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EP-4736957-A2 - METHODS AND KITS FOR GENERATING MIMETIC INNATE IMMUNE CELLS FROM PLURIPOTENT STEM CELLS

EP4736957A2EP 4736957 A2EP4736957 A2EP 4736957A2EP-4736957-A2

Abstract

Human pluripotent stem cells (hPSCs), especially induced pluripotent stem cells (iPSCs) provide a promising starting material to produce mimetic innate immune cells such as natural killer (NK) cells and γδ T-cells for cancer immunotherapy. To facilitate consistent mass production, an overall manufacturing scheme to make mimetic innate immune cells from hPSCs was designed and demonstrated. Particularly, a robust protocol to differentiate hPSCs into NK cells or γδ T-cells through sequential hematopoietic differentiation on stromal cell line deficient in expressing M-CSF and lymphoid commitment on stromal cell line deficient in expressing M-CSF ectopically expressing DLL1 without employing CD34+ cell enrichment and spin embryoid body formation is established. Using this two-stage protocol, the generation of functional mimetic NK cells and functional mimetic γδ NKT-cells was demonstrated from hPSCs, including hESCs, peripheral blood cell-derived iPSCs (PBC-iPSCs), non-T cell-derived iPSCs or γδ T cell-derived iPSCs and the use of these mimetic innate immune cells in killing cancer cells.

Inventors

  • ZENG, JIEMING
  • WANG, SHU

Assignees

  • Agency for Science, Technology and Research

Dates

Publication Date
20260506
Application Date
20180207

Claims (16)

  1. Mimetic innate immune cells comprising cells expressing at least one of CD56, CD16, NKp30, NKp44, NKp46, NKG2D, DNAM-1, FASL, TRAIL, NKG2A/CD94 or a combination thereof and with low or no expression of killer cell immunoglobulin-like receptors (KIR).
  2. The mimetic innate immune cells according to claim 1, further comprising cells expressing CD3.
  3. The mimetic innate immune cells according to claim 1 or 2, further comprising cells expressing γδ TCR.
  4. The mimetic innate immune cells according to claim 3, wherein the γδ TCR comprises a V gamma 9V delta 2 (Vγ9Vδ2).
  5. The mimetic innate immune cells according to any one of claims 1 to 4, for use in a treatment.
  6. The mimetic innate immune cells according to any one of claims 1 to 4, for use in treating cancer.
  7. The mimetic innate immune cells for use in treating cancer according to claim 6, wherein the cancer is a solid tumor cancer or leukemia.
  8. The mimetic innate immune cells for use in treating cancer according to claim 7, wherein the solid tumor cancer comprises breast adenocarcinoma, colorectal adenocarcinoma, breast ductal carcinoma, ovary adenocarcinoma or leukemia.
  9. A kit for generating mimetic innate immune cells, the kit comprising: (a) a human pluripotent stem cell line; (b) a stromal cell line deficient in expressing macrophage colony stimulating factor (M-CSF); (c) a stromal cell line deficient in expressing macrophage colony stimulating factor and ectopically expressing Notch ligand, Delta like 1 (DLL1); (d) a first simple media for co-culturing a human pluripotent stem cells with the stromal cell line deficient in expressing M-CSF; and (e) a second media comprising stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), interleukin 7 (IL7) and/or interleukin 15 (IL15).
  10. The kit according to claim 9, wherein the media further comprises Fetal Bovine Serum (FBS); alpha Minimum Essential Medium (αMEM) or stem cell differentiation medium or lymphocyte culture medium; or a combination thereof.
  11. A method of generating mimetic NK cells, the method comprising: (a) co-culturing human pluripotent stem cells with a stromal cell line deficient in expressing macrophage colony stimulating factor (M-CSF) to generate innate immune cell progenitors; (b) co-culturing the innate immune cell progenitors with a stromal cell line deficient in expressing M-CSF and ectopically expressing Notch ligand, Delta like 1 (DLL1) in a media comprising stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), interleukin 7 (IL7) and/or interleukin 15 (IL15) to generate differentiated mimetic innate immune cells; (c) passaging the differentiated mimetic innate immune cells weekly for 3 to 5 weeks.
  12. A method of generating mimetic gamma delta T- cells, the method comprising: (a) co-culturing human pluripotent stem cells with a stromal cell line deficient in expressing macrophage colony stimulating factor (M-CSF) to generate innate immune cell progenitors; (b) co-culturing the innate immune cell progenitors with a stromal cell line deficient in expressing M-CSF and ectopically expressing Notch ligand, Delta like 1 (DLL1) in a media comprising stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), interleukin 7 (IL7) and/or interleukin 15 (IL15) to generate differentiated mimetic innate immune cells; (c) passaging the differentiated mimetic innate immune cells weekly for 3 to 5 weeks.
  13. Mimetic NK cells comprising cells expressing at least one of CD56, CD16, NKp30, NKp44, NKp46, NKG2D, DNAM-1, FASL, TRAIL, NKG2A/CD94 or combinations thereof;; and with low or no expression of killer cell immunoglobulin-like receptors KIR.
  14. The mimetic NK cells of claim 13, wherein the cells do not express CD3.
  15. Mimetic γδ T cells comprising cells expressing at least of CD56, CD16, NKp30, NKp44, NKp46, NKG2D, DNAM-1, FASL, TRAIL, NKG2A/CD94 or a combination thereof and with low or no expression of killer cell immunoglobulin-like receptors KIR.
  16. The mimetic γδ T cells of claim 15, wherein the mimetic γδ T cells comprise γδ TCR expression.

Description

Cross reference to related applications This application claims the priority to Singapore application No. 10201700937P, filed 7 February 2017 and Singapore application No. 10201705582S, filed 6 July 2017, the contents of which are incorporated herein by reference. Field The present invention relates to methods and kits for generating innate immune cells, preferably natural killer cells or gamma delta T-cells and methods of using the innate immune cells in treatment and methods of generating induced pluripotent stem cells. Background In 2015, about 90.5 million people had cancer and about 14.1 million new cases occur each year (GBD 2015, Lancet. 388 (10053) 1545-1602). Cancer is often treated with some combination of radiation therapy, surgery, chemotherapy, and/or targeted therapy. While there are many treatments available 8.8 million people die from cancer each year and there is a constant need for new treatments. Cancer immunotherapies exploiting innate immune cells are the new hope for cancer treatment (Woo et al. Annual review of immunology 33, 445-474 (2015)). These innate immune cells including natural killer (NK) cells and γδ T cells can recognize a wide range of cancer cells through major histocompatibility complex (MHC)-independent mechanisms (Scheper, et al. Leukemia 28, 1181-1190 (2014). This unique feature allows the use of innate immune cells to treat cancers in many recipients without MHC-restriction of conventional αβ T cell-based strategies. Currently, healthy donor-derived blood cells are the commonly used cell source to generate a large number of innate immune cells (Kondo, et al. Cytotherapy 10, 842-856 (2008)). However, these donor blood cell-dependent platforms are challenging for centralized, standardized and large-scale production due to the use of variable and limited starting materials and the complicated logistics. Gamma delta T-cells (γδ T cells) are innate immune cells that recognize cancer cells via major histocompatibility complex-independent mechanisms. This feature allows the use of donor-derived γδ T cells to treat cancers in different patients. γδ T cells account for 1-10% of peripheral blood lymphocytes. Among these circulating γδ T cells, Vγ9Vδ2 T cells are the major subset that reacts to phosphoantigens through their Vγ9Vδ2 T-cell receptors (TCRs), and thus recognizes infected cells or malignant cells. During in vivo development, somatic recombination of TCRG and TCRD genes is an early crucial step for γδ T cells to obtain their TCR γ chain and δ chain. To generate γδ T cells from iPSCs, a logical strategy would be to accurately recapitulate this process of somatic recombination of TCRG and TCRD genes during in vitro differentiation to produce functional γδ TCRs. But such a strategy remains very difficult for current technology. Natural killer cells (NK cells) are lymphocytes of the innate immune system that are able to kill a broad spectrum of malignant cells and virus-infected cells (Domogala, et al. Frontiers in immunology 6, 264 (2015)). The target recognition and activation of NK cells depend on an array of activating receptors and inhibitory receptors, which are different from the MHC-restricted αβ T-cell receptor (TCR) -dependent mechanism of αβ T cells (Moretta, et al. Frontiers in immunology 5, 87 (2014)). Thus, it is possible to use allogeneic NK cells to treat cancer with less chance of causing graft-versus-host-disease (GVHD) (Leung. Clinical cancer research: an official journal of the American Association for Cancer Research 20, 3390-3400 (2014).). The distinct target recognition mechanism of NK cells significantly extends the potential cell sources that can be used in adoptive immunotherapy for cancer beyond autologous NK cells. In current clinical trials, large dosages of NK cells ranging from 5x106-5x107/kg body weight are required (Lapteva et al. Critical reviews in oncogenesis 19, 121-132 (2014)). One typical approach to derive such large amounts of allogeneic NK cells is NK cell enrichment from large volumes of donor peripheral blood. Starting from donor-derived leukapheresis products, different protocols have been established to produce NK cell products by immunemagnetic depletion of T cells and B cells and selection of CD56+ cells (Koehl, et al. Frontiers in oncology 3, 118 (2013)). The purities of such products are especially crucial for clinical application in an allogeneic setting, in which the residual T cells may cause GVHD and prohibit the infusion of high dosages. Current generation of high-purity NK cell products requires a prolonged procedure that not only compromises the recovery of NK cells but also affects their viability and potency. With a low recovery rate, it is difficult to obtain sufficient NK cells from a single leukapheresis product. Another popular approach to produce NK cell therapeutics is NK cell expansion using feeder cells, such as K562 cells modified with membrane-bound molecules such as interleukin (IL) -15 and 4-1BB ligan