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WO2025055927A1 - Immunothérapies utilisant des cellules modifiées hypoimmunogènes - Google Patents

Immunothérapies utilisant des cellules modifiées hypoimmunogènes Download PDF

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WO2025055927A1
WO2025055927A1 PCT/CN2024/118162 CN2024118162W WO2025055927A1 WO 2025055927 A1 WO2025055927 A1 WO 2025055927A1 CN 2024118162 W CN2024118162 W CN 2024118162W WO 2025055927 A1 WO2025055927 A1 WO 2025055927A1
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cells
engineered
engineered cells
cell
clec2d
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Jiabiao HU
Xiaomeng BIAN
Lina ZHOU
Hongfang PAN
Luhan Yang
Tony Ho
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Qihan Hong Kong Ltd
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Qihan Hong Kong Ltd
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Definitions

  • the invention relates generally to the field of immunotherapies, and more specifically to engineered hematopoietic cells, e.g., natural killer (NK) cells or T-cells, and uses thereof.
  • engineered hematopoietic cells e.g., natural killer (NK) cells or T-cells, and uses thereof.
  • NK natural killer
  • T cells are a type of white blood cell crucial to the immune system. Their multifaceted roles in combating infections, cancer, and autoimmune diseases have positioned them at the forefront of therapeutic innovation. Ongoing research and clinical trials hold great promise for harnessing the full potential of T-cells in the fight against various human diseases, e.g. Cancer Immunotherapy in which T-cells, especially through Chimeric Antigen Receptor (CAR) T-cell therapy, are engineered to target and kill pathological target cell eg. cancer cells , B cells, T cells et al. Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system whose natural function is to kill microbial-infected and/or cancerous cells.
  • CAR Chimeric Antigen Receptor
  • T cell-and NK cell-mediated immunotherapy has been tried in patients with a variety of diseases, including autoimmune diseases and cancer, such as T cell-mediated immunotherapy for the treatment of acute lymphoblastic leukemia and multiple sclerosis, and NK cell-mediated immunotherapy for the treatment of leukemia and other cancers.
  • autoimmune diseases and cancer such as T cell-mediated immunotherapy for the treatment of acute lymphoblastic leukemia and multiple sclerosis, and NK cell-mediated immunotherapy for the treatment of leukemia and other cancers.
  • NK cell-mediated immunotherapy for the treatment of leukemia and other cancers.
  • autologous T-cells or NK cells were used, by isolating hematopoietic cells from the patient, expanding the T-cells or NK cells, and reintroducing the T-cells or NK cells back to the patient.
  • NK cells comprising transgenes to enhance activity and/or reduce immunogenicity have been described, e.g., to provide enhanced IL-15 expression, or CD16 signaling using CD64/CD16A fusion protein, or to express a hypo-immunity regulator polypeptide, e.g., comprising one or more members selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59, or engineered to comprise a heterologous transcription factor (e.g., STAT) coupled with reduced activity of an endogenous cytokine receptor (e.g., endogenous IL receptor, such as IL-17R) , e.g., as described in WO2022095902A1, the contents of which are incorporated herein by reference.
  • a hypo-immunity regulator polypeptide e.g., comprising one or more members selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59, or engine
  • autologous T-cells or NK cells rather than allogeneic T-cells or NK cells, avoids the need for immunosuppressive therapies to prevent rejection of the engineered cells. But, perhaps due to their compatibility with the patient’s immune system, the autologous T-cells or NK cells are also often ineffective against the cancers, e.g., due to inhibitory interactions between the autologous T-cells or NK cells and self-MHC I molecules.
  • An allogeneic T-cells or NK cell product that could be used “off-the-shelf” for patients has been a goal for many years; however, engineering allogeneic T-cells or NK cells for reduced immunogenicity (hypoimmunity) has proven challenging.
  • Strategies are needed to avoid activation of the host immune cells (e.g., host CD8 T-cells and host NK cells) , as well as to avoid fratricide by the other NK cells in the host due to a “missing self” reaction.
  • iPSCs induced pluripotent stem cells
  • HSCs hematopoietic stem cells
  • NK inhibitory molecules such as human leukocyte antigen (HLA) -E/G are introduced to inhibit the killing by the other allogeneic T-cells or NK cells.
  • T-cells or NK cells Due to the heterogeneity of T-cells or NK cells, however, it is difficult to suppress all allogeneic T-cells or NK cells by expressing these NK cell suppressor molecules. So, while the allogeneic T-cells or NK cells are protected from the host’s immune system by the absence of MHC-I and MHC-II, they will nevertheless rapidly dwindle due to ineffective suppression of the fraternal T-cells or NK cells. They may also encounter rejection by host NK cells.
  • iPSCs induced pluripotent stem cells
  • engineered cells expressing certain NK repressor proteins exhibit reduced activation of allogeneic (host) NK cells in response to the engineered cells, but expression of the selected NK repressor protein (s) does not interfere with the differentiation of iPSCs comprising the transgene, into selected hematopoietic cells, e.g., T-cells or NK cellsT-cells, e.g., wherein the transgene expresses an NK repressor protein selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof.
  • NK repressor protein selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof.
  • hematopoietic cells e.g., T-cells or NK cells
  • selected second signaling molecules e.g., one or more of CD58, CD54, CD86, or ICAM1
  • CD47 or tCD64 are knocked in
  • Induced pluripotent stem cells in this context can thus provide hypo-immunogenic cell products, including T-cells or NK cells, which can be prepared in large quantities, with high homogeneity and low cost.
  • the hypo-immunogenic cell products derived from iPSC differentiation can also overcome the limitations of autologous CAR-T cell therapies.
  • Hypo-immunogenic cell products can resist the fratricide effect arising from mixing with endogenous immune cells commonly associated with introducing engineered immune cells into a patient’s body. This, in turn, improves the survival of the product in the body, to correspondingly improve the efficacy of the product. Since the hypo-immunogenic cell products do not provoke an immune rejection, multiple on-demand administrations can be performed.
  • allogeneic primary cells such as primary T cells and primary NK cells
  • allogeneic primary cells can also be stably supplied and produced in large quantities compared to autologous cells, reducing the need for patient-specific collection and processing, thereby reducing costs.
  • the allogeneic primary cells in this context have stronger killing effects on blood tumors and solid tumors, last longer, and have lower immune rejection.
  • hypo-immunogenic engineered cells can enhance efficacy of cell-based immunotherapies, increase access to the therapies by allowing for easier and more cost-effective production of an “off-the-shelf” product, and improve patient experience by eliminating the need for associated immunosuppressing procedures that elevate risk of complications.
  • iPSCs may interfere with the ability of the iPSCs to differentiate into T-cells or NK cells cause other problems. For example, deleting the conserved gene beta-2-microglobulin completely removes surface expression of HLA class I molecules, thereby reducing immunogenic recognition by host CD8 T cells. Moreover, as discussed above, while immunogenic recognition by host CD8 T cells will be reduced by this approach, the complete loss of HLA class I molecules will increase the risk of fratricide by graft NK cells, due to the “missing self” mechanism, and host CD4 T cells can also contribute to rejection of allogeneic cells through recognition of HLA class II molecules.
  • engineered cells including (i) engineered stem cells, e.g. induced pluripotent stem cells (iPSCs) , and (ii) engineered hematopoietic cells, e.g., T-cells or NK cells (which engineered hematopoietic cells may be the progeny of the engineered stem cells) , wherein selected genes are transfected into or knocked out of the cells to render the cells hypo-immunogenic (e.g., wherein a second signaling molecule (e.g., selected from one or more of CD48, CD80, CD86, LAF-1, ICAM 1, VLA4, VCAM 1, CD2, CD58, CD54, B7, CD155, and CD122) is knocked out, and/or wherein a gene for an NK repressor protein (e.g., selected from Clec2d, Nectin-1, CDH1, CD155, CD47, tCD64, and combinations thereof) is transfected into a second signal
  • Figure 1 depicts sensitivity of engineered K562 cells to allogeneic peripheral blood natural killer (PBNK) cells with gene insertions (i.e., knock-ins, “KI” ) of HLA-E, Clec2d, Nectin-1, CD155, CD24, CD72, FASL, SERPINB9, VPX, VPU, or NEF.
  • PBNK peripheral blood natural killer
  • Figure 2 depicts a gene construct inserted into K562 cells to overexpress a Clec2d gene together with a blue fluorescent protein (BFP) reporter gene.
  • BFP blue fluorescent protein
  • Figure 3 depicts flow cytometry histogram results indicating successful overexpression of the Clec2d/BFP construct inserted into K562 cells (top) and expression levels of Clec2d genes derived from various mammalian species relative to a human-derived control (bottom) .
  • Figure 4 depicts relative expression levels of human-derived Clec2d/BFP constructs comprising various ubiquitination site K-to-R mutations.
  • Figure 5 depicts CD107a degranulation assays of human cord blood natural killer (CBNK) cells and human PBNK cells exposed to the engineered K562/Clec2d cells.
  • Unmodified K562 cells K562 are used as a negative control, which are expected to activate degranulation in allogeneic NK cells.
  • K562 cells overexpressing HLA-E K562/HLAE are used as a positive control, paralleling the state-of-the-art in reducing sensitivity in allogeneic NK cells.
  • the presence (+) or absence (-) of NKG2A denotes the CBNK/PBNK cell ability or inability to bind with HLA-E molecules, respectively.
  • FIG. 6 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/Clec2d cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 7 depicts a dose-dependent effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/Clec2d cells, wherein the human-derived Clec2d construct is transfected into K562 cells overexpressing HLA-E/G and confers protection against human allogeneic NK cells with respect to the expression level of Clec2d.
  • Figure 8 depicts protection by human Clec2d in 2KO NK cells from allogeneic human PBNK cytotoxicity.
  • Figure 9 depicts protection by monkey Clec2d in K562 cells from monkey NK cytotoxicity.
  • Figure 10 depicts results of successful differentiation of induced pluripotent stem cells (iPSCs) into embryoid bodies (EBs) after engineered overexpression of Clec2d.
  • iPSCs induced pluripotent stem cells
  • EBs embryoid bodies
  • Figure 11 depicts a gene construct inserted into K562 cells to overexpress a CDH1 gene together with a BFP reporter gene.
  • Figure 12 depicts flow cytometry histogram results indicating successful overexpression of the CDH1/BFP construct inserted into K562 cells.
  • Figure 13 depicts CD107a degranulation assays of CBNK cells and PBNK cells exposed to the engineered K562/CDH1 cells. Again, K562 are used as a negative control and K562/HLAE are used as a positive control.
  • Figure 14 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/CDH1 cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 15 depicts the effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/CDH1 cells, wherein the CDH1 construct is transfected into K562 cells overexpressing HLA-E/G.
  • Figure 16 depicts dose-dependent results of differentiation of iPSCs into EBs after engineered overexpression of CDH1.
  • Figure 17 depicts a gene construct inserted into K562 cells to overexpress a Nectin-1 (NECTIN1) gene together with a BFP reporter gene.
  • NECTIN1 Nectin-1
  • Figure 18 depicts flow cytometry histogram results indicating successful overexpression of the Nectin-1/BFP construct inserted into K562 cells.
  • Figure 19 depicts CD107a degranulation assays of CBNK cells and PBNK cells exposed to the engineered K562/NECTIN1 cells. Again, K562 are used as a negative control and K562/HLAE are used as a positive control.
  • Figure 20 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/NECTIN1 cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 21 depicts the effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/NECTIN1 cells, wherein the Nectin-1 construct is transfected into K562 cells overexpressing HLA-E/G.
  • Figure 22 depicts results of successful differentiation of iPSCs into EBs after engineered overexpression of Nectin-1.
  • Figure 23 depicts a gene construct inserted into K562 cells to overexpress a CD155 gene together with a BFP reporter gene.
  • Figure 24 depicts flow cytometry histogram results indicating successful overexpression of the CD155/BFP construct inserted into K562 cells.
  • Figure 25 depicts CD107a degranulation assays of CBNK cells and PBNK cells exposed to the engineered K562/CD155 cells. Again, K562 are used as a negative control and K562/HLAE are used as a positive control.
  • Figure 26 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/CD155 cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 27 depicts a dose-dependent effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/CD155 cells, wherein the CD155 construct is transfected into K562 cells overexpressing HLA-E/G and wherein a lower expression level of CD155 is found to confer greater protection against activation of allogeneic NK cells.
  • Figure 28 depicts protection by human CD155 in 2KO NK cells from allogeneic human PBNK cytotoxicity.
  • Figure 29 depicts results of successful differentiation of iPSCs into EBs after engineered overexpression of CD155.
  • Figure 30 depicts results of using a single guide ribonucleic acid (sgRNA) to simultaneously knock-out UL16-binding proteins (ULBP) -2, 5, and 6, which encode major histocompatibility complex (MHC) class I-related molecules that bind to the NKG2D receptor that activates NK cell cytotoxicity.
  • This ULBP2/5/6 knock-out in double knock-out endometrial NK cells i.e., SU11
  • Section (A) depicts indel frequency analysis.
  • Section (B) depicts flow cytometry histogram results of ULBP2/5/6 knockout.
  • Section (C) depicts cytotoxicity by PBNK cells.
  • Section (D) depicts cytotoxicity by CBNK cells.
  • Figure 31 depicts CD107a degranulation assays of CBNK and PBNK cells exposed to the engineered SU11 cells with a ULBP2/5/6 knock-out.
  • Figure 32 depicts a comparison of specific gene transductions and their resulting efficacy toward inhibiting cytotoxicity of K562/HLAE/G cells to PBNK cells.
  • the selected genes that most effectively confer protection to K562/HLAE/G cells are Clec2d-30 > Nectin-1-10 > CDH1-30 > CD155-10 > Clec2d-10 > CD155-30 > CD24-10.
  • Figure 33 depicts effects on iPSC ability to differentiate into EBs following single transgene insertion of CDH1 construct in ANB iPSCs.
  • Low (L4/L6) expression levels of CDH1 in iPSCs allows for normal differentiation into EBs; however, medium (M4/M9) and high (H3/H5) expression levels of CDH1 interfere with typical EB formation.
  • Figure 34 depicts flow cytometry histogram results indicating successful knock-out of transforming growth factor (TGF) -beta receptor 2 in K562 cells, which demonstrates potential inhibition of iPSC differentiation following genetic engineering.
  • TGF transforming growth factor
  • Figure 35 depicts flow cytometry analysis of CD86, ICAM1, and CD58 knock-outs, showing that the CRISPR-Cas9 constructs effectively knock-out protein expression of these genes.
  • Figure 36 shows the killing capacity of the NK cells having CD86, ICAM1, or CD58 knocked out, showing that the knockouts do not affect the ability of the NK cells to kill cancer cells (Raji cells in this case) .
  • Figure 37 shows that the CD58, CD86, and ICAM1 knockouts do not affect MHC-1 expression in the eNK cells.
  • Figure 38 shows that the CD58, CD86, and ICAM 1 knockouts exhibit significantly lowered stimulation of CD8+ cells.
  • Figure 39 shows the effect of knocking out various genes and combinations of genes on CD8+ stimulation.
  • Figure 40 depicts the effect of QN-019 eNK cells, containing CD19-CAR, CD16 and IL15 transgenes, with and without ICAM1, CD86, and CD58 knockouts, showing that these knockouts also reduce the stimulation by QN-019 eNK of CD8+ T cells.
  • FIG 41 shows the effect of Clec2d mutation on its expression in 293T cells, and cysteine mutation abolished Clec2d expression.
  • Figure 42 shows the structure of gene construct used to express CLEC family members.
  • Figure 43 shows CLEC family member expression in 293T.
  • Figure 44 shows the structure of different gene constructs used to express Clec2d.
  • Figure 45 shows the transfection efficiency of different gene constructs.
  • Figure 46 shows the effect of different gene constructs on Clec2d expression in 239T.
  • Figure 47 shows the NHP Clec2d could protect 2KO CAR-T from NHP CD161 + PBNK killing
  • Figure 48 shows the expression of HLA-E and CLEC2D at different MOI.
  • Figure 49 shows the effect of promoter and MOI on Clec2d expression.
  • an immune cell generally refers to a differentiated hematopoietic cell.
  • an immune cell can include an NK cell, a T-cell, a monocyte, an innate lymphocyte, a tumor-infiltrated lymphocyte, a macrophage, a granulocyte, etc.
  • T cell or “T lymphocyte” are art-recognized and are intended to include thymocytes, T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • Illustrative populations of T cells suitable for use in the methods of this invention include but are not limited to helper T cells (HTL; CD4 + T cells) , cytotoxic T cells (CTL; CD8 + T cells) , CD4 + CD8 + T cells, or any other suitable subset of T cells.
  • T cells suitable for use include T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR.
  • the population of T cells comprises, consists essentially of, consists of, or is composed substantially of (e.g., more than 90%, 95%, 97%, 98%, 99%) CD8+ T cells.
  • the T cells are obtained from a mammalian subject. In another aspect, the cells are obtained from a primate subject. In a particular aspect, the cells are obtained from a human subject.
  • the population of T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMCs PBMCs
  • a number of cell marker panels e.g., CD2, CD3, CD4, CD8, CD14, CD16, CD19, CD25, CD28, CD45RA, CD45RO, CD61, CD62L, CD66b, CD152, CD127, NK1.1, FOXP3, Foxp3+, CXCR3, CCR4 and HLA-DR, and maintained in T cell culture medium.
  • a population of PBMCs is used to isolate a population of T cells.
  • Specific cell types can be isolated from PBMCs as described herein or by conventional methods.
  • cytotoxic and helper T lymphocytes can be sorted into memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • T cells may be obtained commercially..
  • NK cell generally refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor, i.e., CD3.
  • NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceR ⁇ , and EAT-2.
  • isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and/or DNAM-1.
  • gene, DNA, and/or RNA “expression” is understood to refer to progression along the canonical pathway beginning with DNA, which may be transcribed into RNA, which may be translated into polypeptides/protein. “Overexpression” generally refers to an increased expression level of a polynucleotide and/or polypeptide sequence relative to its expression level in a wild-type state.
  • an “engineered” cell is understood to mean a cell wherein the genome of the cell has a heterologous nucleic acid sequence or an altered nucleic acid sequence because of the application of genetic engineering techniques to the cell or an ancestor of the cell, such that the genome and gene expression of the engineered cell differs from the genome and gene expression of a normal, nonengineered cell.
  • Genetic engineering techniques include DNA cloning technologies; transduction, transformation, and other gene transfer technologies; homologous recombination; site-directed mutagenesis; gene fusion; gene disruption; gene activation; and gene editing.
  • an engineered cell includes, for example, a cell wherein (a) the genome of the cell (or an ancestor of the cell) has been genetically engineered to include a transgene (sometimes referred to as a “knock-in” ) and/or a heterologous promoter for an endogenous gene, and/or (b) the genome of the cell (or an ancestor of the cell) has been genetically engineered to have significantly reduced or eliminated expression of the functional protein expressed by the naturally occurring gene (e.g., using Crispr-Cas9, prime editing, base editing, gene disruption, or other gene editing approach, sometimes referred to as a “knock-out” ) .
  • the disclosure may refer to a coding sequence “operably linked to a heterologous promoter, ” meaning that the promoter is capable of driving expression of the coding sequence (i.e., is “operably linked” to the coding sequence) , but the coding sequence is not naturally associated with (i.e., is “heterologous” to) the promoter.
  • Promoters for use in transgenes as described herein include, for example, constitutive promoters, such as viral promoters and synthetic promoters.
  • the CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian expression vectors.
  • CMV cytomegalovirus
  • A the first exon and the first intron of chicken beta-actin gene
  • G the splice acceptor of the rabbit beta-globin gene [G] .
  • Other strong constitutive promoters include the adenovirus major late promoter, the human cytomegalovirus immediate early promoter (hCMV-IE) , the SV40 and Rous Sarcoma virus promoters, the murine 3-phosphoglycerate kinase promoter, the translation elongation factor 1 ⁇ (EF-1 ⁇ ) promoter, and the human ubiquitin C promoter.
  • the coding sequence operably linked to a heterologous promoter is part of a transgene. In certain embodiments, the coding sequence is an endogenous coding region operably linked to a heterologous promoter to provide elevated or different expression relative to expression when under control of the natural promoter for the coding region.
  • differentiated generally refers to a process by which an unspecialized ( "uncommitted” ) or less specialized cell acquires the features of a specialized cell such as, e.g., an immune cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized ( "committed” ) position within the lineage of a cell.
  • the term “committed” generally refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • pluripotent generally refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) .
  • embryonic stem cells are a type of pluripotent stem cells that can form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm.
  • Pluripotency can be a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell) , which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell) .
  • iPSCs induced pluripotent stem cells
  • differentiated cells e.g., differentiated adult, neonatal, or fetal cells
  • iPSCs reprogrammed stem cells
  • the iPSCs produced do not refer to cells as they are found in nature.
  • iPSCs can be engineered to differentiation directly into committed cells (e.g., natural killer (T or NK) cells.
  • iPSCs can be engineered to differentiate first into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) ) , which can be further induced to differentiate into committed cells (e.g., T or NK cells) .
  • tissue-specific stem cells e.g., hematopoietic stem cells (HSCs)
  • HSCs hematopoietic stem cells
  • ESCs generally refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm.
  • ESCs can be engineered to differentiation directly into committed cells (e.g., T or NK cells) .
  • ESCs can be engineered to differentiate first into tissue-specific stem cells (e.g., HSCs) , which can be further induced to differentiate into committed cells (e.g., T or NK cells) .
  • isolated stem cells generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs) , etc. ) that are isolated from a multicellular organism.
  • HSCs can be isolated from a mammal's body, such as a human body.
  • an embryonic stem cells can be isolated from an embryo.
  • isolated generally refers to a cell or a population of cells, which has been separated from its original environment. For example, a new environment of the isolated cells is substantially free of at least one component as found in the environment in which the "un-isolated" reference cells exist.
  • An isolated cell can be a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample.
  • the term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated from a cell culture or cell suspension.
  • hematopoietic stem and progenitor cells generally refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into T cells or NK cells) and include, multipotent hematopoietic stem cells (hematoblasts) , myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors.
  • HSCs Hematopoietic stem and progenitor cells
  • myeloid monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages T cells, B cells, NK cells
  • HSCs can be CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T cells, NK cells and B cells.
  • immune cell generally refers to a differentiated hematopoietic cell.
  • an immune cell can include a T cell, an NK cell, a monocyte, an innate lymphocyte, a tumor-infiltrating lymphocyte, a macrophage, a granulocyte, etc.
  • the sources of immune cells in this context include, but are not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumor.
  • the immune cells are derived from primary cells.
  • Methods for obtaining immune cells (e.g., T cells or NK cells) from in situ cells can be obtained from public literature (e.g., Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell, 91 (5) , 661-672) .
  • a primary cell sample is obtained from peripheral blood or tissue, and then peripheral blood mononuclear cells (PBMC) are separated by density gradient centrifugation, and specific immune cells, (e.g., T cells or NK cells) are separated by cell sorting methods.
  • PBMC peripheral blood mononuclear cells
  • specific immune cells e.g., T cells or NK cells
  • the obtained immune cells are cultured and expanded in vitro under appropriate culture conditions to obtain a large number of immune cells.
  • the immune cells are derived from induced pluripotent stem cells (iPSCs) .
  • Induced pluripotent stem cells (iPSCs) are directed to differentiate into hematopoietic stem cells under the stimulation of specific differentiation factors, and then further induced to differentiate into immune cells (e.g., T cells or NK cells) .
  • immune cells e.g., T cells or NK cells
  • Commercially available culture media can be selected and the steps in its instructions can be referred to for inducing differentiation of induced pluripotent stem cells.
  • NK cell or “Natural Killer cell” generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3) .
  • NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceR (E ⁇ , and EAT-2.
  • isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-1.
  • the term "gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript.
  • genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5, ⁇ and 3, ⁇ ends.
  • the term encompasses the transcribed sequences, including 5, ⁇ and 3, ⁇ untranslated regions (5, ⁇ -UTR and 3, ⁇ -UTR) , exons and introns.
  • the transcribed region will contain "open reading frames" that encode polypeptides.
  • a “gene” comprises only the coding sequences (e.g., an "open reading frame” or “coding region” ) necessary for encoding a polypeptide.
  • genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes.
  • rRNA ribosomal RNA genes
  • tRNA transfer RNA
  • the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers, and promoters.
  • a gene can refer to an "endogenous gene” or a native gene in its natural location in the genome of an organism.
  • a gene can refer to an "exogenous gene” or a non-native gene.
  • a non-native gene can refer to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer.
  • a non-native gene can also refer to a gene not in its natural location in the genome of an organism.
  • a non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .
  • expression generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides can be collectively referred to as "gene product. " If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.
  • Up-regulated, with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state.
  • Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time.
  • stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell.
  • a selection advantage may be a resistance towards a certain toxin that is presented to the cell.
  • peptide generally refers to a polymer of at least two amino acid residues joined by peptide bond (s) . This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains) .
  • amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component.
  • amino acid and amino acids as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues.
  • Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid.
  • Amino acid analogues can refer to amino acid derivatives.
  • amino acid includes both D-amino acids and L-amino acids.
  • derivative, variant, and fragment, as used herein with reference to a polypeptide, generally refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary) , activity (e.g., enzymatic activity) and/or function.
  • Derivatives, variants, and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions) , truncations, modifications, or combinations thereof compared to a wild type polypeptide.
  • exogenous gene refers to a gene which has been introduced into the cell or an ancestor thereof by genetic engineering, e.g., comprising a coding sequence for a protein of interest operably linked to a heterologous promoter.
  • the exogenous genes used herein may be transiently expressed by viral vectors, e.g., adeno-associated vectors, but in particular embodiments herein, they are stably incorporated into the genome of the engineered cells.
  • the exogenous genes herein may be associated with a selectable or screenable marker, for example a fluorescent marker such as blue fluorescent protein (BFP) .
  • BFP blue fluorescent protein
  • iPSCs induced pluripotent stem cells
  • iPSCs are stably transformed with the exogenous gene of interest, together with a selectable or screenable marker, and differentiated into hematopoietic cells, e.g., T cells or NK cells, which also express the exogenous gene, and which may be introduced into a patient as an allogeneic cell therapy.
  • treating encompasses prophylaxis, mitigation, amelioration of symptoms, and/or delaying the progression of a disease or condition.
  • the present disclosure provides a population of induced pluripotent stem cells (iPSCs) and hematopoietic cells derived therefrom, pharmaceutical compositions comprising said cells, and methods of use of same wherein the cells are transfected with selected genes for reducing the cytotoxicity of allogeneic immune cells.
  • iPSCs induced pluripotent stem cells
  • hematopoietic cells derived therefrom
  • pharmaceutical compositions comprising said cells, and methods of use of same wherein the cells are transfected with selected genes for reducing the cytotoxicity of allogeneic immune cells.
  • the population of hematopoietic cells, pharmaceutical compositions, and methods disclosed are intended to reduce fratricide of immune cells engineered to detect, scavenge, and kill diseased cells within the body of a subject in need thereof, e.g., a human, e.g., a human diagnosed with cancer.
  • Immune cells described herein can be engineered to exhibit enhanced half-life as compared to a control cell, e.g., a non-engineered immune cell.
  • Immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell.
  • Immune cells can be engineered to effectively and specifically target diseased cells, e.g., cancer cells, B cells, T cells or DCs, that a control cell otherwise is insufficient or unable to target.
  • the engineered immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo.
  • the engineered immune cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease, e.g., myeloma, lymphoma, or solid tumors.
  • a disease e.g., myeloma, lymphoma, or solid tumors.
  • the engineered immune cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.
  • PBNK peripheral blood NK cells
  • Clec2d efficiently inhibits allogeneic CBNK/PBNK killing at a dose-dependent manner, and Clec2d does not affect iPSC to T or NK differentiation.
  • CDH1 efficiently inhibits allogeneic PBNK killing; however, high but not low expression of CDH1 in iPSC affects iPSC to T or NK differentiation, indicating an appropriate expression of CDH1 is required for employing CDH1 for making hypoimmunogenic iPSC derived T cells or NK cells.
  • Nectin-1 efficiently inhibits allogeneic PBNK killing, and expression of Nectin-1 in iPSC does not affect iPSC to T or NK differentiation.
  • CD155 expression efficiently inhibits allogeneic CBNK/PBNK killing and expression of CD155 in iPSC does not affect iPSC to T or NK differentiation.
  • Knockout of ULBP2/5/6 KO could protect 2KO eNK (i.e., SU11 with MHC-1 and MHC-II knock-out) from PBNK killing.
  • TGF- ⁇ KO affects iPSC to T or NK differentiation.
  • Cell 1.3 wherein the engineered cells are hematopoietic stem cells.
  • Cell 1.3 wherein the engineered cells are T cells.
  • the engineered cells are T cells, wherein the T cells are selected from Naive T cells (e.g., CD4 + ⁇ and CD8 + ⁇ ) , CTL (Tc cells) , Tregs (e.g., n T-regs, a T-regs, i T-regs, Tr1, Th3, CD8 Treg, NKT cells) , Memory T cells (e.g., Tcm, Tem, Tpm, TRM, T SCM) , Anergic T cells, Exhausted T cells, Th cells (e.g., . Th1, Th2, Th9, Th17, Th22, Tfh) .
  • Naive T cells e.g., CD4 + ⁇ and CD8 + ⁇
  • CTL Tc cells
  • Tregs e.g., n T-regs, a T-regs, i T-regs, Tr1, Th3, CD8 Treg, NKT cells
  • Memory T cells e.g.,
  • Cell 1.6 wherein the engineered cells are T cells, wherein the T cells are secrete one or more cytokines selected from IFN ⁇ , TNF, IL-2, IL-12, IL-18, IL-4, TGF ⁇ and IL-10.
  • Cell 1.6 wherein the engineered cells are T cells, wherein the nucleus of the T cells contain one or more transcription factors selected from EOMES, STAT4, STAT1, FoxP3, STAT5.
  • Cell 1 wherein the engineered cells are primary cells.
  • Cell 1 wherein the engineered cells are derived from primary cells.
  • any foregoing engineered cells comprising an exogenous gene, said exogenous gene comprising a coding region for an NK repressor protein operably linked to a heterologous promoter, wherein expression of the NK repressor protein reduces activation of allogeneic NK cells in response to the engineered cells but does not interfere with the differentiation of iPSCs comprising the exogenous gene into selected hematopoietic cells, e.g., T cells or NK cells,
  • NK repressor protein is selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof.
  • the engineered cells of Cell 1.13 comprising an exogenous gene expressing Clec2d and an exogenous gene expressing an NK repressor protein selected from Nectin-1 CDH1, and CD155.
  • any foregoing engineered cells wherein the Clec2d expressed comprises one or more ubiquitination site K-to-R mutation comprising K9R, K94R, K144R, K186R, K9/94R, K9/144R, K9/186R, K94/144R, K94/186R, K144/186, K9/94/144R, K/9/94/186R, K9/144/186R, K94/144/186R, and/or K9/94/144/186R.
  • any foregoing engineered cells wherein the cells are hematopoietic cells, e.g. T cells or NK cells, and wherein stimulation of heterologous CD8+ T cells by the hematopoietic cells is substantially reduced; e.g. wherein the cells exhibit reduced stimulation of heterologous CD8+ T cells of at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50%reduction relative to a control, e.g., in an in vitro assay as described in the Examples below.
  • the cells are hematopoietic cells, e.g. T cells or NK cells, and wherein stimulation of heterologous CD8+ T cells by the hematopoietic cells is substantially reduced; e.g. wherein the cells exhibit reduced stimulation of heterologous CD8+ T cells of at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50%reduction relative to a control, e.g., in an in vitro assay
  • any foregoing engineered cells wherein the cells are primary cells, e.g. primary T cells or primary NK cells, and wherein stimulation of heterologous CD8+ T cells by the primary cells is substantially reduced; e.g. wherein the cells exhibit reduced stimulation of heterologous CD8+ T cells of at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50%reduction relative to a control.
  • any foregoing engineered cells wherein the cells are derived from primary cells, e.g. derived from primary T cells or primary NK cells, and wherein stimulation of heterologous CD8+ T cells by the cells derived from primary cells is substantially reduced; e.g. wherein the cells exhibit reduced stimulation of heterologous CD8+ T cells of at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50%reduction relative to a control.
  • any foregoing engineered cells e.g., wherein one or more gene encoding second signaling molecule (s) are knocked-out or disrupted
  • the cells are induced pluripotent stem cells (iPSCs)
  • iPSCs induced pluripotent stem cells
  • stimulation of heterologous CD8+ T cells by the induced pluripotent stem cells (iPSCs) is substantially reduced; e.g. wherein the cells exhibit reduced stimulation of heterologous CD8+ T cells of at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50%reduction relative to a control.
  • any foregoing engineered cells wherein the cells are derived from induced pluripotent stem cells (iPSCs) , e.g. T cells or NK cells which derived from the induced pluripotent stem cells (iPSCs) , and wherein stimulation of heterologous CD8+ T cells by the cells derived from induced pluripotent stem cells (iPSCs) is substantially reduced; e.g. wherein the cells exhibit reduced stimulation of heterologous CD8+ T cells of at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50%reduction relative to a control.
  • iPSCs induced pluripotent stem cells
  • iPSCs induced pluripotent stem cells
  • T cells or NK cells which derived from the induced pluripotent stem cells
  • stimulation of heterologous CD8+ T cells by the cells derived from induced pluripotent stem cells (iPSCs) is substantially reduced; e.g. wherein the cells exhibit reduced stimulation of
  • B2M beta-2-microglobulin
  • CIITA MHC class II transactivator
  • ULBP UL16-binding proteins
  • transgenes e.g., transgenes expressing chimeric antigen receptor (CAR) constructs and/or hypoinflammatory antigens or cytokines, e.g., transgenes selected from one or more of CD19-CAR, CD16, and IL15 transgenes.
  • CAR chimeric antigen receptor
  • heterologous promoter is a constitutive promoter, e.g., selected from the CAG promoter, the adenovirus major late promoter, the human cytomegalovirus immediate early promoter (hCMV-IE) , the SV40 and Rous Sarcoma virus promoters, the murine 3-phosphoglycerate kinase promoter, the translation elongation factor 1 ⁇ (EF-1 ⁇ ) promoter, and the human ubiquitin C promoter; e.g., the CAG promoter.
  • constitutive promoter e.g., selected from the CAG promoter, the adenovirus major late promoter, the human cytomegalovirus immediate early promoter (hCMV-IE) , the SV40 and Rous Sarcoma virus promoters, the murine 3-phosphoglycerate kinase promoter, the translation elongation factor 1 ⁇ (EF-1 ⁇ ) promoter, and the human ubiquitin C promoter; e.g., the CAG promoter
  • allogeneic immune cells e.g., endogenous T-cells or NK cells
  • any foregoing engineered cells wherein the cells or their progeny display enhanced resistance against immune rejection e.g., innate immune rejection, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%.
  • immune rejection e.g., innate immune rejection
  • any foregoing engineered cells wherein the enhanced resistance against immune rejection e.g., innate immune rejection
  • a medium comprising at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%human complement.
  • TGF transforming growth factor
  • any foregoing engineered cells which overexpress HLA-E and/or HLA-G e.g., which comprise one or more exogenous genes comprising a coding region for HLA-E and/or HLA-G operably linked to a heterologous promoter, e.g., which comprise an exogenous gene comprising a coding region for HLA-E operably linked to a heterologous promoter and/or which comprise an exogenous genes comprising a coding region for HLA-G operably linked to a heterologous promoter.
  • any foregoing engineered cells which (i) which comprise an exogenous gene, said exogenous gene comprising a coding region for an NK repressor protein operably linked to a heterologous promoter, wherein the NK repressor protein is selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof, and (ii) which do not express one or more genes encoding second signaling molecule (s) are knocked-out or disrupted, wherein the one or more second signaling molecule (s) are selected from one or more of CD86, ICAM1 and CD58; and/or (iii) which do not express UL16-binding proteins (ULBP) -2, 5, and 6.
  • ULBP UL16-binding proteins
  • any foregoing engineered cells which (i) which comprise an exogenous gene, said exogenous gene comprising a coding region for an NK repressor protein operably linked to a heterologous promoter, wherein the NK repressor protein is selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof, and (ii) which do not express one or more genes encoding second signaling molecule (s) are knocked-out or disrupted, wherein the one or more second signaling molecule (s) are selected from one or more of CD86, ICAM1 and CD58; and/or (iii) which do not express UL16-binding proteins (ULBP) -2, 5, and 6.
  • ULBP UL16-binding proteins
  • any foregoing engineered cells which are iPSCs (i) which comprise an exogenous gene, said exogenous gene comprising a coding region for an NK repressor protein operably linked to a heterologous promoter, wherein the NK repressor protein is selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof, and (ii) which do not express one or more genes encoding second signaling molecule (s) are knocked-out or disrupted, wherein the one or more second signaling molecule (s) are selected from one or more of CD86, ICAM1 and CD58; and/or (iii) which do not express UL16-binding proteins (ULBP) -2, 5, and 6.
  • iPSCs which comprise an exogenous gene, said exogenous gene comprising a coding region for an NK repressor protein operably linked to a heterologous promoter, wherein the NK repressor protein is selected from Clec2d
  • NK cells which comprise an exogenous gene, said exogenous gene comprising a coding region for an NK repressor protein operably linked to a heterologous promoter, wherein the NK repressor protein is selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof, and (ii) which do not express one or more genes encoding second signaling molecule (s) are knocked-out or disrupted, wherein the one or more second signaling molecule (s) are selected from one or more of CD86, ICAM1 and CD58; and/or (iii) which do not express UL16-binding proteins (ULBP) -2, 5, and 6; e.g., wherein the NK cells are derived from the iPSCs of the foregoing scope.
  • ULBP UL16-binding proteins
  • (v) optionally, which do not express UL16-binding proteins (ULBP) -2, 5, and 6.
  • ULBP UL16-binding proteins
  • any foregoing engineered cells which are iPSCs, Primary cells, T cells, NK cells, B cells, macrophages, monocytes, cardiomyocytes, islet cells, neural cells, endothelial cells; for example iPSCs, and T cells, NK cells, B cells, macrophages, monocytes, cardiomyocytes, islet cells, neural cells, or endothelial cells derived from said iPSCs or stem cell; for example T cells or NK cells derived from said iPSCs; and Primary cells, and T cells, NK cells, B cells, macrophages, monocytes, cardiomyocytes, islet cells, neural cells, or endothelial cells derived from said Primary cells; for example T cells or NK cells derived from said Primary cells.
  • any foregoing engineered cell comprising an exogenous gene comprising a coding region for Clec2d operably linked to a heterologous promoter, wherein the extracellular domain region of Clec2d (e.g. AA 65-191, e.g. AA 160-181) is mutated to enhance its interaction with CD161 [e.g.
  • the Clec2d includes mutations at one or more of amino acid positions C163, K16, R175, R175, H176, R180, and K181, for example one or more mutations selected from C163S, K169E, R175E, H176C, R180E, and K181E] ; and/or wherein the N-terminal transmembrane domain of the Clec2d is replaced with a membrane protein to enhance constitutive presentation of the Clec2d on the cell membrane [e.g., wherein the extracellular domain of Clec2d (e.g.
  • AA 65-191, e.g. AA 160-181) is fused to the N-terminal transmembrane domain of NKG2, e.g. NKG2A, NKG2C. or NKG2D, or the N-terminal transmembrane domain of other type 2 membrane proteins] .
  • any foregoing engineered cells wherein the gene or genes knocked-in are derived from mammalian genes, e.g., human, monkey, cattle, dog, mouse, or rat, preferably human or monkey.
  • Any foregoing engineered cells in a pharmaceutical composition comprising the engineered cells in a pharmaceutically acceptable carrier, suitable for administration by injection, e.g., via intravenous, intramuscular, intraperitoneal, intrathecal, or intraosseous injection.
  • Any foregoing engineered cells for use in treating cancer e.g., comprising administering a composition comprising any foregoing cells to a patient in need thereof.
  • a composition comprising iPSCs, differentiated immune cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes) , or differentiated immune cells derived from said iPSCs.
  • a composition comprising primary cells or immune cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes) derived from said primary cells.
  • Any forgoing engineered cells for use in treating an autoimmune disease comprising administering a composition comprising iPSCs, differentiated immune cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes) , or differentiated immune cells derived from said iPSCs.
  • differentiated immune cells e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes
  • Any forgoing engineered cells for use in treating an autoimmune disease comprising administering a composition comprising primary cells or immune cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes) derived from said primary cells.
  • primary cells or immune cells e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes
  • Any foregoing engineered cells for use in treating diabetes e.g., comprising administering a composition comprising iPSCs, islet cells, or islet cells derived from said iPSCs.
  • Any foregoing engineered cells for use in treating diabetes e.g., comprising administering a composition comprising primary cells, islet cells, or islet cells derived from said primary cells.
  • Any foregoing engineered cells for use in regenerative medicine treatments e.g., cardiomyocyte transplantation for heart injury or failure, islet cell transplantation for diabetes, neural progenitor cell transplantation for stroke or central nervous system disorders/injury, e.g., comprising administering a composition comprising iPSCs, NK cells, T cells, B cells, macrophages, monocytes, cardiomyocytes, islet cells, neural cells, neural progenitor cells, endothelial cells, mesenchymal cells, or are said cells derived from said iPSCs.
  • a composition comprising iPSCs, NK cells, T cells, B cells, macrophages, monocytes, cardiomyocytes, islet cells, neural cells, neural progenitor cells, endothelial cells, mesenchymal cells, or are said cells derived from said iPSCs.
  • Any foregoing engineered cells for use in regenerative medicine treatments e.g., cardiomyocyte transplantation for heart injury or failure, islet cell transplantation for diabetes, neural progenitor cell transplantation for stroke or central nervous system disorders/injury, e.g., comprising administering a composition comprising primary cells, NK cells, T cells, B cells, macrophages, monocytes, cardiomyocytes, islet cells, neural cells, neural progenitor cells, endothelial cells, mesenchymal cells, or are said cells derived from said primary cells.
  • Any foregoing engineered cells for use in the manufacture of a medicament for use in treating cancer e.g., comprising administering a composition comprising any foregoing cell to a patient in need thereof.
  • Any foregoing engineered cells for use in the manufacture of a medicament for use in treating autoimmune disease e.g., comprising administering a composition comprising any foregoing cell to a patient in need thereof.
  • a pharmaceutical composition comprising engineered cells according to any of the foregoing engineered cells in a pharmaceutically acceptable carrier suitable for injection, e.g., suitable for intravenous infusion, e.g., for use in treating a disease or condition in a human patient, e.g., for use in treating cancer or autoimmune disease.
  • the disclosure provides hypoimmunogenic engineered cells (Cell 2) comprising an exogenous gene, said exogenous gene comprising a coding region for an NK repressor protein operably linked to a heterologous promoter, wherein expression of the NK repressor protein reduces activation of NK cells (e.g.
  • NK cell or fraternal NK cell activation in response to the engineered cells but does not interfere with the differentiation of iPSCs comprising the exogenous gene into selected hematopoietic cells, e.g., NK cells or T cells, e.g., wherein the NK repressor protein is selected from Clec2d, Nectin-1, CDH1, CD155, and combinations thereof.
  • the disclosure provides:
  • Cell 2 wherein the engineered cells are induced pluripotent stem cells (iPSCs) .
  • iPSCs induced pluripotent stem cells
  • Cell 2 wherein the engineered cells are derived from induced pluripotent stem cells (iPSCs) .
  • iPSCs induced pluripotent stem cells
  • the engineered cell is T cells, wherein the T cells are selected from Naive T cells (e.g., CD4 + ⁇ and CD8 + ⁇ ) , CTL (Tc cells) , Tregs (e.g., n T-regs, a T-regs, i T-regs, Tr1, Th3, CD8 Treg, NKT cells) , Memory T cells (e.g., Tcm, Tem) , Anergic T cells, Exhausted T cells, Th cells (e.g., . Th1, Th2, Th9, Th17, Th22, Tfh) .
  • Naive T cells e.g., CD4 + ⁇ and CD8 + ⁇
  • CTL Tc cells
  • Tregs e.g., n T-regs, a T-regs, i T-regs, Tr1, Th3, CD8 Treg, NKT cells
  • Memory T cells e.g., Tcm, Tem
  • the engineered cell is T cells, wherein the T cells surface have one or more markers selected from CD2, CD4, CD8, NK1.1, FOXP3, CD25, CD28, Foxp3+, CD127, CD152, CXCR3 and CCR4.
  • Cell 2.6 or 2.10 wherein the engineered cell is T cells, wherein the T cells secrete one or more cytokines selected from IFN ⁇ , TNF, IL-2, IL-12, IL-18, IL-4, TGF ⁇ and IL-10.
  • NK repressor protein is selected Clec2d, Nectin-1, CDH1, CD155, or a combination thereof.
  • NK repressor protein comprises Clec2d, wherein the extracellular domain region of the Clec2d (e.g. AA 65-191) is mutated to enhance its interaction with CD161 [e.g.
  • the Clec2d includes mutations at one or more of amino acid positions C163, K16, R175, R175, H176, R180, and K181, for example one or more mutations selected from C163S, K169E, R175E, H176C, R180E, and K181E] ; and/or wherein the N-terminal transmembrane domain of the Clec2d is replaced with a membrane protein to enhance constitutive presentation of the Clec2d on the cell membrane [e.g., wherein the extracellular domain of Clec2d (e.g.
  • AA 65-191) is fused to the N-terminal transmembrane domain of NKG2, e.g. NKG2A, NKG2C. or NKG2D, or the N-terminal transmembrane domain of other type 2 membrane proteins] .
  • NK repressor protein comprises Nectin-1.
  • NK repressor protein comprises CD155.
  • B2M beta-2-microglobulin
  • CIITA MHC class II transactivator
  • ULBP UL16-binding proteins
  • transgenes e.g., transgenes expressing chimeric antigen receptor (CAR) constructs and/or hypoinflammatory antigens or cytokines, e.g., transgenes selected from one or more of CD19-CAR, CD16, and IL15 transgenes.
  • CAR chimeric antigen receptor
  • heterologous promoter is a constitutive promoter, e.g., selected from the CAG promoter, the adenovirus major late promoter, the human cytomegalovirus immediate early promoter (hCMV-IE) , the SV40 and Rous Sarcoma virus promoters, the murine 3-phosphoglycerate kinase promoter, the translation elongation factor 1 ⁇ (EF-1 ⁇ ) promoter, and the human ubiquitin C promoter; e.g., the CAG promoter.
  • constitutive promoter e.g., selected from the CAG promoter, the adenovirus major late promoter, the human cytomegalovirus immediate early promoter (hCMV-IE) , the SV40 and Rous Sarcoma virus promoters, the murine 3-phosphoglycerate kinase promoter, the translation elongation factor 1 ⁇ (EF-1 ⁇ ) promoter, and the human ubiquitin C promoter; e.g., the CAG promoter
  • allogeneic immune cells e.g., endogenous T-cells or NK cells
  • any foregoing engineered cells wherein the enhanced resistance against immune rejection e.g., innate immune rejection
  • a medium comprising at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%human complement.
  • the disclosure provides:
  • Cell 3 wherein the engineered cells are derived from induced pluripotent stem cells (iPSCs) .
  • iPSCs induced pluripotent stem cells
  • Cell 3 wherein the engineered cells are primary natural killer (NK) cells.
  • NK primary natural killer
  • Cell 3 wherein the engineered cells are primary T cells.
  • the engineered cells are T cells, wherein the T cells are selected from Naive T cells (e.g., CD4 + ⁇ and CD8 + ⁇ ) , CTL (Tc cells) , Tregs (e.g., n T-regs, a T-regs, i T-regs, Tr1, Th3, CD8 Treg, NKT cells) , Memory T cells (e.g., Tcm, Tem) , Anergic T cells, Exhausted T cells, Th cells (e.g., . Th1, Th2, Th9, Th17, Th22, Tfh) .
  • Naive T cells e.g., CD4 + ⁇ and CD8 + ⁇
  • CTL Tc cells
  • Tregs e.g., n T-regs, a T-regs, i T-regs, Tr1, Th3, CD8 Treg, NKT cells
  • Memory T cells e.g., Tcm, Tem
  • the engineered cells are T cells, wherein the T cells surface has one or more markers selected from CD2, CD4, CD8, NK1.1, FOXP3, CD25, CD28, Foxp3+, CD127, CD152, CXCR3 and CCR4.
  • Cell 3.6 or 3.10 wherein the engineered cells are T cells, wherein the T cells secrete one or more cytokines selected from IFN ⁇ , TNF, IL-2, IL-12, IL-18, IL-4, TGF ⁇ and IL-10.
  • Cell 3.6 or 3.10 wherein the engineered cells are T cells, wherein the nucleus of the T cells contain one or more transcription factors selected from EOMES, STAT4, STAT1, FoxP3 and STAT5.
  • any foregoing engineered cells wherein the one or more second signaling molecule (s) comprise CD58, CD86, and ICAM1.
  • transgenes e.g., transgenes expressing CAR constructs and/or hypoinflammatory antigens or cytokines, e.g., transgenes selected from one or more of CD19-CAR, CD16 and IL15 transgenes.
  • Any foregoing engineered cells in a pharmaceutical composition comprising the engineered cells in a pharmaceutically acceptable carrier, suitable for administration by injection, e.g., via intravenous, intramuscular, intraperitoneal, intrathecal, or intraosseous injection.
  • Any foregoing engineered cells for use in treating cancer e.g., comprising administering a composition comprising any foregoing cells to a patient in need thereof.
  • Any foregoing engineered cells for use in the manufacture of a medicament for use in treating cancer e.g., comprising administering a composition comprising any foregoing cell to a patient in need thereof.
  • Any foregoing engineered cells for use in treating autoimmune diseases e.g., comprising administering a composition comprising any foregoing cells to a patient in need thereof.
  • Any foregoing engineered cells for use in the manufacture of a medicament for use in treating autoimmune diseases e.g., comprising administering a composition comprising any foregoing cell to a patient in need thereof.
  • a pharmaceutical composition comprising engineered cells according to any of the foregoing engineered cells in a pharmaceutically acceptable carrier suitable for injection, e.g., suitable for intravenous infusion, e.g., for use in treating a disease or condition in a human patient, e.g., for use in treating cancer or autoimmune diseases.
  • the disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising hypoimmunogenic engineered cells according to any of Cell 1, et seq., or any of Cell 2, et seq., or any of Cell 3, et seq., in a pharmaceutically acceptable carrier suitable for injection, e.g., suitable for intravenous infusion.
  • the pharmaceutically acceptable carrier suitable for intravenous infusion may be an isotonic saline solution, e.g., 0.9%w/v saline solution, lactated Ringer’s solution, or an isotonic solution formulated for cell culture or therapy, e.g., an isotonic solution comprising physiologically acceptable levels of sodium chloride, dextrose, electrolytes, albumin, and optionally a cryopreservative [e.g.
  • the pharmaceutical composition is frozen during storage and thawed upon administration to the patient.
  • the engineered cells according to any of Cell 1, et seq.
  • the cells include, for example, engineered cells wherein the cells are allogeneic with respect to the patient and wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 15 days, e.g., for at least 30 days, e.g., for at least 60 days; e.g., wherein the population comprises T-cells or NK cells wherein the one or more genes transfected into the cells comprise Clec2d, Nectin-1, CDH1, CD155, or combinations thereof; , and/or wherein the genes expressing UL16-binding proteins (ULBP) -2, 5, and 6 are knocked out; and/or wherein one or more second signaling molecule (s) selected from one or more of CD86, ICAM1 and CD58 is knocked out.
  • ULBP UL16-binding proteins
  • the disclosure provides T cells and T cells derived from iPSCs or primary cells, wherein one or more genes are transfected into said cells, such that recognition by and activation of allogeneic immune cells is reduced, e.g., such that stimulation of heterologous CD8+ T-cells by the T cells or and T cells derived from iPSCs or primary cells is substantially reduced.
  • the disclosure provides NK cells and NK cells derived from iPSCs or primary cells, wherein one or more genes are transfected into said cells, such that recognition by and activation of allogeneic immune cells is reduced, e.g., such that stimulation of heterologous CD8+ T-cells by the NK cells or and NK cells derived from iPSCs or primary cells is substantially reduced.
  • the disclosure provides iPSCs and hematopoietic cells derived therefrom wherein one or more genes are transfected into said cells, such that recognition by and activation of allogeneic immune cells is reduced, e.g., such that stimulation of heterologous CD8+ T-cells by the iPSCs or their progeny is substantially reduced, e.g., wherein the progeny of the iPSCs comprise engineered cells according to any of Cell 1, et seq., or any of Cell 2, et seq., or any of Cell 3, et seq.,
  • the disclosure provides a method of treating cancer, comprising administering engineered cells according to any of Cell 1, et seq., or any of Cell 2, et seq., or any of Cell 3, et seq., or a pharmaceutical composition comprising engineered cells according to any of Cell 1, et seq., or any of Cell 2, et seq., or any of Cell 3, et seq., to a patient in need thereof, e.g., wherein the cells are allogeneic with respect to the patient and wherein the patient’s endogenous NK cells or CD8+ T-cells are not significantly stimulated by the administration; e.g., wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 15 days, e.g., for at least 30 days, e.g., for at least 60 days; e.g., wherein the population comprises T-cells and/or NK cells wherein the one or more gene transfected
  • the disclosure provides a method of treating autoimmune diseases, comprising administering engineered cells according to any of Cell 1, et seq., or any of Cell 2, et seq., or any of Cell 3, et seq., or a pharmaceutical composition comprising engineered cells according to any of Cell 1, et seq., or any of Cell 2, et seq., or any of Cell 3, et seq., to a patient in need thereof, e.g., wherein the cells are allogeneic with respect to the patient and wherein the patient’s endogenous NK cells or CD8+ T-cells are not significantly stimulated by the administration; e.g., wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 15 days, e.g., for at least 30 days, e.g., for at least 60 days; e.g., wherein the population comprises T-cells and/or NK cells wherein the one or more gene trans
  • the disclosure provides a method of making hematopoietic cells wherein (i) one or more genes is transfected into said cells and/or (ii) one or genes is knocked out, such that stimulation of innate immune rejection, e.g., NK cell activation or CD8+T-cell stimulation, is substantially reduced, comprising culturing a population of engineered induced pluripotent stem cells (iPSC) , e.g.
  • iPSC engineered induced pluripotent stem cells
  • engineered iPSCs into engineered hematopoietic cells, e.g., engineered cells according to any of Cell 1, or Cell 1.2, et seq., or any of Cell 2, or Cell 2.2, et seq.
  • the one or more gene transfected into said cells comprises Clec2d, Nectin-1, CDH1, CD155, or combinations thereof; and/or wherein one or more second signaling molecule (s) comprising one or more of CD86, ICAM1, and CD58 is knocked out, and/or wherein the genes expressing UL16-binding proteins (ULBP) -2, 5, and 6 are knocked out; e.g., wherein the hematopoietic cells are T cells and the conditions which induce differentiation of the iPSCs into hematopoietic cells are conditions which further induce differentiation into T cells; or wherein the hematopoietic cells are NK-cells and the conditions which induce differentiation of the iPSCs into hematopoietic cells are conditions which further induce differentiation into NK-cells.
  • ULBP UL16-binding proteins
  • Example 1 Cells expressing NK repressors
  • Candidate NK inhibitory ligands are assessed by transforming K562 cells with the candidate ligands and assessing resistance to cord blood NK cells and peripheral blood NK cells (CBNK/PBNK) .
  • the following single genes are electroporated into K562 cells for overexpression (using the CAG promoter for the genes of interest, and BFP under control of hEF1A promoter as a selectable marker) : Clec2d, Nectin-1, CDH1, CD155, CD24, CD72, FASL, SERPINB9, VPX, VPU and NEF.
  • the resistance to PBNK is measured for (i) the transformed K562 cells with single gene overexpression, (ii) wild-type K562 cells without overexpressing any transgenes (negative control) , and (iii) K562 cells overexpressing HLA-E (positive control) .
  • the results are summarized in Figure 1.
  • Clec2d is a cognate ligand for the inhibitory NK receptors (NKR) -P1B and NKR-P1D (CD161b/d) .
  • NSR inhibitory NK receptors
  • Clec2d is delivered together with a BFP reporter into K562 cells, and flow cytometry is used to analyze transgene expression.
  • Figure 2 depicts a gene construct inserted into K562 cells to overexpress a Clec2d gene under control of a CAG promoter, together with a blue fluorescent protein (BFP) reporter gene under control of a human elongation factor-1 alpha (hEF1a) promoter, the genes being separated by a chromatin insulator (A2CBX) .
  • Figure 3 top, depicts flow cytometry histogram results indicating successful overexpression of the Clec2d/BFP construct inserted into K562 cells;
  • Figure 3, bottom depicts relative expression levels of Clec2d/BFP genes derived from various mammalian species.
  • Figure 4 depicts relative expression levels of human-derived Clec2d/BFP constructs comprising various ubiquitination site K-to-R mutations.
  • Figure 5 depicts CD107a degranulation assays of human cord blood natural killer (CBNK) cells and human PBNK cells exposed to the engineered K562/Clec2d cells. Unmodified K562 cells (K562) are used as a negative control, which are expected to activate degranulation in allogeneic NK cells. K562 cells overexpressing HLA-E (K562/HLAE) are used as a positive control, paralleling the state-of-the-art in reducing sensitivity in allogeneic NK cells.
  • CBNK human cord blood natural killer
  • FIG. 6 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/Clec2d cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 7 depicts a dose-dependent effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/Clec2d cells, wherein the human-derived Clec2d construct is transfected into K562 cells overexpressing HLA-E/G and confers protection against human allogeneic NK cells with respect to the expression level of Clec2d.
  • Figure 8 further demonstrates protection conveyed by Clec2d expression, wherein expression of human Clec2d in 2KO NK cells (i.e., SU11 with MHC-1 and MHC-II knock-out) provides reduced cytotoxicity from allogeneic human PBNK cells.
  • Figure 9 provides a parallel example, wherein monkey Clec2d in K562 cells provides reduced cytotoxicity from monkey NK cells.
  • Engineered K562 do not have killing capacity against cancer cells, so in a therapeutic product, we use engineered NK cells which are the product of differentiation of engineered iPSCs (iPSC-NK cells) .
  • Figure 10 depicts results of successful differentiation of induced pluripotent stem cells (iPSCs) into embryoid bodies (EBs) after engineered overexpression of Clec2d.
  • CDH1 To test whether CBH1 could inhibit NK killing, CDH1 is delivered together with a BFP reporter into K562 cells, and flow cytometry is used to analyze transgene expression.
  • Figure 11 depicts a gene construct inserted into K562 cells to overexpress a CDH1 gene together with a BFP reporter gene.
  • Figure 12 depicts flow cytometry histogram results indicating successful overexpression of the CDH1/BFP construct inserted into K562 cells.
  • Figure 13 depicts CD107a degranulation assays of CBNK cells and PBNK cells exposed to the engineered K562/CDH1 cells. Again, K562 are used as a negative control and K562/HLAE are used as a positive control.
  • FIG 14 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/CDH1 cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 15 depicts the effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/CDH1 cells, wherein the CDH1 construct is transfected into K562 cells overexpressing HLA-E/G.
  • Figure 16 depicts dose-dependent results of differentiation of iPSCs into EBs after engineered overexpression of CDH1.
  • Figure 33 depicts effects on iPSC ability to differentiate into EBs following single transgene insertion of CDH1 construct in ANB iPSCs.
  • Low (L4/L6) expression levels of CDH1 in iPSCs allows for normal differentiation into EBs; however, medium (M4/M9) and high (H3/H5) expression levels of CDH1 interfere with typical EB formation.
  • Nectin-1 Figure 17 depicts a gene construct inserted into K562 cells to overexpress a Nectin-1 (NECTIN1) gene together with a BFP reporter gene.
  • Figure 18 depicts flow cytometry histogram results indicating successful overexpression of the Nectin-1/BFP construct inserted into K562 cells.
  • Figure 19 depicts CD107a degranulation assays of CBNK cells and PBNK cells exposed to the engineered K562/NECTIN1 cells. Again, K562 are used as a negative control and K562/HLAE are used as a positive control.
  • Figure 20 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/NECTIN1 cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 21 depicts the effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/NECTIN1 cells, wherein the Nectin-1 construct is transfected into K562 cells overexpressing HLA-E/G.
  • Figure 22 depicts results of successful differentiation of iPSCs into EBs after engineered overexpression of Nectin-1.
  • Figure 23 depicts a gene construct inserted into K562 cells to overexpress a CD155 gene together with a BFP reporter gene.
  • Figure 24 depicts flow cytometry histogram results indicating successful overexpression of the CD155/BFP construct inserted into K562 cells.
  • Figure 25 depicts CD107a degranulation assays of CBNK cells and PBNK cells exposed to the engineered K562/CD155 cells. Again, K562 are used as a negative control and K562/HLAE are used as a positive control.
  • Figure 26 depicts results of a cytotoxicity study wherein CBNK and PBNK cells are exposed to unmodified K562 cells (K562) , K562 cells overexpressing HLA-E (K562/HLAE) , and the engineered K562/CD155 cells.
  • E T ratios indicate the amount of effector cells, i.e., CBNK/PBNK, relative to the target cells, i.e., K562 cells.
  • Figure 27 depicts a dose-dependent effect of inhibiting CBNK/PBNK cytotoxicity to engineered K562/CD155 cells, wherein the CD155 construct is transfected into K562 cells overexpressing HLA-E/G and wherein a lower expression level of CD155 is found to confer greater protection against activation of allogeneic NK cells.
  • Figure 28 further demonstrates protection conveyed by CD155 expression, wherein expression of human CD155 in 2KO NK cells (i.e., SU11 with MHC-1 and MHC-II knock-out) provides reduced cytotoxicity from allogeneic human PBNK cells.
  • Figure 29 depicts results of successful differentiation of iPSCs into EBs after engineered overexpression of CD155.
  • Figure 32 depicts a comparison of specific gene transfections and their resulting efficacy toward inhibiting cytotoxicity of K562/HLAE/G cells to PBNK cells.
  • the transduction is accomplished using lentiviral vectors at a multiplicity of infection (MOI, i.e., the ratio of the number of virus particles to the number of the host cells in a given infection medium) of 10 or 30.
  • MOI multiplicity of infection
  • the selected genes that most effectively confer protection to K562/HLAE/G cells are Clec2d-30 > Nectin-1-10 >CDH1-30 > CD155-10 > Clec2d-10 > CD155-30 > CD24-10, wherein 10 or 30 refers to the MOI of thetransduction.
  • Selected genes are knocked out using a CRISPR-Cas9 system. These gene knockouts can also be coupled with one or more of the gene knock-ins in the preceding example to provide optimal efficacy in avoiding PBNK stimulation.
  • Figure 30 depicts results of using a single guide ribonucleic acid (sgRNA) to simultaneously knock-out UL16-binding proteins (ULBP) -2, 5, and 6, which encode major histocompatibility complex (MHC) class I-related molecules that bind to the NKG2D receptor that activates NK cell cytotoxicity.
  • sgRNA constructs for this purpose are as follows:
  • SU11 cells are double knock-out endometrial NK cells, which lack beta-2-microglobulin (B2M, a component of MHC class I) , and MHC class II transactivator (CIITA) . Accordingly, the SU11 cells lack MHC-I and MHC-II, rendering them susceptible to “missing self” -induced killing by NK cells.
  • This ULBP2/5/6 knock-out in SU11 cells protects the SU11 cells from cytotoxicity by CBNK and PBNK.
  • Section (A) depicts indel frequency analysis.
  • Section (B) depicts flow cytometry histogram results of ULBP2/5/6 knockout.
  • Section (C) depicts cytotoxicity by PBNK cells.
  • Section (D) depicts cytotoxicity by CBNK cells.
  • Figure 31 depicts CD107a degranulation assays of CBNK and PBNK cells exposed to the engineered SU11 cells with a ULBP2/5/6 knock-out.
  • Figure 34 depicts flow cytometry histogram results indicating successful knock-out of transforming growth factor (TGF) -beta receptor 2 in K562 cells, which demonstrates the potential for inhibition of iPSC differentiation following genetic engineering.
  • TGF transforming growth factor
  • eNK cells are derived from iPSCs and cultured in NK medium including 95%Lymphocyte Serum-Free Medium KBM 581 (Corning, Cat#88581CM) , 5%human AB serum (Access Bio, Cat#515) , 1x Non-Essential Amino Acids Solution (Gibco, Cat#11140050) , 2mM L-Glutamine (Gibco, Cat#25030081) and 200 IU/ml IL2 (R&D, Cat#202-GMP) .
  • the CD86, ICAM1 and CD58 genes in eNK cells are knocked out by a CRISPR/Cas9 system.
  • CRISPR/Cas9 includes two components, Cas9 and sgRNA.
  • Cas9 protein is purchased from Thermo Fisher Scientific (Cat#A36499 and sgRNA is synthesized from Genescript Biotech Corp.
  • Cas9 protein and sgRNA are mixed to form ribonucleoprotein complex (RNP) and delivered into eNK cells using Lonza.
  • the sgRNA is selected from the following sequences:
  • hCD86-sg3, ICAM1-sg1 and hCD58-sg3 have the highest efficiency, with 85.95%, 88.93%and 90.32%, respectively. See Figure 35, showing that CD86, ICAM1, and CD58 are effectively knocked using the CRISPR-Cas9 constructs.
  • the eNK cells with indicated gene knockout are mixed with a cancer cell line (Raji) labeled with GFP at a ratio of 0.3: 1, and the GFP signal is monitored continuously by incucyte.
  • Raji cancer cell line
  • WT wild type
  • MHC-I is tested in these eNKs, and we find that the expression of MHC-I is not affected.
  • MHC-I is a key inhibitory ligand for NK cells, and the presence of MHC-I protects eNK from allogeneic NK cells in patients. See Figure 37, showing that the CD58/CD86/ICAM1 knockouts did not affect MHC-1 expression in the eNK cells.
  • T cell proliferations is used to evaluate the stimulation of the edited eNK cells to T cells.
  • frozen human PBMC purchased from SAILY Bio (Shanghai, China) , is thawed, labeled with Cell Trace Dye Carboxyfluorescein succinimidyl ester (CFSE, Cat#C34554 from Thermo Fisher Scientific) and mixed with indicated eNK in NK medium with 20IU/ml IL2 instead of 200IU/ml for 6 days, medium is refreshed every other day.
  • CFSE Cell Trace Dye Carboxyfluorescein succinimidyl ester
  • CD8+ T cells by NK after knocking out the ICAM1/CD86/CD58 gene is significantly lower than that of WT and SH sg1 (sgRNA targeting to safe harbor, which does not knock out any functional proteins) , where CD58 knockout is comparable to B2M knockout (MHC-I stimulates CD8+ T cells by binding to TCR on CD8+ T cells) .
  • B2M is a component of MHC-I, and B2M knockout causes loss of MHC-I, thereby avoiding stimulation of CD8+ T cells) . See Figure 38, showing that the CD58, CD86, and ICAM 1 knockouts exhibit significantly lowered stimulation of CD8+ cells.
  • CD58/CD86/ICAM1 triple knockout exhibit significantly lower stimulation of CD8+ T cells and CD58+CD86 knockout has the lowest stimulation of the CD8+ T cells.
  • NK repressor genes Clec2d, CDH1, Nectin1, and CD155 are identified herein as NK repressor genes, wherein expression of said genes, or constructs comprising said genes, in whole or in part, convey protection to engineered cells expressing said gene (s) /construct (s) from NK cell-associated cytotoxicity.
  • Exemplary sequences of constructs comprising sequences derived from NK repressor genes of various mammalian species are described in Table 1.
  • the Clec2d gene was mutated as shown in Fig 41, specifically, H176C, C163S. Then, Transient transfection into 293T using Lipofectamine-3000, and FACS detection at 48h post transfection.
  • Fig. 42 The gene constructs shown in Fig. 42 were transiently transfected into 293T cells by Lipofectamine-3000, and each group was repeated twice. The results are shown in Fig. 43.
  • Fig. 44 The gene constructs shown in Fig. 44 were transiently transfected into 293T cells by Lipofectamine-3000, The results are shown in Figs 45-46.
  • NHP PBMCs were isolated from adult cynomolgus peripheral blood by standard protocols, using FicollPaque PLUS (GE Healthcare Bio-Sciences) .
  • Total T cells were isolated from PBMCs using a NHP pan T cell isolation kit per manufacturer ‘sinstructions (Miltenyi Biotec; Cat#130-091-993) .
  • Polyclonal T cells were activated with NHP anti-CD2/anti-CD3/anti-CD28 stimulation beads at a 1: 2 bead to T cell ratio (Miltenyi Biotec, Cat#130-092-919) in CTS TM OpTmizer TM T Cell Expansion SFM (ThermoFisher Scientific, Cat#A1048501) supplemented with 10%AB serum (Access Biologicals) , and recombinant human IL-2 (rhIL-2, 200 U/mL; R&D Systems) .
  • Lentiviral transduction with spinoculation was performed on day 1 or 2 using lentiboost (SIRION Biotech Cat #SB-P-LV-101-01) and ⁇ HIV-Clec2d, ⁇ HIV-CD20 CAR lentiviruses (MOI range: 5-20) .
  • KO were performed using RNP containing synthesized sgRNA (synthesized by Genescript) and Cas9 protein (ThermoFisher Scientific, Cat#A36499) ;
  • NHP PBNK preparation NHP PBMCs were isolated from adult cynomolgus peripheral blood by standard protocols, using FicollPaque PLUS (GE Healthcare Bio-Sciences) .
  • NHP PBNK were sorted from PBMC using CD3-NKG2A+ markers. PBNK were further divided into CD161 -/+ by FACS.
  • NHP Clec2d could protect 2KO CAR-T from NHP CD161 + PBNK killing.
  • C. NHP Clec2d could protect 2KO CAR-T from NHP CD161 + PBNK;
  • Human PBMCs were isolated from adult peripheral blood by standard protocols, using FicollPaque PLUS (GE Healthcare Bio-Sciences) .
  • Total T cells were isolated from PBMCs using a human pan T cell isolation kit per manufacturer ‘sinstructions (Miltenyi Biotec; Cat#130-096-535) .
  • Polyclonal T cells were activated with human T Cell TransAct TM stimulation (Miltenyi Biotec, Cat#130-111-160) in CTS TM OpTmizer TM T Cell Expansion SFM (ThermoFisher Scientific, Cat#A1048501) supplemented with 10%AB serum (Access Biologicals) , and recombinant human IL-2 (rhIL-2, 100 U/mL; R&D Systems) .
  • Lentiviral transduction with spinoculation was performed on day 1 or 2 using polybrene (Sigma Cat #TR-1003-G) and HIV-HLA-E-2A-Clec2d lentiviruses (MOI range: 5-100) . Expression was detected by FACS using corresponding antibody.
  • Fig. 48 shows the expression of HLA-E and CLEC2D at different MOI.
  • Human PBMCs were isolated from adult peripheral blood by standard protocols, using FicollPaque PLUS (GE Healthcare Bio-Sciences) .
  • Total T cells were isolated from PBMCs using a human pan T cell isolation kit per manufacturer ‘sinstructions (Miltenyi Biotec; Cat#130-096-535) .
  • Polyclonal T cells were activated with human T Cell TransAct TM stimulation (Miltenyi Biotec, Cat#130-111-160) in CTS TM OpTmizer TM T Cell Expansion SFM (ThermoFisher Scientific, Cat#A1048501) supplemented with 10%AB serum (Access Biologicals) , and recombinant human IL-2 (rhIL-2, 100 U/mL; R&D Systems) .
  • Lentiviral transduction with spinoculation was performed on day 1 or 2 using polybrene (Sigma Cat #TR-1003-G) and HIV-Clec2d lentiviruses with different promoters (CAGs vs EF1a) (MOI range: 5-20) . Expression was detected by FACS using corresponding antibody.

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Abstract

L'invention concerne des cellules modifiées hypoimmunogènes, comprenant des cellules souches pluripotentes induites (CSPi), des cellules hématopoïétiques dérivées de celles-ci et des cellules primaires, des compositions pharmaceutiques comprenant lesdites cellules, ainsi que des procédés d'utilisation de celles-ci, un ou plusieurs gènes étant transfectés dans lesdites cellules, de telle sorte que l'activation de cellules immunitaires allogéniques est réduite.
PCT/CN2024/118162 2023-09-13 2024-09-11 Immunothérapies utilisant des cellules modifiées hypoimmunogènes Pending WO2025055927A1 (fr)

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017066634A1 (fr) * 2015-10-16 2017-04-20 Fate Therapeutics, Inc. Plate-forme pour l'induction et la maintenance de la pluripotence à l'état fondamental
CN107921148A (zh) * 2015-05-08 2018-04-17 哈佛学院校长同事会 通用供体干细胞和相关方法
CN110869493A (zh) * 2017-07-12 2020-03-06 韦尔赛特公司 通用供体细胞和相关方法
CN113383018A (zh) * 2018-09-05 2021-09-10 波赛达治疗公司 同种异体细胞组合物和使用方法
WO2022012591A1 (fr) * 2020-07-15 2022-01-20 南京北恒生物科技有限公司 Cellule immunitaire modifiée destinée à une allotransplantation
WO2022125439A2 (fr) * 2020-12-07 2022-06-16 The Regents Of The University Of California Silençage de cellules immunitaires innées par un engageur sirp-alpha
WO2023279112A1 (fr) * 2021-07-02 2023-01-05 Fate Therapeutics, Inc. Cellules effectrices protégées et leur utilisation pour des thérapies cellulaires adoptives allogéniques
US20230149461A1 (en) * 2020-04-01 2023-05-18 Nanjing Legend Biotech Co., Ltd. Compositions and methods for reducing graft rejection in allogeneic cell therapy
WO2023093763A1 (fr) * 2021-11-24 2023-06-01 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules
WO2023147777A1 (fr) * 2022-02-07 2023-08-10 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour des immunothérapies améliorées

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107921148A (zh) * 2015-05-08 2018-04-17 哈佛学院校长同事会 通用供体干细胞和相关方法
WO2017066634A1 (fr) * 2015-10-16 2017-04-20 Fate Therapeutics, Inc. Plate-forme pour l'induction et la maintenance de la pluripotence à l'état fondamental
CN110869493A (zh) * 2017-07-12 2020-03-06 韦尔赛特公司 通用供体细胞和相关方法
CN113383018A (zh) * 2018-09-05 2021-09-10 波赛达治疗公司 同种异体细胞组合物和使用方法
US20230149461A1 (en) * 2020-04-01 2023-05-18 Nanjing Legend Biotech Co., Ltd. Compositions and methods for reducing graft rejection in allogeneic cell therapy
WO2022012591A1 (fr) * 2020-07-15 2022-01-20 南京北恒生物科技有限公司 Cellule immunitaire modifiée destinée à une allotransplantation
WO2022125439A2 (fr) * 2020-12-07 2022-06-16 The Regents Of The University Of California Silençage de cellules immunitaires innées par un engageur sirp-alpha
WO2023279112A1 (fr) * 2021-07-02 2023-01-05 Fate Therapeutics, Inc. Cellules effectrices protégées et leur utilisation pour des thérapies cellulaires adoptives allogéniques
WO2023093763A1 (fr) * 2021-11-24 2023-06-01 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules
WO2023147777A1 (fr) * 2022-02-07 2023-08-10 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour des immunothérapies améliorées

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU BO, TIAN LEI, CHEN JING, WANG JING, MA RUI, DONG WENJUAN, LI AIMIN, ZHANG JIANYING, ANTONIO CHIOCCA E., KAUR BALVEEN, FENG MING: "An oncolytic virus expressing a full-length antibody enhances antitumor innate immune response to glioblastoma", NATURE COMMUNICATIONS, vol. 12, no. 1, 1 January 2021 (2021-01-01), UK, pages 1 - 17, XP093130859, ISSN: 2041-1723, DOI: 10.1038/s41467-021-26003-6 *

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