor and “CAR” are used interchangeably to refer to an engineered receptor that grafts an arbitrary specificity onto an immune effector cell, such as a T cell (e.g., cytotoxic T cell).
  • a CAR is typically a fusion protein comprising antigen recognition moieties and cell -activation elements.
  • a CAR may have an extracellular domain (ectodomain), which comprises an antigen-binding domain and a stalk region, a transmembrane domain and an intracellular (endodomain) domain.
  • ectodomain extracellular domain
  • a chimeric antigen receptor is also known as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immunoreceptor.
  • CRISPR/Cas system refers to a group of molecules including RNA-guided nucleases or other effector molecules and guide RNA (gRNA) molecules, which can direct and implement RNA-guided nucleases or other effector molecules to a target nucleic acid.
  • the target nucleic acid is modified by the interaction of the CRISPR/Cas system and a sequence present in the target nucleic acid, for example, to cause cleavage (e.g., hydrolysis of one or more phosphodiester bonds) of the target nucleic acid.
  • the CRISPR/Cas system can be used for introducing genetic alterations into a cell (e.g., adding, disrupting or changing the sequence of specific genes) as well as altering gene regulation of genes.
  • the CRISPR/Cas system can be used to integrate nucleic acid sequences encoding a gene of interest into specific regions of the genome.
  • the CRISPR system comprises gRNA and Cas protein, for example, a Cas9 or a Casl2 protein.
  • a system containing Cas9 or a functional mutant thereof is referred to as the “Cas9 system” or “CRISPR/Cas9 system” in this application.
  • Casl2 system A system containing Casl2 or a functional mutant thereof is referred to as the “Casl2 system” or “CRISPR/Casl2 system” in this application.
  • gRNA molecules and Cas molecules can be complexed to form a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • co-stimulatory ligand or “co-stimulatory molecule” refers to a molecule that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a co-stimulatory signal which, in addition to the primary signal provided by, for instance, binding of a T cell receptor (TCR) an MHC molecule loaded with an antigen, leads to an T cell-mediated response.
  • TCR T cell receptor
  • a co-stimulatory ligand can include, but is not limited to, CD2, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD58, CD70, CD83, CD86, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor (LTBR), 3TR6, ILT3, ILT4, HVEM, BTLA, an agonist or antibody that binds a Toll ligand receptor and a ligand that specifically binds with B7-H3.
  • the term “dosage form” refers to a discrete amount of a composition comprising a predetermined amount of the active ingredient (e.g., a population of cells).
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and any additional ingredients in a pharmaceutical composition will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the dosage form can further include one or more additional pharmaceutically active agents. In some cases, the dosage form is for administration by injection into a subject.
  • the term “differentiation,” and its grammatical equivalents, refers to a process by which a stem cell or progenitor cell alters from one cell type to a more specialized cell type.
  • Each specialized cell type in an organism can express a subset of all the genes that constitute the genome of the cell.
  • Each cell type can be defined by its particular pattern of regulated gene expression. Cell differentiation can thus be described as a transition of a cell from one cell type to another cell type coincident with a switch from one pattern of gene expression to another.
  • the term “encoding” refers to the property of specific sequences of nucleotides in a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (z.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene or at least the exons of a gene encodes a protein (e.g., IL- 12) if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • the term “expression” refers to the transcription and/or translation of a particular nucleotide sequence in a cell.
  • overexpress refers to a level of expression of a gene in a cell under particular conditions that is increased relative to the level of expression in a cell under normal conditions.
  • an enhancement or increase in biological activity can refer to an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.
  • the term “genetic modification” or “genetic alteration” refers to a change at the DNA level of a cell.
  • a genetic modification includes an insertion, deletion, or substitution, typically within a defined sequence or genomic locus.
  • the genetic modification includes the integration of a nucleotide sequence heterologous to the genomic locus.
  • the genetic modification can be at a single nucleotide position or at multiple nucleotides, e.g., 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, e.g., contiguous nucleotides.
  • the expression of the gene can be up-regulated or down-regulated.
  • the term “genetically modified” or “engineered” cell refers to a cell (e.g., an immune cell) that includes one or more genetic modifications, and which is not found in nature.
  • the engineered cells can be made by any method known in the art, such as by manipulating the genome of the cell or inserting a new nucleic acid into the cell.
  • a cell can be modified by integrating a nucleic encoding a gene of interest into a cell using a genome editing technique, such as a CRISPR/Cas9 system.
  • the terms “gRNA molecule,” “guide RNA,” and “gRNA” refer to an RNA that functions as a guide for an endonuclease (e.g., a Cas enzyme), with which it forms a complex.
  • an endonuclease e.g., a Cas enzyme
  • the hybridization of a part of the gRNA with DNA for example, through the gRNA steering domain
  • the binding of a part of the gRNA molecule to RNA-guided nucleases or other effector molecules for example, at least through tracrRNA.
  • the gRNA molecule is composed of a single continuous polynucleotide molecule, referred to herein as a “single guide RNA” or “sgRNA” or the like.
  • heterologous when used in reference to a nucleic acid and/or polypeptide that is introduced to a host cell, refers a nucleic acid and/or polypeptide that is not naturally found in the host cell or naturally found at a given position in the genome of the host cells.
  • a construct is heterologous to a host cell if it contains some homologous sequences arranged in a manner not found in the host cell and/or the construct contains some heterologous sequences not found in the host cell.
  • IL- 12 refers to a protein encoded by an interleukin 12 gene.
  • IL-12 is a heterodimeric cytokine with multiple biological effects on the immune system, such as maturation of antigen presenting cells, increased presentation of antigen, and lymphocyte activation. It is composed of two subunits, p35 and p40, both of which are required for the secretion of the active form of IL-12, p70.
  • the IL-12 can be an engineered IL- 12, such as for example, a variant IL- 12 or an IL- 12 that includes a fusion between the p35 and p40 subunits.
  • the following IL- 12 amino acid sequence has been introduced into the engineered cells:
  • immune cell refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of an immune response in an organism.
  • An immune cell can be part of the innate and/or adaptive immune system.
  • Exemplary immune cells include, but are not limited to, cells of the myeloid lineage (e.g., neutrophils, dendritic cells, eosinophils, mast cells, basophils, monocytes, microglia, and precursors thereof), as well as cells of the lymphoid lineage (e.g., T cells, B cells, natural killer cells, and precursors thereof).
  • immune response refers to the physiological reaction that occurs in an organism in response to exogenous factors, pathogens, injury, or a disease (e.g., cancer).
  • An immune response can include activation of either or both the adaptive and innate immune system.
  • An “adaptive immune response” refers to an immune response specific to a particular antigen (e.g., a tumor antigen) that involves the formation of antibodies and/or the activation of lymphocytes to remove the antigen.
  • induced pluripotent stem cell refers to any pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a “forced” expression of specific genes.
  • a “pluripotent” cell is a cell that has the ability to differentiate into cells from any of the three germ layers of an organism (endoderm, mesoderm or ectoderm), but not into extra-embryonic tissues like the placenta.
  • the pluripotency may be incomplete or partial, in that the pluripotent cell may form cells of all three germ layers but may not exhibit all the characteristics of completely pluripotent cells.
  • the term “instructional material” refers to a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of using the compositions associated with the publication, recording, diagram or other medium of expression.
  • the instructional material of a kit of the disclosure can, for example, be affixed to a container that contains the population of cells and/or pharmaceutical composition of the disclosure, or be shipped together with a container that contains the population of cells and/or pharmaceutical composition.
  • the instructional material can be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.
  • MHC MHC protein
  • MHC molecule MHC molecule
  • An MHC binds to and presents antigens to immune effector cells, such as T cells (e.g., cytotoxic T cells), by interacting with a T cell receptor, and are essential for an adaptive immune response.
  • T cells e.g., cytotoxic T cells
  • the MHC molecule may be a MHC class I molecule or a MHC class II molecule.
  • MHC class I molecules include an a-chain and a P2m chain, and are encoded by the HLA-A, HLA- B, HLA-C, HLA-E, HLA-F and HLA-G genes in humans.
  • MHC class II molecules include an a-chain and a P chain, and are encoded by the HLA-DP, HLA-DQ, and HLA-DR genes in humans.
  • An MHC molecule of the disclose may be an engineered MHC molecule.
  • an engineered MHC molecule comprised of a single chain fusion of the MHC chains.
  • An engineered MHC molecule may also include an antigen-presenting polypeptide covalently or non-covalently bound to the MHC molecule.
  • myeloid progenitor cell refers to a cell capable of dividing and/or undergoing differentiation into one or more mature myeloid cells.
  • a myeloid progenitor may differentiate into one or more myeloid cell types, including, but not limited to, monocytes, microglia, macrophages, dendritic cells, basophils, eosinophils, erythrocytes, mast cells, neutrophils, megakaryocytes, or platelets, or any intermediate progenitor thereof.
  • nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PCR polymerase chain reaction
  • Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • the terms “patient,” “subject,” “individual,” and the like are used interchangeably and refer to any animal, or cells thereof, whether in vitro or in situ, amenable to the compositions and methods described herein. In some instances, the patient, subject or individual is a human.
  • the term “pharmaceutically acceptable excipient, carrier or diluent” refers to any material which, when combined with an active ingredient (e.g., a population of cells), allows the ingredient to retain biological activity and is non-reactive with the subject’s immune system.
  • an active ingredient e.g., a population of cells
  • examples include, but are not limited to, any of the standard pharmaceutical excipients, carriers, or diluents, such as a phosphate buffered saline solution, normal saline, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • progenitor cell refers to a descendant of a stem cell that can further differentiate into specialized cell types within a particular cell lineage.
  • a progenitor cell can be a lymphoid progenitor cell, whereby upon further differentiation give rise to a lymphoblast (the precursor to T cells), or a myeloid progenitor cell, whereby upon further differentiation give rise to a myeloblast (e.g., the precursor to granulocytes).
  • promoter refers to a region of a nucleic acid positioned upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of the gene.
  • the promoter can be the promoter of an endogenous house-keeping gene (e.g., GAPDH).
  • GAPDH house-keeping gene
  • the promoter can be a tissue-specific promoter. For example, a promoter of a gene that is turned on or off in certain cell or tissue types.
  • the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “essentially the same” or “substantially the same” refer a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • STAPLR expands the repertoire of genomic safe harbors where nucleic acids encoding a protein of interest (e.g., IL- 12) can be stably integrated and their expression can be maintained over multiple passages and as the cell changes its phenotype.
  • STAPLR refers to exogenous or heterologous nucleotide sequences introduced to the region.
  • the STAPLR can refer to an intergenic region found between essential genes or genes that are expressed throughout different cell states.
  • the term “sustained transgene expression locus” or “STEL” refer to a locus in the genome of an organism that is resistant to silencing of gene expression.
  • a STEL can be resistant to silencing over time or after changes in cell fate (e.g., differentiation), such that expression of genes contained in the STEL is sustained.
  • Exemplary STEL include, but are not limited to genes encoding ribosomal subunits, mitochondria proteins, actin proteins, eukaryotic translation factors, and histones. Additional exemplary STEL are described in WO2021072329A1, which is incorporated herein by reference.
  • targeting moiety refers to a nucleic acid, polypeptide, peptide, glycoprotein, glycopeptide, proteoglycan, carbohydrate, lipid, small molecule, etc., which is present on the surface of a cell and permits binding of the cell to one or more “receptors,” “targets,” or “markers” associated with a particular organ, tissue, or cell in an organism.
  • Exemplary targeting moieties include, but are not limited to, chimeric antigen receptors (CARs), T-cell receptors (TCRs), and receptors for a cell surface molecule operably linked through at least a transmembrane domain in an internal signaling domain capable of activating a T cell upon binding of the extracellular receptor portion of a protein.
  • CARs chimeric antigen receptors
  • TCRs T-cell receptors
  • receptors for a cell surface molecule operably linked through at least a transmembrane domain in an internal signaling domain capable of activating a T cell upon binding of the extracellular receptor portion of a protein.
  • T cell refers to a type of lymphocyte that plays a central role in cell-mediated immunity.
  • T cells may be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface.
  • B cells B cells and natural killer cells (NK cells)
  • TCR T-cell receptor
  • T cell types include, but are not limited to, conventional adaptive T cells, which include helper CD4+ T cells (also known as helper T cells), CD8+ T cells (also known as cytotoxic T cells), memory T cells, and regulatory CD4+ T cells (also known as T regs), and innate-like T cells including natural killer T cells (also known as NKT cells), mucosal associated invariant T cells, and gamma delta T cells.
  • helper CD4+ T cells also known as helper T cells
  • CD8+ T cells also known as cytotoxic T cells
  • memory T cells also known as regulatory CD4+ T cells (also known as T regs)
  • regulatory CD4+ T cells also known as T regs
  • innate-like T cells including natural killer T cells (also known as NKT cells), mucosal associated invariant T cells, and gamma delta T cells.
  • T cells can be naturally occurring or non-natural, e.g., modified T cells, such as CAR-T cells.
  • T cell activation refers to activation of CD8+ T cells, activation of CD4+ T cells, stimulation of cytotoxic activity of T cells, stimulation of cytokine secretion by T cells, detectable effector functions, modification of the differentiation state of a T cell (e.g., promote expansion and differentiation from T effector to T memory cell), and/or any combination thereof.
  • T cell activation is mediated through stimulation of the TCR receptor by antigen-loaded MHC molecules.
  • activated T cells refers to, among other things, T cells that are undergoing cell division.
  • T cell-mediated immune response refers to an immune response associated with the activation of T cells (e.g., CD8+ T cells) in response to an antigen (e.g., a tumor antigen).
  • an antigen e.g., a tumor antigen
  • cytotoxic T cell or “CD8+ T cell” refers to a T cell that expresses the CD8 glycoprotein in the cell surface and function to destroy virus-infected cells and tumor cells.
  • CD8+ T cells recognize their targets by binding to antigen (e.g., a tumor antigen) associated with MHC class I molecules, which are present on the surface of all nucleated cells.
  • helper T cell or “helper CD4+ T cell” refers to a T cell that expresses the CD4 glycoprotein on the cell surface and assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
  • APCs antigen-presenting cells
  • cytokines that regulate or assist in the active immune response.
  • therapeutic refers to a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
  • the term “treat,” or a grammatical equivalent thereof, refers to a means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder (e.g., a tumor) experienced by a subject.
  • tumor and cancer refer to a group of diseases characterized by the rapid and uncontrolled growth of aberrant cells. Tumor cells or cancer cells can spread locally, or can become metastatic, travelling through the bloodstream and lymphatic system to invade and spread in other parts of the body.
  • cancers include but are not limited to, ACUTE lymphoblastic leukemia (ALL), ACUTE myeloid leukemia (AML), anal cancer, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain tumor (e.g., glioblastoma), breast cancer, cancer of unknown primary, cancer spread to bone, cancer spread to brain, cancer spread to liver, cancer spread to lung, carcinoid, cervical cancer, choriocarcinoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon cancer, colorectal cancer, endometrial cancer, eye cancer, gallbladder cancer, gastric cancer, gestational trophoblastic tumor (GTT), hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma skin cancer, mesothelioma, men’s cancer, molar pregnancy
  • tumor antigen refers to any antigenic substance produced or overexpressed in, by, or on the surface of tumor cells.
  • a tumor antigen can trigger an immune response to the tumor in the host.
  • the tumor antigens can be proteins that are expressed by both healthy and tumor cells, but identify a particular tumor type.
  • a statement that a cell or population of cells is “positive” for a particular marker, or “expresses” a particular marker refers to the detectable presence on or in the cell of a particular marker, for example, a surface marker or an intracellular marker, such as transcription factors.
  • the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
  • FMO fluorescence minus one
  • a statement that a cell or population of cells is “negative” for a particular marker, or fails to express a particular marker or gene refers to the absence of substantial detectable presence on or in the cell of a particular marker for example, a surface marker or an intracellular marker, such as transcription factors.
  • the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, or at a level substantially similar as compared to that for a cell known to be negative for the marker.
  • FMO fluorescence minus one
  • the cytokine is TNFa, IL-IRA, IL-ip, IL- la, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL- 17, IL-18, IL-20, IL-21, IL-23, IL-33, IL-37, TRAIL, MIF, TGFp, IFNa, IFNy, or GM-CSF.
  • the cytokine is interleukin- 12 (IL-12).
  • the population of engineered cells that secrete a cytokine is a population of engineered immune cells that secrete a cytokine. In some embodiments, the population of engineered immune cells that secrete the cytokine is a population of engineered immune cells that secrete IL-12. In some embodiments, the population of engineered immune cells includes one or more of neutrophils, dendritic cells, eosinophils, mast cells, basophils, monocytes, microglia, T cells, B cells, natural killer cells, or any precursors or progenitors thereof.
  • the population of engineered immune cells includes one or more of myeloid progenitor cells, microglia precursor cells, microglial cells, T cells, natural killer cells, or macrophages.
  • the population of engineered cells comprises a population of pluripotent stem cells.
  • the population of engineered cells may comprise a population of induced pluripotent stem cells.
  • the population of engineered immune cells secretes the cytokine at a concentration between 1.0 and 1,000 ng/ml, as measured by homogeneous time resolved fluorescence (HTRF), when cultured (in vitro) at a density of 1 million cells per milliliter for 24 hours.
  • HTRF homogeneous time resolved fluorescence
  • concentration ranges within this spectrum include, but are not limited to: between 1.0 - 10.0 ng/mL, between 10 - 50 ng/mL, between 50 - 100 ng/mL, between 100 - 200 ng/mL, between 200 - 300 ng/mL, between 300 - 400 ng/mL, between 400 - 500 ng/mL, between 500 - 600 ng/mL, between 600 - 700 ng/mL, between 700 -800 ng/mL, between 800 - 900 ng/mL, and between 900 - 1,000 ng/mL.
  • the populations of engineered immune cells secrete the cytokine at a concentration of less than 1.0 ng/ml, when cultured (in vitro) at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells secrete IL-12 at a concentration of between 0.01 - 1.0 ng/mL, between 0.5 - 1.0 ng/mL, or between 0.01 - 0.5 ng/mL.
  • the population of engineered immune cells secrete the cytokine at a concentration of greater than 1,000 ng/ml when cultured (in vitro) at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells secrete the cytokine at a concentration of between 1,000 ng/ml and 2,000 ng/ml.
  • the population of engineered immune cells secrete IL-12 at a concentration of between 1,000 ng/ml and 1,500 ng/ml, or between 1,500 ng/ml and 2,000 ng/ml.
  • the cytokine is TNFa, IL-IRA, IL-1 , IL-la, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-23, IL-33, IL-37, TRAIL, MIF, TGF0, IFNa, IFNy, or GM-CSF.
  • the cytokine is IL-12.
  • Exemplary HTRF assays that can be used to assay the secretion of cytokines, such as IL- 12, and other molecules described here are exemplified in the Examples and well known in the art as described in, for example, Degorce, F., et al. (2009) Chem Genomics 3:22-32; and Achard, S, et al. (2003) Assay and Drug Development Technologies 1.1, Supplement 2: 181-185, which are incorporated herein by reference.
  • the population of engineered immune cells secretes the cytokine at a concentration that is below 1000 ng/ml, as measured by HTRF, when cultured at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells secretes the cytokine at a concentration that is below 900 ng/ml, below 800 ng/ml, below 700 ng/ml, below 600 ng/ml, below 500 ng/ml, below 400 ng/ml, below 300 ng/ml, below 200 ng/ml, below 100 ng/ml, below 50 ng/ml, below 10 ng/ml, or below 1 ng/ml, when cultured at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells secretes the cytokine at a concentration that is below 500 ng/ml, as measured by HTRF, when cultured at a density of 1 million cells per milliliter for 24 hours.
  • the cytokine is IL- 12
  • the population of engineered immune cells secretes IL-12 at a concentration between 1.0 and 1,000 ng/ml, as measured by HTRF, when cultured (in vitro) at a density of 1 million cells per milliliter for 24 hours.
  • concentration ranges within this spectrum include, but are not limited to: between 1.0 - 10.0 ng/mL, between 10 - 50 ng/mL, between 50 - 100 ng/mL, between 100 - 200 ng/mL, between 200 - 300 ng/mL, between 300 - 400 ng/mL, between 400 - 500 ng/mL, between 500 - 600 ng/mL, between 600 - 700 ng/mL, between 700 -800 ng/mL, between 800 - 900 ng/mL, and between 900 - 1,000 ng/mL.
  • the populations of engineered immune cells secrete IL- 12 at a concentration of less than 1.0 ng/ml, when cultured (in vitro) at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells secrete IL-12 at a concentration of between 0.01 - 1.0 ng/mL, between 0.5 - 1.0 ng/mL, or between 0.01 - 0.5 ng/mL.
  • the population of engineered immune cells secrete IL- 12 at a concentration of greater than 1,000 ng/ml when cultured (in vitro) at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells secrete IL- 12 at a concentration of between 1,000 ng/ml and 2,000 ng/ml.
  • the population of engineered immune cells secrete IL-12 at a concentration of between 1,000 ng/ml and 1,500 ng/ml, or between 1,500 ng/ml and 2,000 ng/ml.
  • the population of engineered immune cells secretes IL-12 at a concentration that is below 1,000 ng/ml, as measured by HTRF, when cultured at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered immune cells secretes IL- 12 at a concentration that is below 900 ng/ml, below 800 ng/ml, below 700 ng/ml, below 600 ng/ml, below 500 ng/ml, below 400 ng/ml, below 300 ng/ml, below 200 ng/ml, below 100 ng/ml, below 50 ng/ml, below 10 ng/ml, or below 1 ng/ml, when cultured at a density of 1 million cells per milliliter for 24 hours.
  • the population of engineered cells that secrete the cytokine is a population of engineered myeloid progenitor cells that secrete the cytokine.
  • the population of engineered myeloid progenitor cells has a genetic modification to elicit or enhance an amount of the cytokine (e.g., IL-12) that is secreted by the engineered myeloid progenitor cells as compared to myeloid progenitor cells not comprising the genetic modification.
  • the population of engineered myeloid progenitor cells secrete the cytokine at between 2 and 1000 ng/mL as measured by HTRF, when the cells are cultured at a density of 1 million cells per 24hr.
  • the population of engineered myeloid progenitor cells secrete cytokine at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete the cytokine at below Ing/mL/lM cells/24hr.
  • the population of engineered myeloid progenitor cells that secrete the cytokine is a population of engineered myeloid progenitor cells that secrete IL-12.
  • the population of engineered myeloid progenitor cells has a genetic modification to elicit or enhance an amount of IL-12 that is secreted by the engineered myeloid progenitor cells as compared to myeloid progenitor cells not comprising the genetic modification.
  • the population of engineered myeloid progenitor cells secrete IL-12 at between 2 and 1000 ng/mL as measured by HTRF, when the cells are cultured at a density of 1 million cells per 24hr.
  • the population of engineered myeloid progenitor cells secrete IL-12 at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete IL-12 at below Ing/mL/lM cells/24hr.
  • the population of engineered cells secretes IL- 12 that has an amino acid sequence as set forth in SEQ ID NO: 1.
  • the IL-12 secreted by the population of engineered cells has an amino acid sequence that has at least 75%, 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 is identical to the amino acid sequence set forth in SEQ ID NO: 1.
  • the IL-12 secreted by the population of engineered cells is a human IL-12.
  • the IL-12 secreted by the population of engineered cells is an engineered or variant IL-12.
  • a population of engineered cells described herein e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • the genetic modification enhances the expression, stability or secretion of the cytokine.
  • the genetic modification to elicit or enhance an amount of the cytokine that is secreted by the cells includes an integration of a heterologous nucleic acid encoding the cytokine into the genome of the cells.
  • the integration of a heterologous nucleic acid encoding the cytokine into the genome of the cells results in stable expression and/or secretion of cytokine by the cells after proliferation or differentiation of the cells.
  • the cytokine is IL-12.
  • a population of engineered cells described herein e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • a population of engineered cells described e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • the population of engineered cells having the genetic modification secrete an amount of IL-12 that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher as compared to the amount of IL- 12 secreted by cells not having the genetic modification.
  • the population of engineered cells having the genetic modification secrete an amount of IL-12 that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, or higher as compared to the among of IL- 12 secreted by cells not having the genetic modification.
  • the genetic modification enhances the expression, stability or secretion of the IL-12.
  • the genetic modification to elicit or enhance an amount of IL-12 that is secreted by the cells includes an integration of a heterologous nucleic acid encoding IL-12 into the genome of the cells.
  • the integration of a heterologous nucleic acid encoding IL-12 into the genome of the cells results in stable expression and/or secretion of IL-12 by the cells after proliferation or differentiation of the cells.
  • the genetic modification to elicit or enhance an amount of IL-12 that is secreted by the cells includes an integration of a heterologous nucleic acid encoding the cytokine (e.g., IL-12) into a sustained transgene expression locus (STEL) in the genome of the cells.
  • a heterologous nucleic acid encoding the cytokine e.g., IL-12
  • STL sustained transgene expression locus
  • the STEL is a gene locus that encodes a protein involved in one or more of: ribonucleoprotein complex formation, focal adhesion, cellsubstrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding.
  • the STEL is GAPDH.
  • the STEL is a ribosomal protein gene locus, such as an RPL or RPS gene locus.
  • RPL genes are RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL 18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22.
  • RPS genes are RPS2, RPS 19, RPS 14, RPS3A, RPS 12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS 13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, and RPSII.
  • the STEL is a gene locus encoding a mitochondrial protein, such as MT-CO1, MT-C02, MT-ND4, MT-ND1, and MT-ND2.
  • the STEL is a gene locus encoding an actin protein, such as ACTG1 and ACTB.
  • the STEL is a gene locus encoding a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor such as EIEI.
  • the STEL is a gene locus encoding a histone, such as H3F3A and H3F3B.
  • the STEL is a gene locus selected from FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.
  • the payload candidate’s expression cassette can be governed by an endogenous gene promoter, such as, GAPDH. Consequently, the expression of the cytokine can be linked to an endogenous gene’s expression. The continued activity of the endogenous gene in the engineered cells will then imply that the linked the cytokine transgene expression will remain sustained and constitutive.
  • this disclosure provides the capability to modulate the cytokine’s expression by utilizing an endogenous gene’s expression as the promoter to drive transgene expression.
  • This innovative approach enables precise control over the cytokine’s secretion, allowing for fine-tuning to achieve optimal levels.
  • the disclosure offers a useful mechanism to maintain the cytokine’s secretion at an appropriate and therapeutic level, thereby ensuring effective immune modulation without the risk of excessive or potentially toxic secretion.
  • the genetic modification includes an integration of a heterologous nucleic acid encoding the cytokine (e.g., IL-12) into a sustained transcriptionally active payload region (STAPLR) in the genome of the engineered cells.
  • the STAPLRs can include open chromatin landscape for landing genomic payloads, as well as regulatory regions that enhance expression of the payload, such as promoters or enhancers.
  • the STAPLR can be of any size, such as for example, at least 100 base pairs to at least 100,000 base pairs in length.
  • the STAPLR is in the vicinity of transcriptionally active gene.
  • the STAPLR is near a gene that is specifically expressed in the population of engineered cells.
  • the STAPLR includes the intergenic region between the RPL34 gene and the OSTC gene , the intergenic region between the ACTB gene and the FSCN1 gene , the intergenic region between the AKIRIN1 gene and the NDUFS5 gene, the intergenic region between the PRDX1 gene and the AKR1 Al gene, the intergenic region between the PTGES3 gene and the NACA gene, the intergenic region between the MLF2 gene and the PTMS gene , the intergenic region between the RABI 3 gene and the RPS27 gene, the intergenic region between the JTB gene and the RABI 3 gene, the intergenic region between the AKR1A1 gene and the NASP gene, the intergenic region between the NDUFS5 gene and the MACF1 gene, the intergenic region between the SRSF9 gene and the DYNLL1 gene, the intergenic region between the MYL6B gene and the MYL6 gene, the intergenic region between the GPX1 gene and the RHOA gene, the intergenic region
  • the heterologous nucleic acid encoding IL-12 integrated at a location that is at least 100-5000 base pairs away from the nearest flanking gene of the STAPLR.
  • the exogenous nucleotide sequence has been integrated at a location that is at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 5000, at least 10000, at least 15000, or at least 20000 base pairs away from the nearest flaking gene of the STAPLR.
  • the STAPLR is located near a STEL. For additional information relating to STEL, see International Application No PCT/US2023/66396, which is incorporated by reference. For additional information relating to STAPLR, see International Application No. PCT/US2023/066396, which is incorporated herein by reference.
  • the genetic modification includes an integration of a heterologous nucleic acid encoding the cytokine (e.g., IL-12) into a STAPLR in the genome of the engineered cells.
  • a population of engineered cells described herein e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • the cytokine can be any of a chemokine, interferon, interleukin, lymphokine, and tumor necrosis factor.
  • Exemplary additional cytokines or immunomodulatory soluble factors that can be expressed by the population of engineered cells include, but are not limited to, LILRA3, sCD40L, CCLI, CCL2, CCL3, CCR4, CCL5, CCL7, CCL8/MCP-2, CCL11, CCL13 (also known as MCP-4), HCC-I/CCLI 4, CTAC/CCLI 7, CCLI9, CCL22, CCL23, CCL24, CCL26, CCL27, VEGF, PDGF, lymphotactin (also known as XCLI), Eotaxin, FGF, EGF, IP-IO, TRAIL, FASL, GCP-2 (also known as CXCL6), NAP-2 (also known as CXCL7), CXCL8, CXCL10, IT AC (also known as CXCL11), CXCLI2, CXCL13, CXCLI5, TGFBR11, TGFb, IL-la, ILlb, ILIRn, IL
  • a population of engineered cells described herein is able to present antigens to T cells.
  • the T cells are CD4+ T cells.
  • the T cells are modified T cells.
  • the modified T cells are CAR-T cells.
  • the presentation of tumor antigens by the population of engineered cells results in activation of the T cells.
  • the presentation of tumor antigens by the population of engineered cells results in production of tumor antigen-specific T cells.
  • a population of engineered cells described herein can activate or enhance an immune response when administered to a subject.
  • a population of engineered cells can activate or enhance an immune response to an antigen (e.g., a tumor antigen) when administered to a subject.
  • the population of cells results in the activation or enhancement of an immune response to an antigen (e.g., a tumor antigen) when administered to a subject.
  • the population of engineered cells results in the activation or enhancement of a T-cell mediated immune response to an antigen when administered to a subject.
  • the T cell-mediated immune response is a CD8+ T cell-mediated immune response. In other embodiments, the T cell-mediated immune response is a CD4+ T cell-mediated immune response. In some embodiments, the subject has a tumor, and the antigen is a tumor antigen.
  • the population of engineered cells can further express cell surface proteins that enhance presentation of antigen (e.g., tumor antigen) to T cells and/or the immune triggered by interacting with the T cells.
  • the population of engineered cells can express cell surface proteins associated with antigen presenting cells (APCs) that facilitate presentation of antigens (e.g., tumor antigens) to T cells and B cells, thereby enhancing an immune response to said antigens.
  • APCs antigen presenting cells
  • each of the engineered cells in the populations of engineered cells has a major histocompatibility complex (MHC) molecule on the cell surface.
  • MHC major histocompatibility complex
  • the MHC molecule binds to and presents an antigen to T cells.
  • the MHC molecule is an MHC class I molecule.
  • the MHC molecule is an MHC class II molecule.
  • each of the engineered cells overexpress the MHC molecule on the cell surface.
  • the overexpression of the MHC molecule on the cell surface increases the presentation of the antigens to T cells.
  • each of the engineered cells in the populations of engineered cells has a co-stimulatory molecule on the cell surface.
  • the co-stimulatory molecule can be any co-stimulatory molecule that can interact with a protein in the cell surface of T cells to enhance an immune response.
  • the each of the engineered immune cells or engineered myeloid progenitor cells express a an immune modulatory molecule selected from CD2, CD7, B7-1 (CD80), B7-2 (CD86), 4- 1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD58, CD70, CD80, CD83, CD86, HLA-G, HLA-E, MICA, MICB, HVEM, lymphotoxin beta receptor, 3TR6, ILT3, ILT4, TGFb, IL-IRn, HVEM, BTLA, an agonist or antibody that binds to a Toll ligand receptor, and B7-H3 ligand.
  • an immune modulatory molecule selected from CD2, CD7, B7-1 (CD80), B7-2 (CD86), 4- 1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion
  • the said co-stimulatory molecule is CD80, CD86, or CD40. In some embodiments, the co-stimulatory molecule enhances the immune response triggered by the population of engineered cells. In some embodiments, the co-stimulatory molecule enhances a T cell-mediated immune response by interacting with T cells, e.g., by interacting with a cell surface protein expressed by the T cells. In some embodiments, the T cell-mediated immune response is a CD8+ T cell mediated response. In other embodiments, the T cell-mediated immune response is a CD4+ T cell mediated response.
  • each of the engineered cells in the populations of engineered cells has a targeting moiety on the cell surface.
  • the targeting moiety can allow the engineered immune cells of the myeloid progenitor cells to specifically interact with a target cell, tissue or organ in a subject.
  • the targeting moiety can promote the interaction of the engineered immune cells of the myeloid progenitor cells with T cells, thereby enhancing presentation of antigens (e.g., tumor antigens) and/or the activation of a T cell-mediated immune.
  • the targeting moiety binds to a protein expressed in the cell surface of T cells. In some embodiments, the targeting moiety binds to a protein expressed in the cell surface of CD8+ T cells. In other embodiments, the targeting moiety binds to a protein expressed in the cell surface of CD4+ T cells. In some embodiments, the targeting moiety is an engineered receptor. In some embodiments, the targeting moiety is a CAR.
  • the population of engineered cells is a population of human engineered cells.
  • the method to prepare a population of engineered cells described herein includes introducing a genetic modification to elicit or enhance an amount of the cytokine (e.g., IL- 12) that is secreted by the cells into induced pluripotent stem cells (iPSCs) using a CRISPR/Cas system; and deriving the population of engineered cells from the iPSCs.
  • the genetic modification to elicit or enhance an amount of the cytokine that is secreted by the cells includes is the integration of a heterologous nucleic acid encoding the cytokine.
  • the cytokine is IL- 12.
  • introducing the genetic modification to elicit or enhance an amount of the cytokine (e.g., IL-12) that is secreted by the cells into iPSCs includes integrating the heterologous nucleic acid encoding the cytokine is integrated into the genome of the iPSCs using a CRISPR/Cas system.
  • the CRISPR/Cas system has been used for introducing genetic modifications and gene regulation in a variety of species.
  • a target nucleic acid can be modified by the interaction of the CRISPR/Cas system and a sequence present in the target nucleic acid, for example, to cause cleavage (e.g., hydrolysis of one or more phosphodiester bonds) of the target nucleic acid and introduce the genetic modification.
  • the heterologous nucleic acid encoding IL-12 is integrated into the genome of the iPSCs using a CRISPR/Cas9 system or a CRISPR/Casl2 system.
  • the heterologous nucleic acid encoding the cytokine is integrated into a STEL in the genome of the iPSCs.
  • the STEL is a gene locus that encodes a protein involved in one or more of: ribonucleoprotein complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding.
  • the STEL is GAPDH.
  • the STEL is a ribosomal protein gene locus.
  • the ribosomal protein gene locus is an RPL or RPS gene locus.
  • the RPL gene locus is RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL 12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLPO, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, or RPL22.
  • the RPS gene locus is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS 13, RPSA, RPS5, RPS16, RPS25, RPS 15, RPS20, or RPSII.
  • the STEL is a gene locus encoding a mitochondrial protein.
  • the gene locus encoding a mitochondrial protein is MT-CO1, MT-C02, MT-ND4, MT-ND1, or MT-ND2.
  • the STEL is a gene locus encoding an actin protein.
  • the actin protein gene locus is ACTG1 and ACTB.
  • the STEL is a gene locus encoding a eukaryotic translation elongation factor or a eukaryotic translation initiation factor such.
  • the gene locus encoding the eukaryotic translation elongation factor or the eukaryotic translation initiation factor is EEF1 Al, EEF2, or EIEI.
  • the STEL is a gene locus encoding a histone.
  • the gene locus encoding a histone is H3F3 A or H3F3B.
  • the STEL is a gene locus selected from FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.
  • the heterologous nucleic acid encoding the cytokine (e.g., IL-12) is integrated into a STAPLR loci in the genome of the iPSCs.
  • the STAPLR is in the vicinity of transcriptionally active gene.
  • the STAPLR is near a gene that is specifically expressed in the population of engineered cells.
  • the STAPLR includes the intergenic region between the RPL34 gene and the OSTC gene , the intergenic region between the ACTB gene and the FSCN1 gene , the intergenic region between the AKIRIN1 gene and the NDUFS5 gene, the intergenic region between the PRDX1 gene and the AKR1 Al gene, the intergenic region between the PTGES3 gene and the NACA gene, the intergenic region between the MLF2 gene and the PTMS gene , the intergenic region between the RABI 3 gene and the RPS27 gene, the intergenic region between the JTB gene and the RABI 3 gene, the intergenic region between the AKR1A1 gene and the NASP gene, the intergenic region between the NDUFS5 gene and the MACF1 gene, the intergenic region between the SRSF9 gene and the DYNLL1 gene, the intergenic region between the MYL6B gene and the MYL6 gene, the intergenic region between the GPX1 gene and the RHOA gene, the intergenic region
  • the heterologous nucleic acid encoding IL- 12 further includes one or more regulatory elements.
  • a regulatory element can include a regulatory sequence, which is any DNA sequence that is responsible for the regulation of gene expression, such as promoters and operators.
  • the regulatory element can be a segment of a nucleic acid molecule, which is able to increase or decreasing the expression of specific genes within an organism.
  • the regulatory element is a promoter.
  • a promoter is a nucleotide sequence that directs the transcription of a structural gene.
  • a promoter is in the 5’ non-coding region of a gene, proximal to the transcriptional start site of a structural gene.
  • Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Without being limiting, these promoter elements can include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al., Mol. Endocrinol.
  • CREs cyclic AMP response elements
  • SREs serum response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O’Reilly et al., J. Biol. Chem. 267: 19938 (1992); incorporated by reference in its entirety), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1994); incorporated by reference in its entirety), SPI, cAMP response element binding protein (CREB; Loeken et al., Gene Expr.
  • promoters used herein can be inducible or constitutive promoters.
  • inducible promoters can include, for example, a tamoxifen inducible promoter, tetracycline inducible promoter, or a doxycycline inducible promoter (e.g., tre) promoter.
  • Constitutive promoters can include, for example, SV40, CMV, UBC, elongation factor la short (EFS) promoter, EFlalpha, PGK, or CAGG. Any suitable promoter known in the art for expression of a gene in a population of engineered cells as described herein can be used.
  • the heterologous nucleic acid encoding the cytokine (e.g., IL-12) is introduced into the iPSCs in a vector.
  • the vector can be plasmid, virus, or other nucleic acid designed for introducing a nucleic acid of interest into a cell.
  • the vector is used to introduce a gene of interest into a host cell whereby the vector will interact with polymerases in the cell to express the protein encoded in the vector.
  • the vector can exist in the cell extra-chromosomally or integrated into the genome of the host cell.
  • a vector described herein is integrated into the genome of a cell.
  • the heterologous nucleic acid encoding the cytokine is delivered in the same vector or a separate vector as the CRISPR/Cas system.
  • the iPSCs can be prepared by inducing expression of one or more genes, e.g., POU5F1/OCT4 in combination with, but not restricted to, SOX2, KLF, c-MYC, NANOG, and/or LIN28/LIN28A.
  • Reprogramming factors may be delivered by various means (e.g., viral, non-viral, RNA, DNA, or protein delivery).
  • endogenous genes may be activated by using, e.g., a CRISPR/Cas system to reprogram non-pluripotent cells into iPSCs.
  • the population of engineered cells is derived from the iPSCs.
  • deriving the population of engineered cells from the iPSCs includes differentiating the iPSCs into the engineered cells. Differentiation can be performed using suitable known methods. For example, methods for inducing PSCs into hematopoietic progenitor cells, cells of myeloid lineage, and T lymphocytes are described in, e.g., Kennedy et al., Cell Rep. (2012) 2: 1722-35. Exemplary methods of differentiating iPSCs into myeloid progenitor cells are described in, for instance, U.S.
  • differentiating the iPSCs into the engineered cells includes contacting the iPSCs with one or more differentiation factors.
  • the specific combination of differentiation factors used will depend on the desired cell type(s).
  • differentiating the iPSCs into the engineered cells includes contacting the iPSCs with one or more differentiation factors that drive the commitment and/or differentiation into myeloid progenitor cells.
  • the one or more differentiation factors that drive the commitment and/or differentiation into myeloid progenitor cells includes one or more of bone morphogenetic protein 4 (BMP4), stem cell factor (SCF), Fms-like tyrosine kinase 3 (FLT3/CD135), IL-6, IL-3, granulocyte colony stimulating factor (G-CSF), granulocyte and monocyte stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF).
  • the method for preparing a population of cells described herein further include an isolation step.
  • the isolation step is for isolating cells that have the genetic modification to elicit or enhance an amount of IL-12 that is secreted, from cells lacking the genetic modification prior to deriving the population of engineered cells from the iPSCs.
  • the isolation step is for isolating derived engineered cells from iPSCs, or intermediate cells, after deriving the population of engineered cells. Any suitable method known in the art for selection and isolation of cells of interest can be used, such as centrifugation and fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the method for preparing a population of cells described herein further include expanding the population of engineered cells.
  • the population of engineered cells is expanded after isolating the engineered cells from the iPSCs. Expanding the population of engineered cells can include culturing or contacting the engineered cells with a medium having a cytokine and growth factor mixture permissive for expansion of the engineered cells.
  • the method for preparing a population of cells described herein further includes preserving the population of engineered cells after the deriving.
  • the population of engineered cells can be cryopreserved.
  • the cryopreserved population of engineered cells can be thawed at a later time, and can be diluted for downstream applications.
  • the population of engineered immune cells or the population of engineered myeloid progenitor cells produced by the methods described herein is used in preparing a composition for treating a tumor (e.g., a glioblastoma) in a subject.
  • a tumor e.g., a glioblastoma
  • This disclosure provides a serum- and feeder-free protocol to differentiate stem cells (including human iPSCs) towards the myeloid lineage.
  • stem cells including human iPSCs
  • Various methods known in the art can be employed in connection with the present disclosure. Muffat et.al, Nat Med. 2016 Nov; 22(11): 1358-1367, Pandaya et. al, Nat Neurosci. 2017 May; 20(5): 753-759, Abud et. al, Neuron 2017 Apr 19;94(2):278-293, Douvaras et.
  • Myeloid progenitor cells arise from the yolk sack during embryonic development. In order to mimic the embryonic development of these myeloid progenitor cells, primitive streak-like cells are generated from PSCs, followed by hematopoietic and myelopoietic cocktails in serum-free media.
  • myeloid progenitor cells in the supernatant fraction of the culture that expressed myeloid markers, including but not limited to CD45, CD14, CX3CR1, CD33, CD1 lb. These myeloid cells can continue to be generated in the supernatant fraction of the culture for a considerable amount of time, often reaching 3 to 4 months.
  • Myeloid progenitor cells can be collected and frozen around 30 days or 45 days after initiation of the differentiation protocol (the exact timing of collection is PSC-line dependent and should be experimentally defined based on the day of maximum yield). The yield is usually between 25 and 120 myeloid cells for every starting PSC, and their post-thaw viability is 85 ⁇ 10%.
  • this also provides methods to generate “matured” myeloid progenitor cells.
  • Maturation of the iPSC-derived myeloid progenitor cells is advantageous to imbue these therapeutic cells with an array of enhanced functions.
  • the maturation process can offer the cells resistance to M2 polarization, which typically involves a shift toward tumor-promoting phenotypes, enhancing their potency in cancer-fighting applications.
  • matured myeloid progenitor cells exhibit an improved recovery rate post-cryopreservation, conferring robust stability and longevity to these cells.
  • the maturation process can increase cell viability, increasing recovery rate post-cry opreservation by increasing pro-survival signaling molecules like c-FLIP (cFLAR), MCL-1 and Al (Bfl-1).
  • the matured cells can also be characterized by enhanced migration capacity, facilitating their efficient navigation towards tumors.
  • the cells may harness an elevated ability to phagocytose tumor cells, coupled with an improved chemokine and cytokine expression, which exemplifies their enhanced therapeutic utility.
  • the described maturation process thus, strengthens these cells, rendering them a powerful asset in the field of cancer immunotherapy.
  • the present disclosure provides methods for generating myeloid cells from pluripotent stem cells.
  • the pluripotent stem cells are from any mammalian species, but preferably from humans.
  • the pluripotent stem cells are either induced pluripotent stem cells (“iPS cells” or “iPSCs”), or embryonic stem cells (“ES cells” or “ESCs”).
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • Such methods involve a step where the pluripotent stem cells are cultured under conditions that induce myeloid differentiation, leading to the generation of CD45 +/CD14 + /CX3CR1+ myeloid cells.
  • these cells express Lyz2, CD14, TGM2, SLAMF7, ILl-Rn, GPR34 when evaluated by qPCR.
  • the differentiation medium comprises BMP4, GM-CSF, VEGF, SCF, IL3, TPO, M-CSF and FLT31.
  • the medium further optionally comprises bFGF.
  • the pluripotent stem cells are cultured under conditions that induce myeloid differentiation, leading to the generation of CX3CR1 + myeloid cells. In some of the embodiments, the pluripotent stem cells are cultured under conditions that induce myeloid differentiation, leading to the generation of CD45+ myeloid cells. In some embodiments the pluripotent stem cells are cultured or expanded in a bioreactor. In some embodiments the pluripotent stem cells are cultured in a cell factory under active gassing. By “active gassing” is meant exerting or applying a gas mixture pressure gradient in the cell factory or cell factories. Gas mixtures contemplated by the present disclosure include ratios of about 1% to about 20% CO2 to about 80% to about 99% air, about 3% CO2 to about 97% air and about 5% CO2 to about 95% air.
  • the myeloid cells are cultured from about 1 to about 30 days, from about 2 to about 25 days, from about 2 to about 20 days, from about 2 to about 18 days, from about 2 to about 15 days, from about 2 to about 10 days, from about 2 to about 8 days, from about 2 to about 6 days or from about 2 to about 4 days.
  • the myeloid cells are at least 90% CD45+, at least 91% CD45+, at least 92% CD45+, at least 93% CD45+, at least 94% CD45+, or at least 95% CD45+.
  • the myeloid cells are cultured for about 2 days, for about 4 days, for about 6 days or about 8 days, for about 10 days, for about 12 days for about 14 days, for about 16 days, for about 18 days, for about 20 days, for about 22 days, for about 24 days, for about 26 days, for about 28 days or for about 30 days.
  • the myeloid cells are at least about 70% CD45+/CD14+/CX3CR1+, at least about 71% CD45+/CD14+/CX3CR1+, at least about 72% CD45+/CD14+/CX3CR1+, at least about 73% CD45+/CD14+/CX3CR1+, at least about 74% CD45+/CD14+/CX3CR1+, at least about 75% CD45+/CD14+/CX3CR1+, at least about 80% CD45+/CD14+/CX3CR1+, or at least about 85% CD45+/CD14+/CX3CR1+.
  • a multi-step process is used in which the cells are cultured with different combinations of cytokines and tissue culture media at each stage. Steps in these multi-step processes may result in inducing differentiation of pluripotent stem cells into primitive hemangioblasts, and/or inducing differentiation of primitive hemangioblasts into myeloid progenitors.
  • the methods provided herein for the generation of CD45+/CD14 +/CX3CR1+ myeloid cells from pluripotent stem cells comprise performing one or more of the following steps: First, contacting a cell culture with a first composition comprising BMP4 in a culture medium, wherein when the cell culture is initially contacted with the first composition the cell culture comprises pluripotent stem cells.
  • a small molecule able to activate the same pathway as BMP4 can be used; Second, contacting the cell culture with a second composition comprising one or more of SCF, and VEGF, and optionally bFGF, (for example each of SCF, and VEGF, with or without bFGF) in a hematopoietic cell medium; Third, contacting the cell culture with a third composition comprising one or more of SCF, IL-3, TPO, M-CSF, and FLT3 ligand (for example each of SCF, IL-3, TPO, M-CSF, and FLT3 ligand) in a hematopoietic cell medium; and fourth, contacting the cell culture with a fourth composition comprising one or more of M-CSF , FLT3 ligand, and GM-CSF (for example each of M-CSF, FLT3 ligand , and GM-CSF) in a hematopoietic cell medium. In some embodiments all of the above four steps
  • tissue culture medium suitable for maintenance of stem cells in some embodiments a tissue culture medium suitable for maintenance of stem cells is used, while in other embodiments a tissue culture medium suitable for differentiation of stem cells is used. In the last three of the above four steps, any suitable hematopoietic cell medium can be used.
  • the supernatant when carrying out the methods described above or elsewhere herein for the generation of myeloid cells from pluripotent stem cells, instead of discarding the tissue culture supernatant when performing media changes, the supernatant is centrifuged, and the cells present in the supernatant are recovered and added back to the cell cultures.
  • the cells present in the supernatant are recovered and added back to the cell cultures.
  • cells present in the culture supernatant are recovered and added back to the cell cultures.
  • the present disclosure provides myeloid cells or microglial progenitor cells, such as those produced by the methods described herein.
  • the present disclosure provides a “substantially pure” populations of such cells.
  • myeloid progenitor cells begin to emerge as “floaters”.
  • differentiation of cells into myeloid progenitor cells involves adherent cell culture. However, during differentiation, progenitor cells release from tissue culture dishes in which the cells are cultured and float or suspend in the tissue culture medium.
  • the progenitor cells begin to emerge as floaters around day 8 of differentiation. In some embodiments, the progenitor cells begin to emerge as floaters around day 9 of differentiation. In some embodiments, the progenitor cells begin to emerge as floaters around day 10 of differentiation.
  • progenitor cells prior to day 10, progenitor cells started to emerge as floaters. Accordingly, on Day 10 floating progenitor cells can be collected from the supernatant fraction, pelleted, and resuspended in culture medium (e.g., StemPro-34 SFM medium containing Flt-3, M-CSF, and GM-CSF. In some embodiments, the cells can be further cultured until at about day 30 or day 45, at which point, the generated cells (z.e., myeloid progenitor cells).
  • culture medium e.g., StemPro-34 SFM medium containing Flt-3, M-CSF, and GM-CSF.
  • the cells can be further cultured until at about day 30 or day 45, at which point, the generated cells (z.e., myeloid progenitor cells).
  • myeloid progenitor cells are subjected to a maturation phase.
  • the maturation of the myeloid progenitor cells can be advantageous as maturation can imbue the myeloid progenitor cells with added functionality.
  • the maturation phase can offer the cells resistance to M2 polarization, which typically involves a shift toward tumor-promoting phenotypes, enhancing their potency in cancer-fighting applications.
  • matured myeloid progenitor exhibit an improved recovery rate postcryopreservation, conferring robust stability and longevity to the myeloid progenitor cells by increasing expression of pro-survival signaling molecules like cFLAR, Al, MCL-1.
  • floating progenitor cells are harvested, e.g., harvested on either day 30 or day 45, from the supernatant fraction as described. These cells are then centrifuged to form a pellet and subsequently resuspended in a specially tailored maturation culture medium.
  • the maturation medium is an XVIVO-15 culture blend enriched with GlutaMax and M-CSF.
  • the prepared cells are then seeded onto plastic dishes for cultivation.
  • the myeloid progenitor cells are matured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days. For example, in some embodiments, the myeloid progenitor cells are matured in maturation medium for a duration of 7 days.
  • the myeloid progenitor cells can be evaluated for the expression of XCR1, CD66B, and CD 11b, to confirm maturation.
  • matured myeloid progenitor cells can be identified based on their unique expression pattern: a lack of XCR1 and CD66B expression combined with the presence of CD1 lb expression.
  • myeloid progenitor cells expressed LYZ2, MMP7, CD14, SLAMF7, TGM2, IL-IRn, and GPR34.
  • compositions having any of the populations of engineered cells described herein.
  • the composition is a pharmaceutical composition that includes a population of engineered cells that a cytokine, and a pharmaceutically acceptable excipient, carrier or diluent.
  • the cytokine is TNFa, IL-IRA, IL-ip, IL-la, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-23, IL-33, IL-37, TRAIL, MIF, TGFP, IFNa, IFNy, or GM-CSF.
  • the cytokine is IL-12.
  • the composition (e.g., pharmaceutical composition) includes a population of engineered immune cells that secrete the cytokine (e.g., IL-12).
  • the population of engineered immune cells secrete the cytokine at between 1 and 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per 24 hours.
  • the population of engineered immune cells secrete the cytokine at a concentration that is below 1,000 ng/mL/lM cells/24hr.
  • the population of engineered immune cells secrete the cytokine at between 2 and 1000 ng/mL/lM cells/24hr.
  • the population of engineered immune cells secrete cytokine at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells secrete the cytokine at below Ing/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells includes one or more of myeloid progenitor cells, microglia precursor cells, microglial cells, T cells, natural killer cells, or macrophages. [00166] In other embodiments, the composition (e.g., pharmaceutical composition) includes a population of engineered myeloid progenitor cells that secrete the cytokine (e.g., IL-12).
  • the population of engineered myeloid progenitor cells has a genetic modification to elicit or enhance an amount of the cytokine that is secreted by the engineered myeloid progenitor cells as compared to myeloid progenitor cells not comprising the genetic modification.
  • the population of engineered myeloid progenitor cells secrete the cytokine at between 1 and 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per milliliter for 24 hrs.
  • the engineered myeloid progenitor cells secrete the cytokine at a concentration that is below 500 ng/mL/lM cells/24hr.
  • the population of engineered myeloid progenitor cells secrete the cytokine at between 2 and 1000 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete the cytokine at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete the cytokine at below Ing/mL/lM cells/24hr.
  • the composition (e.g., pharmaceutical composition) includes a population of engineered immune cells that secrete IL-12.
  • the population of engineered immune cells secrete IL-12 at between 1 and 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per 24 hours.
  • the population of engineered immune cells secrete IL-12 at a concentration that is below 1,000 ng/mL/lM cells/24hr.
  • the population of engineered immune cells secrete IL-12 at between 2 and 1000 ng/mL/lM cells/24hr.
  • the population of engineered immune cells secrete IL-12 at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells secrete IL-12 at below Ing/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells includes one or more of myeloid progenitor cells, microglia precursor cells, microglial cells, T cells, natural killer cells, or macrophages.
  • the composition (e.g., pharmaceutical composition) includes a population of engineered myeloid progenitor cells that secrete IL-12.
  • the population of engineered myeloid progenitor cells has a genetic modification to elicit or enhance an amount of IL-12 that is secreted by the engineered myeloid progenitor cells as compared to myeloid progenitor cells not comprising the genetic modification.
  • the population of engineered myeloid progenitor cells secrete IL-12 at between 1 and 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per milliliter for 24 hrs.
  • the engineered myeloid progenitor cells secrete IL-12 at a concentration that is below 500 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete IL-12 at between 2 and 1000 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete IL-12 at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete IL-12 at below Ing/mL/lM cells/24hr.
  • a composition (e.g., pharmaceutical composition) described herein includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that secrete IL- 12 having an amino acid sequence as set forth in SEQ ID NO: 1.
  • the IL- 12 secreted by the population of engineered cells in the composition has at least 75%, 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 is identical to the amino acid sequence set forth in SEQ ID NO: 1.
  • the IL-12 is a human IL-12.
  • the IL-12 is an engineered or variant IL-12.
  • a composition e.g., pharmaceutical composition
  • a composition includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that has a genetic modification to elicit or enhance an amount of the cytokine (e.g., IL-12) that is secreted by the cells.
  • the genetic modification enhances the expression, stability or secretion of the cytokine.
  • the genetic modification includes an integration of a heterologous nucleic acid encoding cytokine into the genome of the cells.
  • the cytokine is IL-12.
  • a composition e.g., pharmaceutical composition
  • a population of engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells expresses one or more additional cytokines.
  • a composition (e.g., pharmaceutical composition) described herein includes a population of engineered cells (e.g, a population of engineered immune cells or a population of engineered myeloid progenitor cells) is able to present antigens to T cells.
  • the T cells are CD8+ T cells.
  • the T cells are CD4+ T cells.
  • the T cells are modified T cells.
  • the modified T cells are CAR-T cells.
  • the presentation of tumor antigens by the population of engineered cells results in activation of the T cells in a subject that has been administered the composition.
  • the presentation of tumor antigens by the population of engineered cells results in production of tumor antigen-specific T cells in a subject that has been administered the composition.
  • a composition e.g., pharmaceutical composition
  • a composition includes a population of engineered cells described herein (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that can activate or enhance an immune response when administered to a subject.
  • the population of engineered cells results in the activation or enhancement of an immune response to an antigen (e.g., a tumor antigen) in a subject that has been administered the composition.
  • the population of engineered cells results in the activation or enhancement of a T-cell mediated immune response to an antigen in a subject that has been administered the composition.
  • the T cell- mediated immune response is a CD8+ T cell-mediated immune response. In other embodiments, the T cell-mediated immune response is a CD4+ T cell-mediated immune response. In some embodiments, the subject has a tumor, and the antigen is a tumor antigen.
  • a composition e.g., pharmaceutical composition
  • includes population of engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • cell surface proteins that enhance presentation of antigen (e.g., tumor antigen) to T cells and/or the immune triggered by interacting with the T cells.
  • each of the engineered cells in the populations of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) included in the composition (e.g., pharmaceutical composition) described herein has a major histocompatibility complex (MHC) molecule on the cell surface.
  • MHC major histocompatibility complex
  • the MHC molecule binds to and presents an antigen to T cells.
  • the MHC molecule is an MHC class I molecule or an MHC class II molecule.
  • each of the engineered cells overexpress the MHC molecule on the cell surface.
  • the overexpression of the MHC molecule on the cell surface increases the presentation of the antigens to T cells.
  • each of the engineered cells in the populations of engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • the composition e.g., pharmaceutical composition
  • the co-stimulatory molecule selected from CD2, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD58, CD70, CD80, CD83, CD86, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3TR6, ILT3, ILT4, HVEM, BTLA, an agonist or antibody that binds a Toll ligand receptor, or B7-H3 ligand.
  • the said co-stimulatory molecule is CD80, CD86, or CD40.
  • the co-stimulatory molecule enhances the immune response triggered by the population of engineered cells. In some embodiments, the co-stimulatory molecule enhances a T cell-mediated immune response by interacting with T cells, e.g., by interacting with a cell surface protein expressed by the T cells. In some embodiments, the T cell-mediated immune response is a CD8+ T cell mediated response. In other embodiments, the T cell-mediated immune response is a CD4+ T cell mediated response.
  • each of the engineered cells in the populations of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) included in the composition (e.g., pharmaceutical composition) described herein has a targeting moiety on the cell surface.
  • the targeting moiety binds to a protein expressed in the cell surface of T cells.
  • the targeting moiety binds to a protein expressed in the cell surface of CD8+ T cells.
  • the targeting moiety binds to a protein expressed in the cell surface of CD4+ T cells.
  • the targeting moiety is an engineered receptor.
  • the targeting moiety is a CAR.
  • the pharmaceutically acceptable excipient, carrier, or diluent can be any excipient, carrier or diluent known in the art.
  • the pharmaceutically acceptable excipient, carrier, or diluent can be a cell culture medium (e.g., one that optionally lacks any animal-derived component), sterilized water, physiological saline, general buffers (e.g., phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, anti-oxidants, surfactants, suspensions, isotonic agents, and/or preservatives may be included in a pharmaceutical composition described herein.
  • the specific excipient, carrier, or diluent will depend on the route of administration intended for the pharmaceutical composition.
  • the pharmaceutically acceptable excipient, carrier, or diluent is an excipient, carrier, or diluent suitable for treatment of a tumor in a subject.
  • the pharmaceutically acceptable excipient, carrier, or diluent is an excipient, carrier, or diluent suitable for administration of the pharmaceutical composition by injection to a subject.
  • the pharmaceutically acceptable excipient, carrier, or diluent is an excipient, carrier, or diluent suitable for administration of the composition by direct injection into the site of a tumor (e.g., a brain tumor).
  • compositions e.g., pharmaceutical compositions
  • a disease such as cancer
  • the pharmaceutical formulation can be used for the treatment of a tumor formed by cancerous cells.
  • the composition is for use in treating a subject having a tumor.
  • the tumor can be, for example, anal cancer, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain tumor (e.g., glioblastoma), breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, endometrial cancer, eye cancer, gallbladder cancer, gastric cancer, gestational trophoblastic tumor (GTT), head and neck cancer, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, mesothelioma, molar pregnancy, mouth and oropharyngeal cancer, nasal and sinus cancers, nasopharyngeal cancer, esophageal cancer, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, rare cancers, rectal cancer, salivary gland cancer, secondary cancers, skin cancer (non-melanoma), stomach cancer, testicular cancer, thyroid cancer, unknown primary cancer, uterine cancer, vaginal cancer, or vulval cancer.
  • the tumor is a leukemia, a carcinoid, a melanoma, a lymphoma, a myeloma, a carcinoid, a sarcoma, a carcinoma, an adenoma, an adenocarcinoma, or a cancer of unknown primary.
  • the tumor can be metastatic or non- metastatic.
  • the composition is for use in treating a brain tumor. In some embodiments, the composition is for use in treating a glioblastoma.
  • compositions e.g., a pharmaceutical composition
  • a population of engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid cells
  • the dosage form can be in any form suitable for administration to a subject by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intracranial, intracerebraor topically.
  • the dosage form is suitable for administration by injection into a subject.
  • kits having a dosage form of a composition (e.g., a pharmaceutical composition) that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid cells) suitable for administration to a subject, including any of the compositions described herein.
  • a composition e.g., a pharmaceutical composition
  • engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid cells
  • the kit includes instructional material for the use of said dosage form.
  • the instructional material includes instructions of preparing the composition or the population of engineered cells for administration into a subject. Such instructions can include, but are not limited to, instructions for preparing or storing the composition, diluting the population of engineered cells, adding additional additives to the treatment, or combining the composition with an additional anti-cancer treatment.
  • the instructional material includes instructions for administering the dosage form into a subject.
  • the instructional material can include instructions for administering the dosage form by injection to the site of a tumor in a subject.
  • the kit further includes an applicator for administering a composition, including a pharmaceutical composition, described here.
  • the applicator can be any device suitable for administration of the pharmaceutical compositions described herein to a subject, including, but not limited to, a hypodermic syringe, a needle, a balloon-dilating catheter, a pipette, and the like.
  • the applicator can also be a single-use or multiple-use administration device, and can be included in the kit as a pre-filled device with the pharmaceutical composition.
  • the device can be selected or modified depending on the route of administration of the composition.
  • the device can be a device for administration by injection into a subject, e.g., a hypodermic syringe.
  • the device can also be an implantable device.
  • the device is a specialized device for delivery of cell therapies into a subject, such as those described in U.S. Patent No. 11,666,710 B2, which in incorporated by reference.
  • the device is pre-filled with the composition.
  • One aspect of the present disclosure provides methods for treating a subject having a tumor, or for stimulating an immune response against a tumor in a subject.
  • the method is a method for stimulating an immune response against a tumor in a subject, the method including administering a composition that includes a population of engineered cells that secrete a cytokine (e.g., IL-12) as described herein, and allowing the population of engineered immune cells to secrete the cytokine, thereby stimulating the immune response against the tumor.
  • a cytokine e.g., IL-12
  • the method is a method for treating a subject having a tumor, the method including administering a composition that includes a population of engineered cells that secrete a cytokine (e.g., IL-12) as described herein, and allowing the population of engineered immune cells to secrete the cytokine, thereby treating the tumor in the subject.
  • a cytokine e.g., IL-12
  • the cytokine is TNFa, IL- 1RA, IL-ip, IL-la, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL- 13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-23, IL-33, IL-37, TRAIL, MIF, TGFp, IFNa, IFNy, or GM-CSF.
  • the cytokine is IL-12.
  • the method includes administering to a subject a composition (e.g., a pharmaceutical composition) that includes a population of engineered immune cells that secrete the cytokine (e.g., IL- 12) as described herein.
  • a composition e.g., a pharmaceutical composition
  • the population of engineered immune cells secrete the cytokine at between 1 and 1,000 ng/mL/lM cells/24hr.
  • the population of engineered immune cells secrete the cytokine at a concentration that is below 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per milliliter for 24 hrs.
  • the population of engineered immune cells secrete the cytokine at a concentration that is below 500 ng/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells secrete the cytokine at between 2 and 1000 ng/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells secrete the cytokine at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells secrete the cytokine at below Ing/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells includes one or more of myeloid progenitor cells, microglia precursor cells, microglial cells, T cells, natural killer cells, or macrophages.
  • the method includes administering to a subject a composition (e.g., a pharmaceutical composition) that includes a population of engineered myeloid progenitor cells that secrete a cytokine (e.g., IL-12) as described herein.
  • a composition e.g., a pharmaceutical composition
  • the population of engineered myeloid progenitor cells has a genetic modification to elicit or enhance an amount of the cytokine that is secreted by the engineered myeloid progenitor cells as compared to myeloid progenitor cells not comprising the genetic modification.
  • the population of engineered myeloid progenitor cells secrete the cytokine at between 1 and 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per milliliter for 24 hrs. In some embodiments, the engineered myeloid progenitor cells secrete the cytokine at a concentration that is below 1000 ng/mL/lM cells/24hr. In some embodiments, the engineered myeloid progenitor cells secrete the cytokine at a concentration that is below 500 ng/mL/lM cells/24hr.
  • the population of engineered myeloid progenitor cells secrete the cytokine at between 2 and 1000 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete the cytokine at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete the cytokine at below Ing/mL/lM cells/24hr.
  • the method includes administering to a subject a composition (e.g., a pharmaceutical composition) that includes a population of engineered immune cells that secrete IL- 12 as described herein.
  • a composition e.g., a pharmaceutical composition
  • the population of engineered immune cells secrete IL-12 at between 1 and 1,000 ng/mL/lM cells/24hr.
  • the population of engineered immune cells secrete IL-12 at a concentration that is below 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per milliliter for 24 hrs.
  • the population of engineered immune cells secrete IL-12 at a concentration that is below 500 ng/mL/lM cells/24hr.
  • the population of engineered immune cells secrete IL-12 at between 2 and 1000 ng/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells secrete IL-12 at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells secrete IL-12 at below Ing/mL/lM cells/24hr. In some embodiments, the population of engineered immune cells includes one or more of myeloid progenitor cells, microglia precursor cells, microglial cells, T cells, natural killer cells, or macrophages.
  • the method includes administering to a subject a composition (e.g., a pharmaceutical composition) that includes a population of engineered myeloid progenitor cells that secrete IL-12 as described herein.
  • a composition e.g., a pharmaceutical composition
  • the population of engineered myeloid progenitor cells has a genetic modification to elicit or enhance an amount of IL-12 that is secreted by the engineered myeloid progenitor cells as compared to myeloid progenitor cells not comprising the genetic modification.
  • the population of engineered myeloid progenitor cells secrete IL-12 at between 1 and 1,000 ng/mL, as measured by HTRF, when the cells are cultured at a density of 1 million cells per milliliter for 24 hrs. In some embodiments, the engineered myeloid progenitor cells secrete IL-12 at a concentration that is below 1000 ng/mL/lM cells/24hr. In some embodiments, the engineered myeloid progenitor cells secrete IL-12 at a concentration that is below 500 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete IL-12 at between 2 and 1000 ng/mL/lM cells/24hr.
  • the population of engineered myeloid progenitor cells secrete IL-12 at between 1 and 2 ng/mL/lM cells/24hr. In some embodiments, the population of engineered myeloid progenitor cells secrete IL-12 at below Ing/mL/lM cells/24hr.
  • the method includes administering to a subject a pharmaceutical composition that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that secrete IL-12 having an amino acid sequence as set forth in SEQ ID NO: 1.
  • the IL-12 secreted by the population of engineered cells in the pharmaceutical composition has at least 75%, 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 is identical to the amino acid sequence set forth in SEQ ID NO: 1.
  • the IL-12 is a human IL-12.
  • the IL-12 is an engineered or variant IL-12.
  • the population of engineered cells secretes IL-12 constitutively.
  • the method includes administering to a subject a pharmaceutical composition that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that has a genetic modification to elicit or enhance an amount of the cytokine (e.g., IL-12)that is secreted by the cells.
  • the genetic modification enhances the expression, stability or secretion of the cytokine.
  • the genetic modification includes an integration of a heterologous nucleic acid encoding the cytokine into the genome of the cells.
  • the cytokine is IL-12.
  • the method includes administering to a subject a pharmaceutical composition that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) expresses one or more additional cytokines.
  • a population of engineered cells e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells
  • the method includes administering a pharmaceutical composition that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that is able to present antigens to T cells.
  • the T cells are CD8+ T cells.
  • the T cells are CD4+ T cells.
  • the T cells are modified T cells, e.g., CAR-T cells.
  • the presentation of tumor antigens by the population of engineered cells results in activation of the T cells in the subject.
  • the presentation of tumor antigens by the population of engineered cells results in production of tumor antigen-specific T cells in the subject.
  • the method includes administering a pharmaceutical composition that includes a population of engineered cells described herein (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) the stimulation of an immune response against a tumor antigen in the subject.
  • the immune response is a T-cell mediated immune response.
  • the T cell-mediated immune response is a CD8+ T cell-mediated immune response.
  • the T cell-mediated immune response is a CD4+ T cell- mediated immune response.
  • the immune response comprises secretion of pro-inflammatory cytokines by T regs.
  • the pro- inflammatory cytokines comprise TFNy, TNFa, or both IFNy and TNFa.
  • the method includes administering a pharmaceutical composition that includes population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that can further express cell surface proteins that enhance presentation of antigen (e.g., tumor antigen) to T cells and/or the immune triggered by interacting with the T cells.
  • each of the engineered cells in the populations of engineered cells has a major histocompatibility complex (MHC) molecule on the cell surface.
  • the MHC molecule binds to and presents an antigen to T cells.
  • the MHC molecule is an MHC class I molecule or an MHC class II molecule.
  • each of the engineered cells overexpress the MHC molecule on the cell surface, thereby increasing the presentation of the antigens to T cells.
  • each of the engineered cells in the populations of engineered cells has a co-stimulatory molecule on the cell surface.
  • the co-stimulatory molecule selected from CD2, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD58, CD70, CD80, CD83, CD86, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3TR6, ILT3, ILT4, HVEM, BTLA, an agonist or antibody that binds a Toll ligand receptor, or B7-H3 ligand.
  • the said co-stimulatory molecule is CD80, CD86, or CD40.
  • the co-stimulatory molecule enhances a T cell-mediated immune response (e.g., a CD8+ T cell-mediated immune response) by interacting with T cells, e.g., by interacting with a cell surface protein expressed by the T cells.
  • a T cell-mediated immune response e.g., a CD8+ T cell-mediated immune response
  • each of the engineered cells in the populations of engineered cells has a targeting moiety on the cell surface.
  • the targeting moiety binds to a protein expressed in the cell surface of T cells.
  • the targeting moiety is an engineered receptor, e.g., a CAR.
  • the method includes administering a pharmaceutical composition that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that is allogenic to the subject. In some embodiments, the method includes administering a pharmaceutical composition that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that is autologous to the subject.
  • a pharmaceutical composition that includes a population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells) that is autologous to the subject.
  • the stimulation of an immune response against the tumor results in the improvement of at least one symptom associated with the tumor, e.g., as determined by responsiveness/non-responsiveness, or indicators known in the art and described herein.
  • Non-limiting examples of the common medical technologies and methods used to examine and diagnose a tumor in a subject include, but are not limited to, X-rays, computed tomography (CT) scan, magnetic resonance imaging (MRI), positron emission tomography (PET), bone scintigraphy (Bone Scan), biopsy, EQ-5D questionnaire for assessing quality of life, PERCIST criteria for assessing disease progression, numeric rating scale for assessing pain degree.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • Bone Scan bone scintigraphy
  • biopsy EQ-5D questionnaire for assessing quality of life
  • PERCIST criteria for assessing disease progression
  • numeric rating scale for assessing pain degree.
  • the method further includes examining the subject for responsiveness to the pharmaceutical composition that includes the population of engineered cells (e.g., a population of engineered immune cells or a population of engineered myeloid progenitor cells).
  • the responsiveness of the subject can be determined as an improvement of at least one parameter of disease progression (e.g., tumor progression).
  • the subject can have partial response or complete response to a treatment.
  • the response to a treatment can be determined based on methods known in the art. A person skilled in the art can determine the proper methods based on the type of diseases being evaluated. Non-limiting examples of the methods include RECIST criteria, ICDS criteria and PERCIST criteria.
  • subject has a tumor selected from anal cancer, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain tumor (e.g., glioblastoma), breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, endometrial cancer, eye cancer, gallbladder cancer, gastric cancer, gestational trophoblastic tumor (GTT), head and neck cancer, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, mesothelioma, molar pregnancy, mouth and oropharyngeal cancer, nasal and sinus cancers, nasopharyngeal cancer, esophageal cancer, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, rare cancers, rectal cancer, salivary gland cancer, secondary cancers, skin cancer (non-melanoma), stomach cancer, testicular cancer, thyroid cancer,
  • a tumor selected from anal cancer, bile duct cancer, bladder cancer, bone cancer,
  • the subject has a leukemia, a carcinoid, a melanoma, a lymphoma, a myeloma, a carcinoid, a sarcoma, a carcinoma, an adenoma, an adenocarcinoma, or a cancer of unknown primary.
  • the tumor is metastatic or non-metastatic.
  • the tumor is a brain tumor.
  • the brain tumor is a glioblastoma.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • composition e.g., a pharmaceutical composition
  • the method includes administering the composition that includes a population of engineered cells described herein orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically.
  • the composition is administered intratum orally.
  • the composition is administered by direct injection into the site of the tumor.
  • the composition can be administered to the subject at one time or over a series of administrations and may be administered to the patient at any time from diagnosis of the tumor onwards.
  • the composition can be administered as one or more doses over the course of a treatment.
  • the doses can be administered using the same or a different route of administration, and can contain the same amount or a different amount of the composition.
  • a dose of the composition is administered about once every day, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every week, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 2 weeks, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 3 weeks, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 4 weeks, about once every 29 days, about once every 30 days, about once every 31 days, about once every 32 days, about once every 33 days, about once every 34 days, about once every 5 weeks, about once every 36 days, about once every 37 days, about once every 38 days, about once every 39 days, about once every 40 days,
  • Such doses can be for a specified period of time.
  • the dose can be for about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 15 weeks, about 18 weeks, about 22 weeks, about 24 weeks or six months, about 7 months, about 8 months about 9 months, about 10 months, about 11 months, about 12 months or more than 12 months.
  • the duration of administration can depend on the route of administration of the composition.
  • the composition is administered to a subject for over less than 5 minutes, about 5 minutes, about 10 minutes, about 15 minutes about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 150 minutes, about 180 minutes, or more than 180 minutes.
  • the methods describe herein include administering an additional anti-cancer therapy to the subject.
  • the additional anti-cancer therapy can be administered before, after or simultaneous to the pharmaceutical composition that includes the population of engineered cells (e.g., the population of engineered immune cells or the population of engineered myeloid progenitor cells).
  • the additional anti-cancer therapy includes a hormone blocking therapy, chemotherapy, an immune checkpoint inhibitor, a kinase inhibitor, a therapeutic antibody or binding fragments thereof, electromagnetic field therapy, a cell therapy, radiation therapy, surgery (e.g., resection surgery), or any combination thereof.
  • the additional anti-cancer therapy includes a hormone blocking therapy.
  • hormone blocking therapies include, but are not limited to, tamoxifen, anastrozole, and letrozole.
  • the additional anti-cancer therapy includes a chemotherapy, of such chemotherapies include platinum-based chemotherapy drugs (e.g., cisplatin, carboplatin); electochemotherapy; alkylating agents; taxanes (e.g., paclitaxel (Taxol®), docetaxel (Taxotere®), EndoTAG-PM (a formulation of paclitaxel encapsulated in positively charged lipid-based complexes; MediGene), Abraxane® (a formulation of paclitaxel bound to albumin)); tyrosine kinase inhibitors (e.g., imatinib/Gleevec®, sunitinib/Sutent®, dasatinib/Sprycel®);
  • platinum-based chemotherapy drugs e.g
  • the additional anti-cancer therapy includes a kinase inhibitor.
  • kinase inhibitors include, but are not limited to Imatinib mesylate (approved for chronic myelogenous leukemia, gastrointestinal stromal tumor and some other types of cancer), Gefitinib (Iressa, also known as ZD 1839), Erlotinib (marketed as Tarceva), Sorafenib, Sunitinib (Sutent), Dasatinib (Srycel), Lapatinib (Tykerb), Nilotinib (Tasigna), Bortezomib (Velcade); Janus kinase inhibitors, ALK inhibitors, crizotinib; Bcl-2 inhibitors, obatoclax, navitoclax, gossypol, PARP inhibitors, Iniparib, Olaparib, PBK inhibitors, perifosine, Apatinib, VEGF
  • the kinase inhibitor can also include serine/threonine kinase inhibitors.
  • examples include Temsirolimus (Torisel), Everolimus (Afinitor), Vemurafenib (Zelboraf), Trametinib (Mekinist) or Dabrafenib (Tafinlar).
  • the additional anti-cancer therapy includes an immune checkpoint inhibitor.
  • An “immune checkpoint” refers to inhibitory pathways hardwired into the immune system that are crucial for maintaining self-tolerance and modulating the immune responses in peripheral tissues in order to minimize collateral tissue damage. Immune checkpoint molecules can be stimulatory or inhibitory to an immune checkpoint. An immune checkpoint inhibitor inhibits inhibitory immune checkpoint molecules.
  • the immune checkpoint inhibitor includes an inhibitor of one or more immune checkpoint molecules selected from PD-1, PD-L1, CTLA-4, VISTA, PD-L2, IDO, ARG1, B7-H3, B7- H4, LAG3, 2B4, BTLA, TIM3, A2aR, KIR, IL4i 1, VEGF, LILRB2, and LILRB4.
  • the immune checkpoint inhibitor is an antibody that binds an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, VISTA, PD-L2, IDO, ARG1, B7- H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, KIR,IL4il, VEGF, LILRB2, and LILRB4.
  • the additional anti-cancer therapy includes a therapeutic antibody or binding fragment thereof.
  • the therapeutic antibody or binding fragment thereof can be an agonistic antibody (ie., activates a target protein) or an antagonistic antibody (ie., inhibits a target protein).
  • Exemplary antibody or binding fragments thereof include, but are not limited, anti-EGFR antibodies, anti-VEGF antibodies, such as bevacizumab (Avastatin), anti-ErbB2 antibodies, such as trastuzumab and pertuzumab, as well as antibodies that bind and inhibit immune checkpoint molecules, such as anti-PD-1 antibodies, anti-PD-Ll antibodies, and anti-CTLA-4 antibodies.
  • the additional anti-cancer therapy includes a cell therapy.
  • the cell therapy is a modified T cell, e.g., CAR-T cell, therapy.
  • the additional anti-cancer therapy includes a CD28 agonist.
  • the CD28 agonist is an anti-CD28 antibody.
  • the additional anti-cancer therapy does not comprise a CD28 agonist.
  • the CD28 agonist is an anti-CD28 antibody.
  • the additional anti-cancer therapy includes an electric field therapy.
  • the electric field therapy is an alternating electric field therapy (e.g., a tumor treating field or TTField).
  • Example 1 Methods for the generation of myeloid progenitor cells from human induced pluripotent stem cells (iPSCs)
  • the following example describes methods to generate myeloid progenitor cells from human induced pluripotent stem cells (iPSCs). These methods, enabled by the unique properties of stem cells to self-renew and their potential to differentiate into any cell type, allow for a more reliable and scalable production of myeloid progenitor cells as compared to existing autologous cell therapies. Furthermore, the methods described include certain “maturation” steps that endow iPSC-derived myeloid progenitor cells with additional functions, for example, an improved recovery rate following cry opreservation and a more suitable profile for anti-tumor therapy, for example, by expressing low levels of certain immune modulatory molecules, such as, IL-6, relative to bone marrow monocyte derived myeloid cells.
  • iPSCs human induced pluripotent stem cells
  • iPSCs were plated in tissue culture dishes with Essential 8 medium containing Y-27632 and cultured for 1 day, before cultures were changed to Essential 8 medium (without Y-27632) and cultured for additional 2 days with daily medium change to allow expansion. Then, cultures were changed to Essential 6 medium containing BMP-4 and cultured for 4 days with daily media change to start differentiation. After this period, the cultures were changed to StemPro-34 SFM medium containing GlutaMAX supplemented with SCF, VEGF, and bFGF for 2 days with daily medium changes.
  • the culture media was replaced every 2 days with StemPro-34 SFM medium containing SCF, IL-3, M-CSF, Flt3 ligand and TPO.
  • cells in the supernatant fraction were re-inoculated into the culture every 2 or 3 days, along with fresh StemPro-34 SFM medium containing Flt-3, M-CSF and GM-CSF until approximately day 30, at which point, the generated cells in the culture supernatant (z.e., myeloid progenitor cells) were subjected to a maturation phase, which imparts the myeloid progenitor cells with added functionality.
  • the floating progenitor cells harvested on approximately day 30 were gathered from the supernatant fraction of the differentiation culture dishes, transferred to cell culture dishes and cultured for 7 days in maturation medium containing XVIVO-15 culture blend supplemented with GlutaMax and M-CSF.
  • the cells underwent evaluation for the expression of XCR1, CD66B, and CD1 lb. Identification of the matured myeloid progenitor cells was achieved based on their unique expression pattern: a lack of XCR1 and CD66B expression combined with the presence of CD1 lb expression.
  • an improved rate of recovery cry opreservation was observed in the matured myeloid progenitor cells as compared to myeloid progenitor cells that were not subjected to the maturation step.
  • FIG. 1 illustrates an exemplary cell differentiation scheme employed to generate myeloid progenitor cells from iPSCs.
  • iPSCs were differentiated into myeloid progenitor cells within a span of 30 to 45 days following induction.
  • the progenitor cells underwent a subsequent maturation process to acquire certain desired cellular characteristics, after which they were subjected to cry opreservation to maintain viability during storage.
  • cryopreservation the cells were isolated and counted with an automated cell counter prior to freezing. Cells were resuspended in freezing medium and transferred to cryogenic vials. Cryogenic vials with cells were frozen with a controlled rate freezer and stored in liquid nitrogen vapor. At time of use, cells from the cryogenic vial were transferred to a centrifuge tube resuspended in cell-culture medium in a drop-wise fashion. Cells were centrifuged and resuspended in the appropriate assay or culture medium prior to utilization in experiments.
  • Example 2 Methods to genetically engineer iPSCs for the stable and sustained expression of IL-12
  • This example describes methods used to genetically engineer iPSCs for the stable and sustained expression of a cytokine transgene.
  • this example describes experimental work that was conducted to integrate heterologous nucleic acids encoding IL- 12 transgenes into the GAPDH STEL site of iPSCs.
  • the resulting iPSCs, genetically engineered to drive increased IL- 12 expression, were then subjected to a myeloid progenitor cell differentiation process as outlined in Example 1.
  • FIG. 2 is an illustration of the cloning strategy used to integrate IL- 12 transgenes into the GAPDH STEL site.
  • the IL- 12 transgenes were integrated into the GAPDH STEL site by using a 2 A sequence replacing the GAPDH stop codon to integrate the IL- 12 transgene cassette into the GAPDH 3’ UTR.
  • the IL- 12 transgenes were integrated using CRISPR-based gene editing systems as described below.
  • IL-12 cassettes were employed to genetically engineer the iPSCs.
  • a first cassette contained IL-12 (SEQ ID NO: 1:
  • the IL-12 cassettes were integrated using the CRISPR gene editing system. To achieve engineering, Cas RNPs were pre-complexed with guide RNAs specific to the GAPDH locus. The resulting protein complexes were electroporated together with a homology-directed DNA repair (HDR) template, containing either IL-12 or CD19t-IL-12 into the iPSCs. Cells having the desired genetic modifications were selected and expanded using methods described herein. In some instances, the IL- 12 only cassette was integrated into two alleles (biallelic) of GAPDH. In some instances, the IL- 12 only cassette was integrated into a single allele (monoallelic) of GAPDH.
  • HDR homology-directed DNA repair
  • the payload candidate’s expression cassette was governed by an endogenous gene promoter, z.e., GAPDH. Consequently, the payload candidate’s expression was tied to the endogenous gene’s expression. The continued activity of the gene in the engineered cells implied that the linked payload transgene’s expression would remain sustained and constitutive.
  • PCR polymerase chain reaction
  • Example 3 Characterization of iPSC-derived myeloid progenitor cells genetically engineered with an IL-12 transgene
  • This example describes the characterization of iPSC-derived myeloid progenitor cells genetically engineered to secrete IL-12.
  • iPSCs were genetically engineered to express IL-12 from the GAPDH locus and subsequently directed along a specific differentiation pathway to produce genetically engineered myeloid progenitor cells.
  • the genetically engineered myeloid progenitor cells were subjected to the maturation process described in Example 1. Subsequently, the genetically engineered myeloid progenitor cells underwent a thorough characterization process. This process was aimed at evaluating cellular features, determining their cell capabilities, and ensuring the cells possessed expected functionality arising from the genetic modifications and the subsequent differentiation and maturation stages.
  • FACS fluorescence-activated cell sorting
  • FIGS. 3A-3D show exemplary FACS data collected to characterize iPSC-derived myeloid progenitor cells genetically engineered to secrete IL-12.
  • FIGS. 3A and 3B show genetically engineered cells express well-established myeloid progenitor cell markers, CD45+, CD1 lb+, and CD14+.
  • FIG. 3C shows an exemplary FACs profile that confirm the myeloid progenitor cells do not express neutrophil marker CD66b, supporting the purity of the myeloid progenitor cell population.
  • FIG. 3D is an exemplary FACS profile showing the cells do not express proliferation markers Ki67 and pHH3.
  • An advantageous aspect of the genetically engineered myeloid progenitor cells described herein is their potential to express Ml markers, which can offer significant benefits when employed for the treatment of cancer, e.g., glioblastoma.
  • Ml markers By expressing Ml markers, the myeloid progenitor cells exhibit enhanced anti-tumor effects through the release of pro- inflammatory cytokines and chemokines that activate immune responses against tumor cells.
  • the ability of myeloid progenitor cells to penetrate tumor tissues coupled with their potential to modulate certain immune responses and inhibit tumor-associated angiogenesis, further augments their therapeutic utility. Accordingly, the upregulation of Ml markers from the genetically engineered myeloid progenitor cells was investigated.
  • FIG. 4 shows exemplary experimental results confirming iPSC-derived myeloid progenitor cells genetically engineered to secrete IL-12 upregulate Ml markers, z.e., HLADR, and CD86.
  • the results are bar graphs based on FACS data collected from myeloid progenitor cell lines that were co-cultured with T cells. These data show that engineered myeloid progenitor cells (CD19t-IL-12, IL-12 monoallelic, and IL-12 biallelic cell lines), but not control cells (non-edited myeloid progenitor cells), upregulated Ml markers when cocultured in the presence of T cells.
  • the upregulation of the Ml markers highlights the therapeutic efficacy of the iPSC-derived myeloid progenitor cells as the Ml markers are involved in the cells’ ability to evoke a potent immune response.
  • the HLADR marker a major histocompatibility complex class II cell surface receptor, plays a role in presenting antigens to T cells, thereby contributing to the activation of the immune response and anti-tumor activity.
  • CD86 is a significant co-stimulatory molecule that enhances T-cell activation, promoting a robust immune reaction.
  • FIG. 5 shows exemplary experimental results demonstrating M2 markers (CD206, CD 163, and MerTK) were downregulated by the genetically engineered myeloid progenitor cells after co-culture with T cells.
  • the results are bar graphs based on FACS data collected from myeloid progenitor cell populations co-cultured with T cells. These data show that the engineered myeloid progenitor cells (CD19t-IL-12, IL-12 monoallelic, and IL-12 biallelic), but not control cells (non-edited myeloid progenitor cells), downregulated genes that are typically present in tumor-associated macrophages and M2 macrophages, which are known to suppress immune responses and promote tissue remodeling, aiding tumor growth and survival.
  • the engineered myeloid progenitor cells CD19t-IL-12, IL-12 monoallelic, and IL-12 biallelic
  • control cells non-edited myeloid progenitor cells
  • CD206 a mannose receptor associated with alternative macrophage activation (M2 -type)
  • M2 -type mannose receptor associated with alternative macrophage activation
  • CD 163 another M2 macrophage marker involved in anti-inflammatory responses
  • MerTK a receptor tyrosine kinase that can mediate efferocytosis and contribute to immune evasion by tumors
  • the lowered expression of these markers suggests the skewing of genetically engineered myeloid progenitor cells away from an M2 phenotype and towards a more tumoricidal Ml profile.
  • cytokine secretion profiles of iPSC-derived myeloid progenitor cells genetically engineered to secrete IL- 12 was evaluated. Evaluation of the cytokine secretion profiles provides insight into the functional characteristics of the cell populations - specifically, their ability to produce and release certain cytokines involved in modulating an immune response.
  • cytokine production To evaluate cytokine production, flow-cytometry based approaches were employed. In particular, spent medium was harvested from populations of the genetically engineered myeloid progenitor cells cultured for 5 days and treated with fluorescent markers specific to cytokines of interest, e.g., IL-4, IL-2, IP-10, IL-1 beta, TNF-alpha, MCP-1, ILI A, IL-6, IL-10, IFN-gamma, IL-12, IL-8, and TGF-beta 1. The presence or absence of the cytokines was then quantified by flow cytometry.
  • fluorescent markers specific to cytokines of interest e.g., IL-4, IL-2, IP-10, IL-1 beta, TNF-alpha, MCP-1, ILI A, IL-6, IL-10, IFN-gamma, IL-12, IL-8, and TGF-beta 1.
  • the presence or absence of the cytokines was then quantified by flow cytometry.
  • FIG. 6 shows exemplary experimental results of cytokine profiles for iPSC derived myeloid progenitor cells genetically engineered to secrete IL-12 and control cells.
  • the results are bar graphs based on flow-cytometry experiments performed to detect cytokines of interest. These data show a side-by-side comparison of cytokine secretion profiles for two genetically engineered myeloid progenitor cell lines (CD19t-IL-12, and IL- 12 monoallelic) and control cells (non-edited myeloid progenitor cells).
  • the cytokines assayed included IL-4, IL-2, IP- 10, IL-1 beta, TNF-alpha, MCP-1, IL- 17 A, IL-6, IL- 10, IFN-gamma, IL-12, IL-8, and TGF-beta 1.
  • IL-12 output from the genetically modified myeloid progenitor cells was conducted.
  • an assay that made use of Forster Resonance Energy Transfer (FRET) technology was employed.
  • FRET Forster Resonance Energy Transfer
  • HTRF homogeneous time resolved fluorescence
  • Samples of the spent medium were processed using manufacture’s protocols.
  • donor and acceptor antibodies were incubated in the spent medium for periods ranging from 2 to 24 hours, followed by analysis on a Clariostar microplate reader (BMG Labtech).
  • the readout from this analysis was based on the ratio of acceptor and donor emission signals, providing a measure known as the delta ratio.
  • This parameter calculated as the difference between the ratio of a standard or sample and the zero-standard ratio, serves as a reliable indicator of IL- 12 secretion.
  • FIG. 7 provides exemplary experimental results showing IL-12 output of iPSC- derived myeloid progenitor cells genetically engineered to secrete IL-12.
  • these exemplary data show rates of IL-12 production of control cells (non-edited myeloid progenitor cells) and myeloid progenitor cells generated from iPSCs genetically engineered to secrete IL-12 with three different engineering strategies.
  • the rate of IL-12 produced is identified on the y-axis in nanograms per milliliter from cells cultured at a density of one million cells per twenty-four hours (IL-12 ng/mL/lM/24hr).
  • the control and genetically engineered myeloid progenitor cell lines are identified on the x-axis.
  • This example describes experimental work that was performed to explore the impact of iPSC-derived myeloid progenitor cells, genetically engineered to secrete IL-12, on tumor cell killing.
  • the genetically engineered myeloid progenitor cells were generated as described in Example 1 and Example 2.
  • these experimental results show genetically engineered myeloid progenitor cells, in combination with T cells, trigger an immune response that causes the successful killing of tumor cells. Accordingly, this example provides experimental evidence that genetically engineered myeloid progenitor cells can elicit tumor cell killing, thus providing valuable strategies for therapeutic applications in cancer treatment.
  • U251NucRed tumor cells were prepared and put into tissue culture plates. Next, engineered myeloid cells and control cells were added at varying densities into the wells. Afterwards, a batch of expanded CD8 T cells sourced from a healthy donor were introduced into the co-cultures, along with suitable stimulation. From Day 0 to Day 5, the co-cultures were evaluated for IL-12 secretion and T cell derived IFN-gamma secretion. At Day 5, the co-cultured cells were stained with the soluble dye Casp-3/7 Green to differentiate cellular viability. Post staining, images of the co-culture were collected and analyzed using the IncuCyte system.
  • FIG. 8 shows exemplary experimental results demonstrating iPSC-derived myeloid progenitor cells genetically engineered to secrete IL-12 effectively kill tumor cells and inhibit tumor cell growth in vitro.
  • FIG. 8 is exemplary data taken from cell killing assays that compare varying quantities of two genetically engineered myeloid progenitor cell lines — one line with CD19t-IL-12 integration, the other with monoallelic, IL- 12 integration — and control cells (non-edited myeloid progenitors).
  • the y-axis shows the number of apoptotic U251 cells.
  • the x-axis identifies the cells (and cell populations) present in the corresponding wells.
  • FIGS. 9A-9B show exemplary experimental results demonstrating the ability of genetically engineered myeloid progenitor cells to kill tumor cells leading to increased tumor cell death.
  • These figures show bar graphs quantifying the growth of U251 tumor cells in coculture with CD8 T cells and varying quantities of control cells (non-edited myeloid progenitor cells) (FIG. 9A), as compared with genetically engineered myeloid progenitor cell lines having a monoallelic IL-12 (FIG. 9B).
  • the x-axis represents cell numbers for each cell type, while the y-axis corresponds to U251 tumor cell growth. Measurements were taken after a 5-day co-culture period.
  • the data reveals that IL-12 engineered myeloid progenitor cells effectively inhibited cancer growth over a range of cells/well, while the nonedited myeloid progenitor cells (parental cell line) had negligible impact on cancer growth.
  • IL-12 engineering enhances myeloid progenitor mediated tumor killing by T cells.
  • Example 5 Genetically engineered myeloid progenitor cells demonstrate resistance to the tumor microenvironment and drive an enhanced immune response
  • This example describes experimental results that was collected to evaluate the impact of the tumor microenvironment on the ability of engineered myeloid progenitor cells to elicit a pro-inflammatory, anti-tumor response from CD8 T cells.
  • the exemplary results described below provide evidence as to the capabilities of iPSC-derived myeloid progenitor cells to overcome an immunosuppressive tumor microenvironment (evaluated by M2 polarization) and elicit CD8 T cell activity, an important component of an anti-tumor immune response.
  • FIG.10 illustrates an exemplary workflow that was used to evaluate the impact of M2 polarization on CD8 T cell activity mediated by genetically modified myeloid progenitor cells.
  • Myeloid progenitor cells derived from iPSCs genetically engineered to secrete IL-12 and control cells (myeloid progenitor cells derived from non-edited iPSCs) were cultured, separately, under conditions that simulate the tumor microenvironment. This stimulation involved culturing cell populations in the presence of TGF-beta and IL-10 to induce M2 skewing.
  • CD8 T cells were separated from donor PBMCs using a commercially available CD8 T cell isolation kit.
  • the isolated CD8 T cells were co-cultured with M2-skewed myeloid progenitor cells.
  • the tissue culture dishes containing populations of modified progenitor cells were evaluated to measure the levels IL-12, IFN-gamma, TNF-alpha, IL-2, Perforin, Granzyme A, Granzyme B, and Granulysin in the cell supernatant. Exemplary experimental results of this workflow are reported in FIGS. 11 A, 11B, and FIGS. 12A-12G.
  • FIGS. 11A and 11B are exemplary experimental results that show myeloid progenitor cells rescue IFN-gamma secretion from CD 8 T cells despite M2 polarization.
  • FIG. 11A shows the amounts of IFN-gamma (y-axis) that was detected by flow cytometry following the co-culture of T cells with varying quantities of M2 polarized myeloid progenitor cells (indicated on x-axis). This data demonstrate that the secretion of IFN-gamma by T cells is rapidly elevated by the addition of genetically engineered myeloid progenitor cells despite M2 polarization.
  • FIG. 11B is exemplary data showing the amounts of IL-12 (y axis) that was detected by varying quantities of M2 polarized myeloid progenitor cells (identified along x-axis).
  • FIGS. 12A-12G are exemplary experimental results showing myeloid progenitor cells genetically engineered to secrete IL-12 activate CD8 T cell cytolytic activity despite M2 polarization. More particularly, FIG. 12A shows the amount of IL-12 (indicated on y-axis) that was detected from the cell populations indicated on the x-axis. FIGS. 12B-12G show the amounts of CD8 T cell cytolytic cytokines (TNF-alpha, IL-2, Perforin, Granzyme A, Granzyme B, and Granulysin - as indicated) in that was detected from corresponding cell populations.
  • CD8 T cell cytolytic cytokines TNF-alpha, IL-2, Perforin, Granzyme A, Granzyme B, and Granulysin - as indicated
  • This example describes experimental work that was performed to evaluate the therapeutic capacity of genetically engineered myeloid progenitor cells to treat a tumor, in vivo.
  • This example also provides exemplary experimental data that present compelling evidence of significant tumor suppression that was achieved with myeloid progenitor cells genetically engineered to secrete IL- 12 as described herein.
  • GBM glioblastoma
  • huPBMCs human peripheral blood mononuclear cells
  • I Intratumoral
  • FIG. 13 provides exemplary data showing the significant tumor suppressive capabilities of genetically engineered myeloid progenitor cells, in vivo. Specifically, FIG. 13 shows line graphs of tumor volumes (indicated on y-axis) that were detected over time (in days, indicated on x-axis) from mice across different treatment groups. The upper panel shows all mice in the study and the lower panels shows individual mice per treatment group. These exemplary data demonstrate the anti-tumor efficacy of genetically engineered myeloid progenitor cells. Notably, the data indicate that myeloid progenitor cell lines engineered to express IL-12 (IL-12 CD19t and IL-12 biallelic) exhibited superior tumor suppressive capabilities when compared to no treatment or control myeloid progenitor cell treatment groups.
  • IL-12 IL-12 CD19t and IL-12 biallelic
  • FIGS. 14A and 14B show exemplary experimental results further demonstrating the in vivo therapeutic efficacy of genetically engineered myeloid progenitor cells.
  • FIG. 14A shows exemplary data that was obtained by collecting tumor measurements from pre-clinical mouse models of glioblastoma (GBM) that had been subjected to the indicated treatments (shown on the x-axis). These exemplary data clearly demonstrate a significantly lower tumor growth inhibition ratio (y-axis) when using myeloid progenitor cells genetically engineered to secrete IL-12 compared to treatment with control non-edited myeloid progenitor cells.
  • GBM glioblastoma
  • FIG 14B shows representative images of the tumors which were harvested from the pre-clinical mouse model described in FIG 14A at humane end points.
  • the tumors were cleared of surrounding fat and muscle tissue to ensure accurate size measurements.
  • the images demonstrate the animals treated with genetically engineered myeloid progenitor cells exhibited the smallest tumor sizes among all the treatment groups.
  • the data indicate that myeloid progenitor cell lines engineered to express IL- 12 (CD19t-IL-12 and IL- 12 monoallelic) exhibited superior tumor suppressive capabilities when compared to aPDl monoclonal antibody (mAB) treatment.
  • mAB monoclonal antibody
  • intratumoral (IT) treatment with genetically engineered myeloid progenitor cells led to a greater reduction in tumor growth compared to aPDl treatment. This provides visual evidence that further supports the exceptional tumor-suppressive capabilities of the genetically engineered myeloid progenitor cells in comparison to the other treatment approaches.
  • FIG. 15 shows IL-12 levels detected in the serum of mice with flank GBM tumors over a 20-day period. Serum samples were collected from the animals at specific time points as indicated, and IL- 12 secretion was analyzed using bead-based Luminex assay. The data demonstrate that animals treated with genetically engineered myeloid cells exhibited detectable levels of IL- 12 in their serum. These findings indicate the sustained and durable expression of IL- 12 for a minimum of 20 days following the administration of the myeloid progenitor cells to the tumor site. These data highlight the extended presence of IL-12, which holds significant implications for the therapeutic capacity and longevity of the treatment approach utilizing genetically engineered myeloid progenitor cells. Furthermore, these data demonstrate that IT treatment with genetically engineered myeloid progenitor cells results in more IL- 12 production than systemic aPDl mAh treatment as reported herein.
  • FIGS 16A-16C provide exemplary results showing serum cytokine levels associated with cytokine release syndrome (CRS), in mice treated with genetically engineered myeloid progenitor cells or control cells as compared to treatment with anti-human PD-1 antibody (aPDl).
  • CRS cytokine release syndrome
  • the evaluation was performed on the IT treatment with genetically engineered myeloid progenitor cells (CD19t-IL-12) in comparison to the benchmark monoclonal antibody (mAb) treatment aPDl administered intraperitoneally (IP).
  • mAb monoclonal antibody
  • IP monoclonal antibody
  • the objective was to compare the levels of cytokines in the serum known to drive CRS following the respective treatments.
  • the data were obtained from humanized mice, where human flank GBM tumors were established, followed by the transfer of human peripheral blood mononuclear cells (huPBMCs).
  • FIG. 16A shows levels of IL-6 (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL-12), and aPDl.
  • FIG. 16B shows levels of IL-8 (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL-12), and aPDl.
  • FIG. 16A shows levels of IL-6 (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL-12), and aPDl.
  • FIG. 16B shows levels of IL-8 (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL
  • 16C shows levels of IL 1 -beta (pg/ml) on the Y axis compared across treatment groups: untreated, vehicle, control cells, genetically engineered myeloid progenitor cells (CD19t-IL- 12), and aPDl.
  • Example 7 Genetically engineered myeloid progenitor cells reshape the tumor microenvironment
  • TME tumor microenvironment
  • This example describes experimental work that was conducted to evaluate the capacity of genetically engineered myeloid progenitor cells to re-polarize the tumor microenvironment (TME).
  • TME tumor microenvironment
  • this example describes experimental results that shed light onto the effects of administering genetically engineered myeloid progenitor cells on the TME.
  • the results obtained from our experimental analyses provide evidence of a notable phenomenon: the administration of genetically engineered myeloid progenitor cells led to a significant increase in the infiltration of CD3 and CD8 T cells into the TME.
  • FIG. 17A-17D show exemplary experimental results that demonstrate the remodeling of the tumor microenvironment (TME) towards an anti-tumor cytolytic T lymphocyte (CTL) response following the administration of myeloid progenitor cells.
  • TME tumor microenvironment
  • CTL anti-tumor cytolytic T lymphocyte
  • FIG. 17A experiment data showing the ratios of anti-tumor cytolytic CD8 T cells to protumor immunosuppressive FoxP3+ T cells (Tregs) in the tumor after treatment with myeloid progenitor cells genetically engineered to secrete IL-12 or control cells (non-edited myeloid progenitor cells).
  • Tregs protumor immunosuppressive FoxP3+ T cells
  • FIGS. 17B-17D Further evidence of the TME repolarization is provided by FIGS. 17B-17D.
  • FIG. 17B demonstrates an infiltration of T cells (CD3+) within the TME with an overall decreased exhaustion phenotype (PD1 negative and TIM3 negative), indicating a shift from immunosuppressive to pro-inflammatory phenotypes, supporting the notion of repolarization.
  • FIG. 17B shows an increased frequency of total T cells within the TME
  • FIGS. 17C and 17D show a decrease in exhaustion markers (PD1/TIM3) within CD4 and CD8 subsets in the TME.
  • Example 8 Enhanced anti-tumor responses from myeloid progenitor cells derived from iPSCs ex-vivo
  • myeloid progenitor cells were generated from iPSCs, as described in Example 1, and polarized to an M2 phenotype by the addition of TGFp and IL- 10 to cell cultures.
  • the myeloid progenitor cells were then cultured with CD8 T cells under conditions in which an anti-CD-28 monoclonal antibody (mAb) was either present or absent from the cultures.
  • mAb monoclonal antibody
  • FIGS. 18A and 18B demonstrate that the myeloid progenitor cells rescue CD8 T cell effector cytokines without agonistic CD28 mAb.
  • FIGS. 18A and 18B demonstrate that engineered M2 polarized, myeloid progenitor cells can restore both CD8 IFNy (FIG. 18A) and TNFa (FIG. 18B) to a level of pro-inflammatory cytokine rescue that is comparable to CD8 T cells stimulated with or without anti-CD28 mAb.
  • FIG. 18A show that engineered M2 polarized, myeloid progenitor cells can restore both CD8 IFNy (FIG. 18A) and TNFa (FIG. 18B) to a level of pro-inflammatory cytokine rescue that is comparable to CD8 T cells stimulated with or without anti-CD28 mAb.
  • FIG. 18A shows that levels of IFNy secreted from CD8 T cells is comparable to when the CD8 T cells are cultured with M2 myeloid progenitor cells with or without anti-CD28 mAB relative to CD8 T cells only or CD8 T cells with anti-CD3 mAB alone and without anti-CD28 mAh.
  • FIG. 18B shows that levels of TNF-a secreted from CD8 T cells is comparable to when the T cells are cultured with M2 myeloid progenitor cells with and without anti-CD28 mAB relative to CD8 T cells only or CD8 T cells with anti-CD3 and without anti-CD28 mAh controls.
  • myeloid progenitor cells can enhance CD8 anti-tumor cytokine production even in the presence of an M2 phenotype, demonstrating their usefulness in tumor therapy and the impact of the intrinsic myeloid progenitor cell, which provides co-stimulation through CD28 activation, resulting in comparable levels of T cell activation to exogenous anti-CD28 mAb.
  • CD8 T cells were cultured with U251 tumor spheroids in a monolayer with the myeloid progenitor cells or control cells under the stimuli indicated.
  • FIGS. 19A and 19B Exemplary results are shown in FIGS. 19A and 19B.
  • Myeloid progenitor cells were able to induce CD8 tumor cell killing in the presence of anti-CD3 alone (FIG. 19A).
  • Treatment with anti-CD3 and anti-CD28 resulted in comparable levels of CD8 tumor cell killing by myeloid progenitor cells as treatment with anti-CD3 alone.
  • These data together with the observation that myeloid progenitor cells increase CD28 ligands CD80/CD86 on its surface, suggest that myeloid progenitor cells, generated ex vivo from iPSCs as described in Example 1, provide CD28 engagement on T cells, through CD80/86, to drive CD8 tumor killing activity.
  • These data also demonstrate the therapeutic efficacy of the myeloid progenitor cells on tumor size and highlight the ability of the myeloid progenitor cell to activate anti-tumor effector responses.
  • iPSC-derived myeloid progenitor cells can promote pro-inflammatory cytokine (IFNy and TNFa) production from Tregs. Briefly, varying amounts of the myeloid progenitor cells (either 17,000, 35,000, or 70,000) were co-cultured with or without a set number of Tregs.
  • IFNy and TNFa pro-inflammatory cytokine
  • FIGS. 20A and 20B Exemplary results are shown in FIGS. 20A and 20B. These results demonstrate myeloid progenitor cells, derived ex-vivo from iPSCs as described in Example 1, promote pro-inflammatory cytokine (IFNy and TNFa) production from Tregs. Co-culturing of Tregs with myeloid progenitor cell stimulated higher production of IFNy and TNFa from Tregs than when the Tregs were cultured alone.
  • IFNy and TNFa pro-inflammatory cytokine
  • any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

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Abstract

L'invention concerne des populations de cellules modifiées comportant une modification génétique pour déclencher ou augmenter la quantité d'IL-12 sécrétée par les cellules. La population de cellules modifiées peut être une population de cellules immunitaires modifiées (par exemple, des cellules progénitrices myéloïdes), ou une population de cellules souches modifiées. L'invention concerne également des compositions pharmaceutiques, des kits et des dispositifs comprenant les populations de cellules modifiées. L'invention concerne également des méthodes pour traiter un sujet atteint d'une tumeur ou stimuler une réponse immunitaire contre une tumeur chez un sujet, comprenant l'administration d'une population de cellules modifiées décrites ici.
EP24832807.2A 2023-06-26 2024-06-25 Cellules progénitrices myéloïdes modifiées Pending EP4731776A2 (fr)

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