Population of in vitro-derived T cells, method for generating them, and method for using them.

Abstract

This disclosure provides a method for generating CD4-CD8+ T cells in vitro from hematopoietic stem cells / progenitor cells. A population of cells produced using this method and methods for using such population are also provided.

Description

Technical Field

[0001] Field Broadly speaking, the present invention relates to an in vitro method for generating a population of T cells from stem cells and / or progenitor cells, a population of T cells generated in vitro, and their use.

Background Art

[0002] Background of the Invention Methods based on various feeder cells are available for generating T-lineage cells from stem cells / progenitor cells. However, the expression of cell surface proteins by supportive stromal cells in feeder cell-based systems is heterogeneous, leading to diverse results. Furthermore, feeder cell-based systems cannot be easily scaled up to meet the needs of clinical manufacturing.

[0003] Immobilized Notch signaling ligands, such as DL4, etc., have been shown to promote the in vitro generation of progenitor T cells in feeder-free and serum-free culture systems in combination with VCAM-1 (Shukla et al., 2017). Microbeads modified to present DL4 have also been shown to support the in vitro differentiation of T cell lineage cells, although progression to mature lineages such as CD4-CD8+ T cells is limited (Trotman-Grant et al., 2021). Short-term stimulation of T cell precursors with anti-CD3 antibodies in the absence of Notch signaling, followed by maturation without anti-CD3 antibodies, has been shown to promote the generation of CD8αβ+ T cells (Iriguchi et al., 2021). It is desirable to remove or reduce one or more of the above drawbacks.

Summary of the Invention

[0004] Summary of the Invention A first aspect of this disclosure provides a method for generating a population of CD4-CD8+ T cells. This method includes generating a population of progenitor T cells by contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand at a first ligand concentration; generating a population of CD4+CD8+ cells by contacting a population of progenitor T cells with an immobilized Notch signaling ligand at a second ligand concentration lower than the first ligand concentration; and contacting a population of CD4+CD8+ cells with a T cell activator in the absence of the Notch signaling ligand.

[0005] In one embodiment, the immobilized Notch signaling ligand is DL4.

[0006] In one embodiment, the step of contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand at a first ligand concentration further includes contacting the population of hematopoietic stem cells / progenitor cells with immobilized VCAM-1.

[0007] In one embodiment, the first ligand concentration is 3.15 × 10⁻⁶ 11 ~1.26×10 12 The concentration is molecules / mL, and the second ligand concentration is 3.94 × 10⁻⁶. 10 ~6.31×10 11 It is molecules / mL.

[0008] In one embodiment, the method further comprises contacting a population of CD4+CD8+ cells with an immobilized Notch signaling ligand at a third ligand concentration lower than a second ligand concentration, before contacting the population of CD4+CD8+ cells with a T cell activator.

[0009] In one embodiment, the third ligand concentration is 3.94 × 10⁻⁶ 10 ~6.31×10 11 It is molecules / mL.

[0010] In one embodiment, the method further includes the step of enriching CD4+CD8+ cells with respect to CD8α or CD8β.

[0011] In one embodiment, the T cell activator is a CD3 stimulator and an integrin ligand.

[0012] In one embodiment, the CD3 stimulating factor is an anti-CD3 / anti-CD28 / anti-CD2 reagent, an anti-CD3 reagent, or a peptide major histocompatibility complex (pMHC) tetramer, and the integrin ligand is laminin, ICAM, fibronectin, fibronectin fragment, VCAM-1, or ICOS-L.

[0013] In one embodiment, the CD3 stimulating factor is an anti-CD3 / anti-CD28 / anti-CD2 reagent, and the integrin ligand is a fibronectin fragment.

[0014] In one embodiment, the T cell activator is a chimeric antigen receptor (CAR) activator.

[0015] In one embodiment, the chimeric antigen receptor activator is an antigen immobilized on a substrate.

[0016] In one embodiment, the antigen is the CD19 antigen, and the substrate is particles.

[0017] In one embodiment, the step of contacting a population of CD4+CD8+ cells with the T cell activator in the absence of Notch signaling ligands is carried out in a cell culture medium containing IL-7, IL-15, and IL-21.

[0018] In one embodiment, the step of contacting a population of CD4+CD8+ cells with the T cell activator in the absence of a Notch signaling ligand is carried out in a cell culture medium containing one or more of IL-7, IL-15, and IL-21.

[0019] In one embodiment, the step of contacting a population of CD4+CD8+ cells with the T cell activator in the absence of Notch signaling ligand is performed in the absence of IL-2.

[0020] In one embodiment, the method further includes contacting a population of CD4-CD8+ T cells with T cell activators as well as IL-7, IL-15, and IL-21.

[0021] In one embodiment, the method further comprises contacting a population of CD4-CD8+ T cells with a T cell activator and one or more of IL-7, IL-15, and IL-21.

[0022] In one embodiment, the T cell activator is a CD3 stimulator.

[0023] In one embodiment, the CD3 stimulating factor is an anti-CD3 / anti-CD28 / anti-CD2 reagent, an anti-CD3 reagent, or a peptide major histocompatibility complex (pMHC) tetramer.

[0024] In one embodiment, the method further includes increasing the cell density of a population of precursor T cells by contacting them with an immobilized Notch signaling ligand at a second ligand concentration, and / or increasing the cell density of a population of CD4+CD8+ cells by contacting them with a T cell activator and an integrin ligand in the absence of the Notch signaling ligand.

[0025] In one embodiment, hematopoietic stem cells / progenitor cells are derived from pluripotent stem cells.

[0026] In one embodiment, a population of CD4-CD8+ T cells includes a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous T cell receptor (TCR).

[0027] In one embodiment, the population of CD4-CD8+ T cells is CD3+.

[0028] In one embodiment, the population of CD4-CD8+ T cells is TRAC- / -.

[0029] In one embodiment, the population of CD4-CD8+ T cells is enriched with respect to CD8αβ+ cells.

[0030] In one embodiment, the population of CD4-CD8+ T cells includes subpopulations of TCRγδ+, CD49c+, and / or CD31+ cells.

[0031] A second aspect of this disclosure provides a population of CD4-CD8+ T cells prepared according to the method of the first aspect.

[0032] In one embodiment, a population of CD4-CD8+ T cells contains a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous TCR.

[0033] In one embodiment, the population of CD4-CD8+ T cells is CD3+.

[0034] In one embodiment, the population of CD4-CD8+ T cells is TRAC- / -.

[0035] A third aspect of this disclosure provides a population of CD4-CD8+CD3+TRAC- / - cells, which are induced in vitro from pluripotent stem cells.

[0036] In one embodiment, the CD4-CD8+CD3+TRAC- / - cell population of the third embodiment includes a nucleic acid sequence encoding a chimeric antigen receptor or exogenous TCR.

[0037] In one embodiment, the population of CD4-CD8+ T cells includes subpopulations of TCRγδ+, CD49c+, and / or CD31+ cells.

[0038] A fourth aspect of this disclosure provides a pharmaceutical composition comprising a population of CD4-CD8+ T cells and a pharmaceutically acceptable carrier. The population of CD4-CD8+ T cells is CD3+ and TRAC- / -.

[0039] In a fifth aspect of the present disclosure, a method of treating a target disease or condition is provided. The method includes generating a population of CD4-CD8+ T cells according to the method of the first aspect, and administering an effective amount of the population of CD4-CD8+ T cells to a subject that needs it.

[0040] In one embodiment, the population of CD4-CD8+ T cells is CD3+ and / or TRAC- / - .

[0041] In one embodiment, the disease or condition is a hematological malignancy, and the CD4-CD8+ T cells include a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous T cell receptor (TCR).

[0042] In a sixth aspect of the present disclosure, the use of a population of CD4-CD8+ T cells in the manufacture of a medicament for treating a disease or condition is provided. The population of CD4-CD8+ T cells is generated according to the method of the first aspect.

[0043] In a seventh aspect of the present disclosure, a method of generating a population of CD4+CD8+ cells is provided. The method includes generating a population of progenitor T cells by contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand at a first ligand concentration, and contacting the population of progenitor T cells with the immobilized Notch signaling ligand at a second ligand concentration lower than the first ligand concentration.

[0044] In one embodiment, the immobilized Notch signaling ligand is DL4.

[0045] In one embodiment, the first ligand concentration is 3.15×10 11 ~1.26×10 12 molecules / mL, and the second ligand concentration is 3.94×10 10 ~6.31×10 11 molecules / mL.

[0046] In one embodiment, the population of CD4+CD8+ cells is CD1a+CD28+ and ICOS+.

[0047] In one embodiment, a population of CD4+CD8+ cells contains a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous TCR.

[0048] In an eighth aspect of this disclosure, a population of CD4+CD8+ cells is provided. The population of CD4+CD8+ cells is produced according to the method of the seventh aspect.

[0049] In a ninth aspect of this disclosure, a population of CD4+CD8+ cells induced in vitro from pluripotent stem cells is provided. The CD4+CD8+ cells are CD1a+CD28+ and ICOS+.

[0050] A tenth aspect of this disclosure provides a method for generating a population of CD4-CD8+ T cells. This method includes generating a population of progenitor T cells by contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand; generating a population of CD4+CD8+ cells by contacting the population of progenitor T cells with a Notch signaling inhibitor and an immobilized Notch signaling ligand; and contacting a population of CD4+CD8+ cells with a T cell activator in the absence of a Notch signaling ligand.

[0051] In one embodiment, the activity of the immobilized Notch signaling ligand in the step of contacting a population of progenitor T cells with a Notch signaling inhibitor and an immobilized Notch signaling ligand is reduced compared to the activity of the immobilized Notch signaling ligand in the step of contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand.

[0052] In one embodiment, the Notch signaling inhibitor is a gamma secretase inhibitor.

[0053] In one embodiment, the gamma secretase inhibitor is provided in a quantity of 0.1 to 1 micromolar (μM).

[0054] In one embodiment, the Notch signaling inhibitor, in the step of contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand, produces 3.15 × 10⁻¹⁰ units. 11 ~1.26×10 12 Provided at a first ligand concentration of molecules / mL, and / or, the Notch signaling inhibitor is used in the step of contacting a population of precursor T cells with the Notch signaling inhibitor and immobilized Notch signaling ligand, at a concentration of 3.15 × 10⁻¹⁶. 11 ~2.52 × 10 12 It is supplied at a second ligand concentration of molecules / mL.

[0055] An eleventh aspect of this disclosure provides a method for generating a population of CD4+CD8+ cells. This method comprises generating a population of progenitor T cells by contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand, and contacting the population of progenitor T cells with a Notch signaling inhibitor and an immobilized Notch signaling ligand.

[0056] In one embodiment, the activity of the immobilized Notch signaling ligand in the step of contacting a population of progenitor T cells with a Notch signaling inhibitor and an immobilized Notch signaling ligand is reduced compared to the activity of the immobilized Notch signaling ligand in the step of contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand.

[0057] In one embodiment, the Notch signaling inhibitor is a gamma secretase inhibitor.

[0058] In one embodiment, the gamma secretase inhibitor is provided in a quantity of 0.1 to 1 micromolar (μM).

[0059] In one embodiment, the immobilized Notch signaling ligand is used in the step of contacting a population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand, resulting in a volume of 3.15 × 10⁻¹⁰ cells. 11~1.26×10 12 The first ligand concentration is provided at 3.15 × 10⁶ molecules / mL, and / or the immobilized Notch signaling ligand is provided in the step of contacting a population of precursor T cells with the Notch signaling inhibitor and the immobilized Notch signaling ligand. 11 ~2.52 × 10 12 It is supplied at a second ligand concentration of molecules / mL.

[0060] A twelfth aspect of this disclosure provides a method for treating a disease or condition of interest. The method comprises generating a population of CD4-CD8+ T cells according to the method of the eleventh aspect, and administering an effective amount of the CD4-CD8+ T cell population to a subject in need.

[0061] In one embodiment, the population of CD4-CD8+ T cells is CD3+ and / or TRAC- / -.

[0062] A thirteenth aspect of this disclosure provides the use of a population of CD4-CD8+ T cells in the manufacture of a pharmaceutical product for treating a disease or condition. The CD4-CD8+ T cells are generated according to the method of the eleventh aspect. [Brief explanation of the drawing]

[0063] Brief explanation of the drawing To facilitate understanding of the present invention, non-limiting embodiments are shown in the accompanying drawings.

[0064] [Figure 1A] This graph quantifies the expression percentage (left axis) and median fluorescence intensity (MFI, right axis) of chimeric antigen receptors (CARs) after lentiviral transduction or gene editing of iPSCs.

[0065] [Figure 1B] This is a microscopic image of the G-band karyotype analysis of the generated iPSC clone.

[0066] [Figure 2A] This graph shows the purity of CD34+ (left) and CD34+CD43+ (right) cells generated from four iPSC cell lines.

[0067] [Figure 2B] This graph shows the yield of CD34+ (left) and CD34+CD43+ (right) cells generated from four iPSC cell lines.

[0068] [Figure 3A] This is a flow cytometry plot showing pluripotency markers (OCT4, SOX2, TRA-160, SSEA-4) and chimeric antigen receptor expression of iPSC cells generated after thawing and proliferation ("Stage 1," culture day 10).

[0069] [Figure 3B] This is a flow cytometry plot characterizing marker expression in hematopoietic stem cells / progenitor cells generated from iPSCs ("Stage 2," culture day 0).

[0070] [Figure 3C] This is a flow cytometry plot showing marker expression in precursor T cells generated from iPSCs ("Stage 3," culture day 10).

[0071] [Figure 3D] This is a flow cytometry plot showing marker expression in CD4+CD8+ double-positive (DP) T lineage cells generated from iPSCs ("Stage 4," culture day 28).

[0072] [Figure 3E] This is a flow cytometry plot showing marker expression in T cell lineages, including DP and CD8+ single-positive (CD8 SP) cell populations generated from iPSCs ("Stage 5," culture day 35).

[0073] [Figure 4A]This graph quantifies the proportion of CD4+CD8-immature single-positive (ISP), CD4-CD8-double-negative (DN), DP, and CD4-CD8α+ single-positive (CD8a SP) cell populations generated from iPSC-derived HSPCs with various doses of manipulated thymic niche (ETN) beads (0.1x, 0.5x, 1x bead doses) and cell densities (2×10⁶, 1×10⁶, 5×10⁵ cells / mL).

[0074] [Figure 4B] This graph shows the correlation between the ETN bead-to-cell ratio (beads:cells) and the percentage of CD4+CD8+ cells (%DP) on day 21.

[0075] [Figure 4C] This is a flow cytometry plot showing the expression of typical cell markers, as quantified in Figure 4A.

[0076] [Figure 5] This is a flow cytometry plot showing marker expression in cells cultured on day 15 under two ETN culture conditions.

[0077] [Figure 6A] This graph quantifies the percentage of CD56+CD7+ cells generated from iPSC-derived HSPCs on day 21 using various doses of ETN beads (0.1x, 0.5x, 1x bead dose) and cell densities (4×10⁶, 2×10⁶, 1×10⁶ cells / mL).

[0078] [Figure 6B] This graph quantifies the percentage of CD3+ cells generated from iPSC-derived HSPCs with various doses of ETN beads (0.1x, 0.5x, 1x bead dose) and cell densities (4×10⁶, 2×10⁶, 1×10⁶ cells / mL).

[0079] [Figure 7A]This graph quantifies the proportions of CD4+ISP, DN, DP, and CD8α SP cell populations generated from iPSC-derived HSPCs with various doses of manipulated ETN beads (0.1x, 0.5x, 1x bead doses) and cell densities (4×10⁶, 2×10⁶, 1×10⁶ cells / mL).

[0080] [Figure 7B] This graph shows the correlation between the bead-to-cell ratio (beads:cells) and the percentage of CD4+CD8+ double-positive cells (%DP) on day 23.

[0081] [Figure 7C] This is a flow cytometry plot showing the expression of typical cell markers, as quantified in Figure 7A.

[0082] [Figure 8A] This graph quantifies the percentage of CD56+CD7+ cells at day 23 generated from iPSC-derived HSPCs with various doses of ETN beads (0.1x, 0.5x, 1x bead dose) and cell densities (4×10⁶, 2×10⁶, 1×10⁶ cells / mL).

[0083] [Figure 8B] This graph quantifies the percentage of CD3+ cells at day 23 generated from iPSC-derived HSPCs with various doses of ETN beads (0.1x, 0.5x, 1x bead dose) and cell densities (4×10⁶, 2×10⁶, 1×10⁶ cells / mL).

[0084] [Figure 9] This is a schematic diagram of the stages of in vitro differentiation of CD8+ cells from iPSCs, including a summary of the culture conditions at each stage.

[0085] [Figure 10]This graph quantifies cell viability and cumulative growth rate throughout 28 days of differentiation from iPSC-derived HSPCs under 2D or 3D differentiation processes. Three cell lines are shown: NTX4A1 (top), NTX6A1 (middle), and NTX4B3 (bottom).

[0086] [Figure 11] Flow cytometry plots of marker expression in cells cultured for 17 or 24 days under 2D differentiation conditions. Two cell lines are shown: NTX4A1 (top) and NTX6A1 (center).

[0087] [Figure 12] Flow cytometry plots of marker expression in NTX4A1 cells cultured under 3D differentiation conditions for 17, 24, or 28 days.

[0088] [Figure 13] Flow cytometry plots of marker expression in NTX6A1 cells cultured under 3D differentiation conditions for 17, 24, or 28 days.

[0089] [Figure 14] This is a flow cytometry plot of marker expression in NTX4B3 cells cultured for 17 or 24 days under 2D differentiation conditions.

[0090] [Figure 15] Flow cytometry plots of marker expression in NTX4B3 cells cultured under 3D differentiation conditions for 17, 24, or 28 days.

[0091] [Figure 16] This graph quantifies cell viability (top) and cumulative growth rate (bottom) after culturing iPSC-derived DP with either ImmunoCult® human CD3 / CD28 / CD2 T cell activator (Immunocult) or antigen beads. Three cell lines are shown: NTX4A1, NTX6A1, and NTX4B3.

[0092] [Figure 17] These are flow cytometry plots of marker expression in NTX4A1 cells (top) and NTX6A1 cells (bottom) cultured with CD3 stimulation on day 35 or 37.

[0093] [Figure 18] This is a flow cytometry plot of marker expression in NTX4B3 cells cultured with CD3 stimulation (top) or antigen beads (bottom) on day 35.

[0094] [Figure 19] This graph quantifies the results of in vitro serial restimulation assays of primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated by antigen-coated beads (top) or CD3 stimulation (bottom). The normalized green region (vertical axis) represents the number of CD19+ / + or CD19- / - target cells for each culture condition.

[0095] [Figure 20A] This graph quantifies the cumulative proliferation rate of primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells during an in vitro serial restimulation assay.

[0096] [Figure 20B] This graph quantifies tumor necrosis factor α (TNFα) secretion by primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells after four stimulations in an in vitro serial restimulation assay.

[0097] [Figure 20C] This graph quantifies the secretion of granzyme B by primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells after four stimulations in an in vitro serial restimulation assay.

[0098] [Figure 21]This graph quantifies the cumulative cytotoxicity and proliferation rate of primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads ("3D Canonical CD19 Beads") or CD3 stimulation ("3D Canonical Immunocult") during an in vitro serial restimulation assay.

[0099] [Figure 22A] This graph quantifies the proportion of T cell memory cells (T stem cell memory (TSCM), CD62 L+CD45RA+CD95+; T central memory (TCM), CD62 L+CD45RA-CD45RO+; T effector memory (TEM), CD62 L-CD45RA-CD45RO+; and terminally differentiated effector memory cells (TEMRA), CD62 L-CD45RA+CD45RO+) reexpressing CD45RA, in primary CD8+CAR-T cell populations and iPSC-derived CD8+CAR-T cell populations before (left) and after (right) an in vitro serial restorative assay.

[0100] [Figure 22B] This graph quantifies the proportions of TIM3+LAG3+, LAG3+, TIM3+, and TIM3-LAG3- cells in primary CD8+ CAR-T cell populations and iPSC-derived CD8+ CAR-T cell populations generated using antigen-coated beads ("CD19 beads") or CD3 stimulation ("Immunocult") before (left) and after (right) an in vitro serial restimulation assay.

[0101] [Figure 23A]This graph quantifies the percentage and RNA expression intensity of cell marker profiles for iPSC-derived progenitor T cells (ProT), iPSC-derived CD4+CD8+ double-positive cells (DP), and iPSC-derived CD8 SP (iPSC CD8-T) cells. According to the reference dataset (Park et al., 2020), cell marker profiles corresponding to each cell type are annotated: early DN cells, DN(early); proliferating DN cells, DN(P); resting DN cells, DN(Q); proliferating DP cells, DP(P); resting DP cells, DP(Q); CD8+ T cells, CD8-T; CD8αα+ cells, CD8-αα; and CD8+ memory T cells, CD8-Tmem.

[0102] [Figure 23B] These are homogeneous manifold approximation and projection (UMAP) plots of iPSC-derived progenitor T cells (ProT), iPSC-derived CD4+CD8+ double-positive cells (DP), and iPSC-derived CD4-CD8+ (iPSC CD8-T) cell populations.

[0103] [Figure 23C] This graph quantifies the percentage and expression intensity of the genes shown on the vertical axis for iPSC-derived progenitor T cells (ProT), iPSC-derived CD4+CD8+ cells (DP), and iPSC-derived CD4-CD8+ T cells (iPSC CD8-T).

[0104] [Figure 24] This is a flow cytometry plot of marker expression in iPSC-derived cells.

[0105] [Figure 25A] This graph quantifies the RNA expression of the genes shown on the vertical axis for primary T cells.

[0106] [Figure 25B]This graph quantifies the percentage of primary CD8+ T cells corresponding to the RNA expression profiles of DN (initial), DN(P), DN(Q), DP(P), DP(Q), γδT cells (GD T), and CD8-T cells, annotated from the reference dataset (Park et al., 2020).

[0107] [Figure 26A] This graph quantifies the RNA expression of the genes shown on the vertical axis for iPSC-derived CD8+ cells from five cell banks.

[0108] [Figure 26B] This graph quantifies the percentage of iPSC-derived CD8+ cells corresponding to DN(initial), DN(P), DN(Q), DP(Q), and CD8-T cell RNA expression profiles, annotated from the reference dataset (Park et al., 2020).

[0109] [Figure 27A] This is a flow cytometry plot of marker expression in TCR-modified iPSC-derived cells (clone TCR-172) on day 25.

[0110] [Figure 27B] This graph quantifies marker expression in TCR-modified iPSC-derived cells (clone TCR-172) on day 25.

[0111] [Figure 27C] This is a flow cytometry plot of marker expression in TCR-modified iPSC-derived cells (clone TCR-172) on day 32.

[0112] [Figure 27D] This graph quantifies marker expression in TCR-modified iPSC-derived cells (clone TCR-172) on day 32.

[0113] [Figure 28A] This is a flow cytometry plot of marker expression in TCR-modified iPSC-derived cells (clone TCR-174) on day 25.

[0114] [Figure 28B] This graph quantifies marker expression in TCR-modified iPSC-derived cells (clone TCR-174) on day 25.

[0115] [Figure 28C] This is a flow cytometry plot of marker expression in TCR-modified iPSC-derived cells (clone TCR-174) on day 32.

[0116] [Figure 28D] This graph quantifies marker expression in TCR-modified iPSC-derived cells (clone TCR-174) on day 32.

[0117] [Figure 29A] This is a flow cytometry plot of marker expression in TCR-manipulated iPSC-derived cells (clone TCR-172) after proliferation.

[0118] [Figure 29B] Figure 29A shows a flow cytometry plot of marker expression in post-proliferation TCR-manipulated iPSC-derived cells (clone TCR-172) that are gated on CD3+ cells.

[0119] [Figure 29C] This graph quantifies marker expression in TCR-manipulated iPSC-derived cells (clone TCR-172) after proliferation.

[0120] [Figure 30A] This is a flow cytometry plot of marker expression in TCR-manipulated iPSC-derived cells (clone TCR-174) after proliferation.

[0121] [Figure 30B] Figure 30A shows a flow cytometry plot of marker expression in post-gated TCR-manipulated iPSC-derived cells (clone TCR-1724) that are gated on CD3+ cells.

[0122] [Figure 30C] This graph quantifies marker expression in TCR-manipulated iPSC-derived cells (clone TCR-174) after proliferation.

[0123] [Figure 31] This graph quantifies the in vitro cytotoxicity of TCR-modified iPSC-derived cells (TCR-172 and TCR-174) and unmodified iPSC-derived cells (NTX4A1) at various effector:target (E:T) ratios.

[0124] [Figure 32A] This graph quantifies the proportions of DN, DP, CD4 ISP, and CD8 SP cell populations in iPSC-derived cells cultured with 3D ETN and various concentrations of gamma secretase inhibitors (GSI) administered once during the culture period ("1 GSI dose").

[0125] [Figure 32B] This graph quantifies the proportions of DN, DP, CD4 ISP, and CD8 SP cell populations in iPSC-derived cells cultured with 3D ETN and gamma-secretase inhibitors (GSIs) at various concentrations three times during the culture period ("3 GSI doses").

[0126] [Figure 33] This graph quantifies the percentage of CD4+CD8+DP cells (top), cell viability (bottom left), and proliferation rate (bottom right) of iPSC-derived cells cultured with 3D ETN and various concentrations of gamma-secretase inhibitors.

[0127] [Figure 34] These are flow cytometry plots showing marker expression in iPSC-derived cells cultured with 3D ETN and various concentrations of gamma-secretase inhibitors (GSIs): 0.1x bead dose, no GSI (top); 2x bead dose, 1 dose of 1 μM GSI (middle); 2x bead dose, 3 doses of 0.3 μM GSI (bottom).

[0128] [Figure 35] This graph quantifies the percentage of CD4+CD8a+DP cells after culturing two cell lines using 2D or 3D ETN and various concentrations of GSI: NTX4B3 (top) and NTX4A1 (bottom).

[0129] [Figure 36] This graph quantifies the proportions of the above-mentioned CD4 ISP, DN, DP, and CD8a SP cell populations after 17 days of culture in two cell lines, NTX4B3 ("4B3") and NTX4A1 ("4A1"), using 3D ETN or 2D ETN and GSI at various concentrations and doses.

[0130] [Figure 37A] This graph quantifies the cell viability over time for three cell lines cultured using 3D ETN.

[0131] [Figure 37B] Figure 37A is a graph that quantifies the cumulative growth rate of the three labeled cell lines.

[0132] [Figure 38] This is a flow cytometry plot of marker expression in TCR-modified cells on day 28.

[0133] [Figure 39A] This graph quantifies the cell viability of TCR-modified cells under various conditions on day 0 and day 7 after transition to SP cells.

[0134] [Figure 39B] This graph quantifies the proliferation rate of TCR-modified cells under various conditions on day 0 and day 7 after transition to SP cells.

[0135] [Figure 40A] This is a flow cytometry plot of marker expression in TCR-modified cells after culture with ImmunoCult® CD3 / CD28 / CD2 stimulation ("1, IC3 / 28 / 2") or ImmunoCult® CD3 / CD28 stimulation ("3, CD3 / CD28").

[0136] [Figure 40B] These are flow cytometry plots of marker expression in TCR-modified cells after culturing with MAGE-A4 tetramer and soluble anti-CD28 ("2, MAGE-A4 Tet.+sol.CD28"), MAGE-A4 tetramer and soluble anti-41BB ("5, MAGE-A4 Tet.+sol.41BB"), or MAGE-A4 tetramer alone ("4, MAGE-A4 Tet.").

[0137] [Figure 40C] These are flow cytometry plots of marker expression in TCR-modified cells after culturing with HIV-gag tetramer and soluble anti-CD28 ("6, HIV gag Tet.+sol.CD28"), HIV-gag tetramer and soluble anti-41BB ("7, HIV gag Tet.+sol.41BB"), or HIV-gag tetramer alone ("8, HIV gag Tet.").

[0138] [Figure 41]The expression of memory markers in TCR-modified cells after culture under various conditions was quantified using graphs (1: ImmunoCult® activator, anti-CD3 / CD28 / CD2; 2: MAGE-A4 tetramer and soluble anti-CD28; 3: ImmunoCult® activator, anti-CD3 / CD28; 4: MAGE-A4 tetramer alone; 5: MAGE-A4 tetramer and soluble anti-41BB; 6: HIV gag tetramer and soluble anti-CD28; 7: HIV gag tetramer and soluble anti-41BB; 8: HIV gag tetramer only), and evaluated by flow cytometry.

[0139] [Figure 42] The expression of co-stimulatory ("Co-stimulation") markers in TCR-modified cells after culture under various conditions was quantified using graphs (1: ImmunoCult® activator, anti-CD3 / CD28 / CD2; 2: MAGE-A4 tetramer and soluble anti-CD28; 3: ImmunoCult® activator, anti-CD3 / CD28; 4: MAGE-A4 tetramer alone; 5: MAGE-A4 tetramer and soluble anti-41BB; 6: HIV gag tetramer and soluble anti-CD28; 7: HIV gag tetramer and soluble anti-41BB; 8: HIV gag tetramer only), and evaluated by flow cytometry.

[0140] [Figure 43A] Graphs quantifying the cell viability of TCR-modified cells after a single proliferation cycle under various conditions (1: ImmunoCult® activator, anti-CD3 / CD28 / CD2; 2: MAGE-A4 tetramer and soluble anti-CD28; 3: ImmunoCult® activator, anti-CD3 / CD28; 4: MAGE-A4 tetramer alone; 5: MAGE-A4 tetramer and soluble anti-41BB; 6: HIV gag tetramer and soluble anti-CD28; 7: HIV gag tetramer and soluble anti-41BB; 8: HIV gag tetramer only).

[0141] [Figure 43B]Graphs quantifying the proliferation rate of TCR-modified cells after a single proliferation cycle under various conditions (1: ImmunoCult® activator, anti-CD3 / CD28 / CD2; 2: MAGE-A4 tetramer and soluble anti-CD28; 3: ImmunoCult® activator, anti-CD3 / CD28; 4: MAGE-A4 tetramer alone; 5: MAGE-A4 tetramer and soluble anti-41BB; 6: HIV gag tetramer and soluble anti-CD28; 7: HIV gag tetramer and soluble anti-41BB; 8: HIV gag tetramer only).

[0142] [Figure 44A] This is a flow cytometry plot of marker expression in a TCR-modified cell line ("Clone 172") after culture under proliferation conditions induced by ImmunoCult® activator, CD3 / CD28 / CD2 stimulation ("1, IC3 / 28 / 2"), and either the anti-CD3 antibody OKT3, the fibronectin fragment RetroNectin®, and either soluble 41BB antibody (Condition B) or soluble ICOS ligand (Condition C).

[0143] [Figure 44B] This is a flow cytometry plot of marker expression in a TCR-modified cell line ("Clone 174") after culture using ImmunoCult® activator, CD3 / CD28 / CD2 stimulation ("1, IC3 / 28 / 2") and the anti-CD3 antibody OKT3, fibronectin fragment RetroNectin®, and soluble CD28 antibody (Condition A), soluble 41BB antibody (Condition B), or soluble ICOS ligand (Condition C), or T Cell TransAct® CD3 / CD28 stimulation (Condition D).

[0144] [Figure 44C]This is a flow cytometry plot of marker expression in a TCR-modified cell line ("Clone 172") cultured in MAGE-A4 tetramer and soluble anti-CD28 ("2, MAGE-A4 Tet.+sol.CD28") and then grown with anti-CD3 antibody OKT3, fibronectin fragment RetroNectin®, and soluble 41BB antibody (condition B) or soluble ICOS ligand (condition C).

[0145] [Figure 44D] This is a flow cytometry plot of marker expression in a TCR-modified cell line ("Clone 174") after culturing with MAGE-A4 tetramer and soluble anti-CD28 ("2, MAGE-A4 Tet.+sol.CD28"), followed by growth with anti-CD3 antibody OKT3, fibronectin fragment RetroNectin®, and soluble CD28 antibody (Condition A), soluble 41BB antibody (Condition B), or soluble ICOS ligand (Condition C), or after growth with T Cell TransAct™ CD3 / CD28 stimulation (Condition D).

[0146] [Figure 45A] This graph quantifies the expression of memory markers in TCR-modified cell lines ("Clone 172") after culture under various conditions, as evaluated by flow cytometry.

[0147] [Figure 45B] This graph quantifies the expression of memory markers in TCR-modified cell lines ("Clone 174") after culture under various conditions, as evaluated by flow cytometry.

[0148] [Figure 46A] This is a schematic diagram illustrating the generation and characterization of cloned iPSC strains possessing the MAGE-A4 TCR integrated into the TRAC gene locus.

[0149] [Figure 46B]This graph quantifies TCR expression at the end of clonal production for four selected biallele iPSC clones. The vector copy number (VCN) values ​​above the bar graph indicate the genetic characteristics of copy number incorporation at the TRAC locus.

[0150] [Figure 46C] This is a scatter plot of TCR expression for unedited (left) and selected clones (174, right).

[0151] [Figure 46D] This is a micrograph of the G-band karyotype analysis of clone 174.

[0152] [Figure 46E] This table summarizes the results of the iCS-digital™ assay (Stem Genomics).

[0153] [Figure 47A] This is a schematic diagram of in vitro T cell differentiation divided into four stages. HPC stands for hematopoietic progenitor cell, Pro-T cell stands for progenitor T cell, DP stands for double-positive cell, and SP stands for single-positive cell.

[0154] [Figure 47B] This is a flow cytometry plot showing marker expression in TCR-modified cells on day 0 of differentiation (stage 1 in the schematic diagram in Figure 47A).

[0155] [Figure 47C] This is a flow cytometry plot showing marker expression in TCR-modified cells on day 10 of differentiation (stage 2 in the schematic diagram in Figure 47A).

[0156] [Figure 47D] This is a flow cytometry plot showing marker expression in TCR-modified cells on day 28 of differentiation (stage 3 in the schematic diagram in Figure 47A).

[0157] [Figure 47E] This is a flow cytometry plot showing marker expression in TCR-modified cells subgated with CD4+CD8α+ cells at day 28 of differentiation (stage 3 in the schematic diagram in Figure 47A).

[0158] [Figure 47F] This is a flow cytometry plot showing marker expression in TCR-modified cells (top) and TCR-modified cells sub-gated with CD4+CD8α+ cells (bottom) at day 35 of differentiation (stage 4 in the schematic diagram of Figure 47A).

[0159] [Figure 47G] This is a flow cytometry plot showing marker expression in TCR-modified cells subgated with CD4+CD8α+ cells at day 35 of differentiation (stage 4 in the schematic diagram of Figure 47A).

[0160] [Figure 47H] This heatmap quantifies the expression of co-stimulatory markers in TCR-modified cells sub-gated with CD4+CD8α+ cells at day 35 of differentiation (stage 4 in the schematic diagram of Figure 47A).

[0161] [Figure 48] This graph quantifies MAGE-A4 TCR expression during T cell differentiation. Pro-T represents progenitor T cells, DP represents double-positive cells, and SP represents single-positive cells.

[0162] [Figure 49A] This is a schematic diagram of a biphasic iPSC-T proliferation culture protocol consisting of an activation phase and a maintenance phase. OKT3 is the anti-CD3 antibody OKT3, and pb is plate-bound.

[0163] [Figure 49B] This graph quantifies cell viability (left axis) and proliferation rate (right axis) in a 7-day proliferation culture protocol.

[0164] [Figure 49C]This is a flow cytometry plot showing marker expression in TCR+iPSC-derived cells at the end of a 7-day proliferation culture protocol.

[0165] [Figure 49D] This is a flow cytometry plot showing marker expression in TCR+ iPSC-derived cells sub-gated with CD8A+ cells at the end of a 7-day proliferation culture protocol.

[0166] [Figure 50A] This is a schematic diagram of an in vitro serial restimulation assay.

[0167] [Figure 50B] This graph quantifies the expression of inhibitory receptors (IRs) PD-1, TIGIT, LAG3, TIM3, and CD39 in iPSC-derived TCR+CD8+ or primary TCR-transduced CD8+ effector cells after in vitro exposure to target cells, at effector-to-target (E:T) ratios of 2:1, 1:1, and 0.5:1 (left panel), or at baseline, or after one or three antigen exposures (right panel).

[0168] [Figure 50C] This graph quantifies tumor cytotoxicity in TCR-transduced primary CD8+ cells and iPSC-derived TCR+CD8+ cells at various E:T ratios. AUC is the area under the curve, and ET50 is the corresponding E:T ratio to reach a relative AUC of 50%.

[0169] [Figure 50D] This graph quantifies tumor cytotoxicity in transduced primary CD8+ cells and iPSC-derived TCR+CD8+ cells over four antigen exposures at a 2:1 E:T ratio.

[0170] [Figure 50E]This graph quantifies the time course of tumor cytotoxicity in primary CD8+ cells and iPSC-derived TCR+CD8+ cells transduced with TCRs, co-cultured with antigen-negative or antigen-positive tumor target cells.

[0171] [Figure 51A] The following are homogeneous manifold approximation projection (UMAP) plots based on CITEseq data (mRNA + surface protein at single-cell resolution) for various cell populations. PBMC-CD8T represents peripheral blood-derived CD8+ T cells, PBMC-CD4-T represents peripheral blood-derived CD4+ T cells, PBMC-T activation represents activated peripheral blood-derived T cells, PBMC-NK represents peripheral blood-derived natural killer cells, and iPS-TCR represents iPSC-derived TCR+CD8+ cells.

[0172] [Figure 51B] This graph quantifies the proportion of iPSC-derived TCR+CD8+ cells (iPS-TCRs) and activated primary T cells (PBMC-T activated) corresponding to the RNA expression profiles of proliferating double-negative cells (DN(P)), CD8+ T cells (CD8+T), NK-T cells (NKT), and regulatory T cell (Treg) cells, annotated from the reference dataset (Park et al., 2020).

[0173] [Figure 51C] Figure 51A is a bubble plot quantifying the RNA expression of the gene shown on the vertical axis in a population of cells. [Modes for carrying out the invention]

[0174] Detailed description of the invention Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0175] Broadly speaking, this disclosure provides methods for generating populations of CD4-CD8+ and CD4+CD8+ cells from stem cells / progenitor cells, populations of CD4-CD8+ and CD4+CD8+ cells generated by the methods disclosed herein, pharmaceutical compositions comprising populations of CD4-CD8+ cells generated by the methods disclosed herein, and the use of populations of CD4-CD8+ cells in the manufacture of pharmaceuticals for treating diseases or conditions. To date, no methods have been reported for controlling the in vitro emergence of mature T cell lineage populations through the temporal regulation of Notch signaling and T cell activation. There have also been no reports of the unique phenotypes of cells emerging from such processes.

[0176] definition As used herein, the term “stem cell” refers to a cell that can differentiate into more specialized cells and has the ability to regenerate itself. Stem cells include pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), as well as multipotent stem cells, such as mobilized peripheral blood-derived CD34+ stem cells, umbilical cord blood stem cells, and adult stem cells, which are found in various tissues. Methods for obtaining, inducing, or producing stem cells are known in the art.

[0177] As used herein, the term “progenitor cell” refers to a cell that can differentiate into one or more types of cells, but typically has limited self-renewal capacity. Progenitor cells are derivatives of stem cells, and their differentiation potential is more limited compared to their corresponding source stem cells. For example, hematopoietic stem cells (HSCs), which are present in adult bone marrow, peripheral blood (in fewer numbers), and umbilical cord blood, have the ability to produce all other blood cells. Hematopoietic progenitor cells (HPCs) are pluripotent or lineage-restricted cell lineages derived from HSCs that have the ability to produce more limited or specific types of blood cells. Hematopoietic stem cells / progenitor cells (HSPCs) typically exist as heterogeneous populations in vivo and have use as heterogeneous populations as described herein. HPCs and HSPCs may be characterized by the expression of one or more CD34, CD43, CD31, and CD45.

[0178] As used herein, the terms “progenitor T cell” and “proT cell” refer to cells derived from pluripotent stem cells or CD34+ hematopoietic stem cells and / or progenitor cells, expressing at least CD7+, and having the ability to differentiate into one or more types of immature and mature T cells. Examples of progenitor T cells include, but are not limited to, CD7+ cells, CD7+CD5+ cells, CD7+CD5+CD34+ cells, CD7+CD5+CD45RA+ cells, and / or CD7+CD5+CD1a+ cells.

[0179] As used herein, “immature T cells” or “mature T cells” are T lineage cells derived from progenitor T cells. T cell differentiation can be characterized by the progressive expression of cell surface receptors, particularly CD4 and CD8. In vivo, T lineage cells progress from progenitor T cells to the CD4-CD8- (double negative, DN), CD4+CD8- (CD4 immature single positive, CD4ISP), early CD4+CD8+ (double positive, DP), late DP, CD4-CD8+ (CD8 single positive, CD8SP), and CD4 single positive (CD4SP) stages. Late DP is characterized by the presence of CD4+ / CD8A+ / CD8B+ / CD3+ and TCRαβ+. The fate of TCRγδ is not opened in the late DP stage, cell size decreases, and the cell is metabolically quiescent. In CD8 SP, CD8 may be expressed as a heterodimer of CD8α and CD8β, resulting in CD8αβ+ cells, or as a homodimer of CD8αα, resulting in CD8αα+ cells. CD4-CD8+ cells may also be characterized by the cell surface expression of CD3 and either TCRγδ (γδ T cells) or TCRαβ (αβ T cells).

[0180] As used herein, “serum-free medium” refers to a cell culture medium that lacks animal serum. Serum-free medium may contain certain known serum components isolated from animals (including human animals), such as bovine serum albumin (BSA).

[0181] As used herein, “Notch signaling ligand” refers to any ligand that can interact with the Notch protein receptor for the restriction and regulation of T cell lineage differentiation. Examples of Notch signaling ligands include delta-like 4 (DL4), delta-like 1 (DL1), delta-like 3 (DL3), Jagged1, and Jagged2.

[0182] As used herein, Notch signaling ligands, e.g., “delta-like 4” and “DL4,” refer to the proteins encoded by the DLL4 gene in humans. DL4 is a member of the Notch signaling pathway and is also referred to in the art as “delta-like ligand 4” and “DLL4.” In this specification, references to DL4 are not limited to the entire DL4 protein but include at least the signaling peptide portion of DL4. For example, a commercially available product (Sino Biologicals) in which the extracellular domain (Met 1-Pro 524) of human DL4 (full-length DL4 accession number NP_061947.1, SEQ ID NO: 1) is fused at the C-terminus to the Fc region of human IgG1 is a DL4 protein suitable for use in the methods described herein.

[0183] As used herein, Notch signaling ligands also include variants of known Notch signaling ligands, such as DL4. A variant Notch signaling ligand refers to a protein molecule whose amino acid sequence differs from that of the wild type by one or more additions, deletions, and / or substitutions, and which retains the desired Notch signaling activity of wild-type DL4. Variants such as polypeptides, oligopeptides, peptides, and proteins that have amino acid sequence identity with a given polypeptide, oligopeptide, peptide, or protein are also included in the definition. The percentage of identity may be at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with a given polypeptide, oligopeptide, peptide, or protein, for example, over a certain length, for example, over the entire length of the polypeptide.

[0184] As used herein, “vascular cell adhesion molecule 1” and “VCAM-1” refer to the protein encoded by the VCAM1 gene in humans. VCAM-1 is a cell surface sialycoglycoprotein, a type I membrane protein and a member of the lg superfamily. VCAM-1 is also referred to in the art as “vascular cell adhesion protein 1 and differentiation cluster 106” (CD106). In this specification, references to VCAM-1 are not limited to the entire VCAM-1 protein, but include at least the signaling peptide portion of VCAM-1 (QIDSPL (SEQ ID NO: 2) or TQIDSPLN (SEQ ID NO: 3)). For example, a commercially available mouse VCAM-1-Fc chimeric protein (R&D) containing the (Phe25-Glu698) region of mouse VCAM-1 (full-length mouse VCAM-1 accession number CAA47989; SEQ ID NO: 4) fused with the Fc region of human IgG1 is a VCAM-1 protein suitable for use herein. The use of at least a portion of human VCAM-1 (full-length human VCAM-1 accession numbers P19320, NP001069, EAW72950; SEQ ID NO: 5) may also be suitable for use in the manner provided herein. In this specification, references to VCAM-1 also include variants that differ in amino acid sequence from the wild-type VCAM-1 by one or more additions, deletions, and / or substitutions, while retaining the desired activity of wild-type VCAM-1. Variants such as polypeptides, oligopeptides, peptides, and proteins having amino acid sequence identity with a given polypeptide, oligopeptide, peptide, or protein are also included in the definition. The identity percentage can be at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with a given polypeptide, oligopeptide, peptide, or protein, for example, over a specific length, or over the entire length of the polypeptide. VCAM-1 has been shown to synergistically increase Notch signaling in combination with DL4 (e.g., Shukla et al., 2017).

[0185] As used herein, “integrin ligand” refers to a peptide or protein capable of binding to an integrin, such as the VLA-1-VLA-5 family of integrins. Examples of integrin ligands are known in the art and include, for example, fibronectin, the fibronectin fragment retronectin®, VCAM, RGD peptide, laminins including laminin 211, laminin 511 and laminin 332, cell adhesion molecules (ICAM including ICAM-1), and inducible costimulator ligands (ICOS-L).

[0186] As used herein, “two-dimensionally manipulated thymic niche (2D ETN)” refers to a two-dimensional substrate immobilized with a Notch signaling ligand, e.g., DL4, and optionally VCAM-1. The two-dimensional (2D) substrate may include, for example, a tissue culture plate. Methods for immobilizing Notch signaling ligands on 2D substrates are known in the art and are described, for example, in Shukla et al., 2017.

[0187] As used herein, “three-dimensionally manipulated thymic niche (3D ETN)” or “ETN beads” refers to a three-dimensional substrate immobilized with Notch signaling ligands, such as DL4 and optionally VCAM-1.

[0188] As used herein, “antigen beads,” “antigen particles,” “antigen-coated beads,” or “antigen-coated particles” refer to antigens immobilized on a substrate, for example, on a three-dimensional substrate such as particles or beads. Antigens may be immobilized on a substrate via covalent or non-covalent interactions, affinity-based interactions, or other suitable forms of interaction. For example, the antigen may be a CD19 antigen (“CD19-coated beads,” “CD19 beads”).

[0189] Three-dimensional (3D) substrates may include, for example, micron-sized particles (or beads) having or not having a magnetic core, coated with one or more complete proteins, protein domains (e.g., extracellular, intracellular, or other domains), peptides, or protein fragments. To produce protein-coated particles, several approaches may be used individually or in combination, e.g., physicoadsorption driven by protein affinity to the particle material, chemical conjugation by reaction with amine, carboxyl, thiol, epoxy, or azide reactive groups, in particular, or chemical conjugation by coating with suitable ligands to capture the protein of interest by affinity. Examples of affinity tags include, but are not limited to, polyhistidine (His), Fc, biotin, halo, aldehyde, Snap, Spy-Catcher, and VIPER. The particles or beads may consist of, for example, polystyrene, iron oxide, polystyrene with magnetizable iron oxide (magnetic polystyrene), gold, or other suitable materials known in the art. ETNs and antigen beads can be used to culture cells on tissue culture plates, flasks, or other containers used for culturing cells.

[0190] As used herein, “immobilized” or “surface-bound” means that a ligand, antigen, peptide, or protein, such as a Notch signaling ligand or integrin ligand, is bound to a substrate via covalent or non-covalent interaction, affinity-based interaction, or other appropriate form of interaction.

[0191] As used herein, “T cell activator” refers to a reagent that activates T cells or T-lineage cells. Methods and reagents for T cell activation are known in the art and include, for example, antibody-based stimulation of intracellular CD3, CD3 and CD28, or CD3, CD28 and CD2, or stimulation of T cell receptors or chimeric antigen receptors ("CAR activators") via peptide major histocompatibility complex (pMHC) tetramers, antibodies, or antigen presentation.

[0192] As used herein, "CD3 stimulant" refers to a reagent that stimulates cells by binding or activating CD3 in the cells. Methods and reagents for CD3 stimulation are known in the art and include, for example, antibody-based stimulation of CD3 intracellularly, CD3 and CD28, or CD3, CD28 and CD2.

[0193] As used herein, "Notch signaling inhibitor" refers to a reagent that inhibits Notch signaling in the cells. A Notch signaling inhibitor can be a small molecule inhibitor. One type of Notch signaling inhibitor is a gamma-secretase inhibitor (γ-secretase inhibitor, GSI) such as N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Further reagents for inhibiting Notch signaling include antibodies against the Notch1 receptor and soluble (i.e., non-immobilized) DL4.

[0194] As used herein, a "concentrated" population of cells refers to a population of cells containing one or more cell phenotypes (e.g., CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), CD4-CD8- (DN)) that shows a higher absolute number or a ratio of a higher cell phenotype (e.g., CD4-CD8+ (CD8SP)) compared to other cell phenotypes, and at least 25% of the population of cells consists of a single cell phenotype.

[0195] As used herein, the term "subject" refers to a vertebrate, preferably a mammal (e.g., a non-human mammal), more preferably a primate, and even more preferably a human. Mammals include, but are not limited to, humans, non-human primates, livestock, sport animals, pets, etc.

[0196] As used herein, the terms "treatment", "treating", or "treatment" refer to an approach for obtaining a beneficial or desired clinical outcome. For the purposes of the present disclosure, beneficial or desired clinical outcomes include, but are not limited to, an increase in immune response, an increase in T cell response, a decrease in the degree of damage from a disease, condition or disorder, a decrease in the duration of a disease, condition or disorder, and / or one or more of a decrease in the number, degree or duration of symptoms associated with a disease, condition or disorder. The term includes administration of a compound, reagent, drug or pharmaceutical composition of the present disclosure to prevent or delay the onset of one or more symptoms, complications or biochemical markers of a disease or condition, reduction or improvement of one or more symptoms; shortening or reduction of the duration of symptoms; or preventing or inhibiting further onset of a disease, condition or disorder. Treatment can be prophylactic (to prevent or delay the onset of a disease, condition or disorder, or to prevent the manifestation of its clinical or subclinical symptoms) or therapeutic suppression or alleviation after the manifestation of a disease, condition or disorder. Beneficial or desired clinical outcomes can be an increase or decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (as appropriate), compared to a suitable control, such as a subject not receiving treatment.

[0197] As used herein, the terms "administer" or "administration" refer to the placement of a reagent, drug, compound, or pharmaceutical composition disclosed herein into a subject by a method or route that results in at least partial delivery of the composition to a desired site. The compounds and pharmaceutical compositions disclosed herein can be administered by any suitable route that provides an effective treatment in a subject. Routes of administration of the compounds and pharmaceutical compositions disclosed herein include, but are not limited to, intravenous or intraperitoneal routes of administration, or combinations thereof.

[0198] The terms “effective dose” or “therapeutic effective dose,” for example, the effective dose or therapeutic effective dose of a T-cell lineage population as used herein, are amounts sufficient to produce any one or more beneficial or desired outcomes. In more specific embodiments, an effective dose may alleviate or improve one or more symptoms of a disease; shorten the duration of time one or more symptoms of a disease are present in a subject; or increase the survival rate of a subject with the disease. In the case of prophylactic use, beneficial or desired outcomes may include eliminating or reducing the risk of disease, reducing its severity, or delaying the onset of disease, including the biochemical and / or histological manifestations of infection, its complications, and intermediate pathological phenotypes that appear during the development of the disease. In the case of therapeutic use, beneficial or desired outcomes may include clinical outcomes such as alleviation of one or more symptoms of a disease; a reduction in the dose or duration of administration of other drugs required to treat the disease; enhancement of the effect and / or reduction of toxicity of another reagent; delaying the progression of disease in a subject; shortening the duration of time one or more symptoms of a disease are present in a subject; and / or increasing the overall survival rate of a subject with the disease. An effective dose may be administered in one or more doses, in a series of treatments, or in a single dose.

[0199] For the purposes of this disclosure, an effective dose of a population of cells or a pharmaceutical composition is an amount sufficient to achieve prophylactic treatment or therapeutic action, either directly or indirectly. As understood in a clinical context, an effective dose of a compound or pharmaceutical composition may or may not be achieved in combination with other reagents, drugs, compounds, or pharmaceutical compositions. Thus, “effective dose” may be considered in relation to the administration of one or more therapeutic agents, and a single agent may be considered given in an effective dose if, when combined with one or more other reagents, the desired outcome can or can be achieved. The amount may vary from subject to subject and may depend on one or more factors, such as the subject’s sex, age, weight, health history, and / or the underlying cause of the disease, condition, or disorder being prevented, inhibited, and / or treated.

[0200] As used herein, the term “pharmaceutically acceptable carrier, diluent or excipient” includes any substance that, when combined with an active ingredient, enables that ingredient to retain its biological activity and does not react with the target immune system. Examples include, but are not limited to, any standard pharmaceutical carriers such as phosphate-buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or ordinary (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (e.g., Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0201] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0202] The phrase "and / or" should be understood to mean "either or both" of the elements thus combined, that is, elements that exist associatively in some cases and disjunctively in others. Therefore, as a non-restrictive example, a reference to "A and / or B," when used in combination with open-ended language such as "equipped with," may in one embodiment refer to A only (optionally including elements other than B); in another embodiment refer to B only (optionally including elements other than A); in yet another embodiment refer to both A and B (optionally including other elements), and so on.

[0203] Where used herein, the phrase “one or more” in relation to a list of elements means at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase “one or more” refers, whether or not they are related to the specifically identified elements. Thus, as an unrestricted example, “one or more of A and B” (or equivalently, “one or more of A or B,” or equivalently, “one or more of A and / or B”) may, in one embodiment, refer to at least one, possibly two or more, A, in which B is absent (and may contain elements other than B). In another embodiment, may include at least one, possibly two or more, B, in which A is absent (and may contain elements other than A). In yet another embodiment, there may be at least one A, which may include two or more, and at least one B, which may include two or more (and optionally other elements); etc.

[0204] When the term "approximately" is used with a numerical range, it modifies that range by extending the upper and lower boundaries of those numbers. Generally, the term "approximately" is used herein to modify numbers above and below a given value with a variance of 20%, 10%, 5%, or 1%. In certain embodiments, the term "approximately" is used to modify numbers above and below a given value with a 10% variance. In certain embodiments, the term "approximately" is used to modify numbers above and below a given value with a 5% variance. In certain embodiments, the term "approximately" is used to modify numbers above and below a given value with a 1% variance.

[0205] Where ranges of values ​​are listed herein, it is intended to include each value and subrange within that range. For example, “1–5 mL” is intended to include 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 1–2 mL, 1–3 mL, 1–4 mL, 1–5 mL, 2–3 mL, 2–4 mL, 2–5 mL, 3–4 mL, 3–5 mL, and 4–5 mL.

[0206] It will be further understood that the terms “comprises,” “comprising,” “includes,” and / or “including,” as used herein, specify the presence of the described features, integers, processes, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof.

[0207] As used herein, the term "consisting of" and its derivatives are intended to be closed terms that specify the presence of the described features, integers, processes, operations, elements, and / or components, and exclude the presence or addition of one or more other features, integers, processes, operations, elements, and / or components. General technology

[0208] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0209] Unless otherwise stated, the implementation of this disclosure will utilize prior arts in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art of those skilled in the art. Such techniques are described in literature such as: Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture (RIFreshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PER Oberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCales, eds., 1987); Current Protocols in Molecular Biology (FMAusubel eta / ., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEThis is described in Coligan et al., eds., 1991; Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley and Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); and lmmunobiology (CA Janeway and P. Travers, 1997).

[0210] Population of hematopoietic stem cells / progenitor cells In a broad sense, the in vitro methods for generating populations of CD4+CD8+ and CD4-CD8+ cells provided herein include culturing hematopoietic stem cells / progenitor cells in the presence of Notch signaling ligands under conditions and for a duration suitable for differentiation into T cell lineage populations.

[0211] Hematopoietic stem cells / progenitor cells (HSPCs) typically exist as heterogeneous populations in vivo and have heterogeneous uses as described herein. HPCs and HSPCs may be characterized by the expression of one or more CD34, CD43, CD31, and CD45.

[0212] In one embodiment, HSPCs may be obtained from umbilical cord blood, peripheral blood, or bone marrow, or may be induced in vitro from pluripotent stem cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or other intermediate stem cells. In a preferred embodiment, the stem cells and / or progenitor cells are human cells. In one embodiment, the stem cells are recruited peripheral blood-derived CD34+ cells. In a preferred embodiment, HSPCs are induced in vitro from iPSCs. Methods for generating HSPCs from iPSCs are known in the art, for example, differentiation under appropriate culture conditions (e.g., Trotman-Grant et al., 2021).

[0213] Appropriate techniques for analyzing cell surface markers are known to those skilled in the art, and may include, for example, flow cytometry or immunocytochemistry as used herein. Cell number and cell viability can be analyzed by techniques well known to those skilled in the art, for example, the use of automated cell counters disclosed herein.

[0214] Cell culture system Cells may be cultured in cell culture systems of the type known in the art, such as bioreactors including cell culture plates, culture dishes, and agitated tank reactors (STRs), agitated bag bioreactors, and other suitable cell culture formats. Cell culture may be carried out under static conditions, dynamic or agitated conditions, or a combination of static and dynamic conditions. The bioreactor may be any type known in the art, and any type of processing / culture conditions and methods may be used, such as batch processes, fed-batch processes, and perfusion culture methods and conditions.

[0215] Notch Ligand Substrate In one embodiment, cells are cultured in a two-dimensional culture system utilizing a suitable 2D substrate, which may include, for example, a standard culture plate coated with a Notch signaling ligand, such as DL4. The culture plate may be coated with VCAM-1.

[0216] In one embodiment, the cells are cultured in a three-dimensional culture system that utilizes a suitable 3D substrate, e.g., micron-sized particles (or beads) with or without a magnetic core, coated with one or more complete proteins, protein domains (e.g., extracellular, intracellular, or other domains), peptides, or protein fragments to activate Notch signaling.

[0217] In one example, a Notch signaling ligand, e.g., DL4, can be conjugated to polystyrene microbeads either alone or in combination with VCAM-1 as described in Trotman-Grant et al., 2021 and International Publication No. WO 2019 / 157597.

[0218] In another example, 3D ETN beads can be produced by affinity capturing DL4 and VCAM-1 carrying appropriate affinity tags on streptavidin or protein G-coated beads. The beads are diluted to 0.1% solids in Dulbecco's phosphate-buffered saline (DPBS) without Ca2+ or Mg2+ supplemented with 0.05% BSA and incubated with a protein solution (0.1× to 20× protein molar excess) with continuous stirring at room temperature for 60 minutes. At the end of the incubation period, the excess free protein is removed by magnetic separation, followed by buffer exchange. This procedure is repeated four more times, and then the 3D ETN beads are concentrated 10-fold for storage.

[0219] Quantification of protein immobilization can be performed according to methods known in the art, e.g., the bicinchoninic acid (BCA) assay, immunofluorescence assay, or other known detection methods.

[0220] T cell therapy T cells have a wide range of therapeutic applications. T cells can be modified by conventional gene editing approaches, such as nuclease editing or viral vector transduction, to express chimeric antigen receptors (CARs) and / or exogenous T cell receptors (TCRs) to generate engineered T cell therapies (Weber et al., 2020). T cells derived from progenitor cells, including pluripotent stem cells, can be genetically engineered at the pluripotent stem cell or progenitor cell stage. Engineered T cell therapies are applicable, for example, in oncology and autoimmune disorders. In oncology, engineered T cell therapies are applicable, for example, in hematological malignancies, such as B-cell lymphoma, B-cell acute lymphoblastic leukemia and other B-cell malignancies, multiple myeloma and other hematological malignancies, as well as in solid tumors, such as mesothelioma, adenocarcinoma, glioma and sarcoma (Weber et al., 2020). In autoimmune disorders, engineered T-cell therapy is applicable, for example, in type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and other autoimmune disorders or conditions (Weber et al., 2020).

[0221] Engineered T-cell therapies can target antigens known to be expressed on target cell types, including tumor cells or tumor tissue. Chimeric antigen receptors (CARs) can be designed to target surface antigens or polyvalent soluble antigens. The targeting external domain of a CAR may be a single-chain variable fragment (scFv), a single-domain antibody (single variable domain on a heavy chain, VHH), a nanoantibody, or another antigen-binding domain (Qu et al., 2022). CAR-T cell therapies can be directed to multiple antigens using various CAR designs or multiple CARs (Qu et al., 2022). Exemplary tumor antigens and corresponding cancer types for CAR-T cell therapy are listed in Table 1 below (Qu et al., 2022; Guha et al., 2022; Drougkas et al., 2023; Want et al., 2023). [Table 1] TIFF2026519876000003.tif254170TIFF2026519876000004.tif253170TIFF2026519876000005.tif254170TIFF2026519876000006.tif226170

[0222] TCR-T cell therapy targets antigens expressed as peptide-human leukocyte antigen (HLA) complexes on the surface of target cells. These targets may include tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) (Baulu et al., 2023). Exemplary tumor antigens and corresponding cancer types for TCR-T cell therapy are listed in Table 2 below (Baulu et al., 2023; Sun et al., 2021; Want et al., 2023). [Table 2] TIFF2026519876000008.tif13170

[0223] T cell therapies, including stem cell-derived T cell therapy, can be genetically modified to eliminate endogenous TCR expression, for example, by knocking out the T cell receptor α constant (TRAC) locus (TRAC- / -).

[0224] Populations of cells induced using the methods provided herein are intended to be included in pharmaceutical compositions.

[0225] Furthermore, a population of cells obtained using the methods described herein is considered usable to treat a disease or condition of interest. “To treat” means administering an effective amount of cells provided herein to a subject under conditions suitable for increasing the number of T cells of that subject, which may result in the prevention, inhibition, and / or therapeutic treatment of a medical condition. “Effective amount” means a therapeutic effective amount, e.g., a sufficient quantity of cells to achieve the intended purpose (e.g., treatment) at the time of administration to a subject. The amount may vary depending on the subject and may depend on one or more factors, e.g., the subject’s sex, age, weight, health history, and / or the underlying cause of the condition being prevented, inhibited, and / or treated.

[0226] For example, subjects suffering from oncological or autoimmune diseases, conditions, or disorders may benefit from administration of a population of CD4-CD8+ cells as described herein.

[0227] The pharmaceutical compositions provided herein may be administered to a subject to alleviate or improve one or more symptoms of a disease, to reduce the duration for which one or more symptoms of a disease are present in the subject; or to increase the survival rate of a subject having a disease.

[0228] The pharmaceutical compositions provided herein may be administered to subjects to treat a target cancer or autoimmune disorder.

[0229] The pharmaceutical compositions provided herein may be administered to a subject in an effective or therapeutically effective dose. Those skilled in the art can determine such dose based on factors such as the size of the subject (e.g., body weight), age and / or sex, the severity of the subject's symptoms, the specific composition or route of administration selected, and will also know how to select an appropriate route of administration and how to administer the compounds and compositions provided herein.

[0230] The dosage of the pharmaceutical compositions of this disclosure will vary depending on many factors, including the pharmacodynamic properties of the composition, the mode of administration, the recipient's age, health status and weight, the nature and severity of the symptoms, the frequency of treatment and, if any, the type of concurrent treatment, and the clearance rate of the compound in the target being treated. Those skilled in the art can determine an appropriate dosage based on the above factors. In some embodiments, the pharmaceutical composition is initially administered at an appropriate dosage that depends on the clinical response and is adjusted as necessary.

[0231] kit The present invention also provides kits comprising the pharmaceutical compositions described herein. The kits of the present invention comprise one or more containers comprising the pharmaceutical compositions described herein, and instructions for use according to any of the methods of the present invention described herein. Generally, these instructions include instructions for administering the pharmaceutical compositions for the therapeutic treatments described above. In some embodiments, kits for preparing single-dose units are provided.

[0232] Instructions for the use of a pharmaceutical composition generally include information regarding the dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions supplied within the kit of the present invention are typically written instructions on a label or accompanying document (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions delivered on a magnetic or optical storage disk) are also acceptable.

[0233] This disclosure will be described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Therefore, this disclosure should not be construed as being limited in any way to the following examples, but rather as encompassing all possible modifications that become apparent as a result of the teachings provided herein.

[0234] Example 1: Materials and Method Manufacturing of DL4 and VCAM-1 Recombinant DL4-Fc fusion proteins were purchased from Sino Biological or manufactured in-house using HEK-293T cells and purified using HiTrap® Protein G affinity columns (GE Healthcare) as previously described (e.g., Trotman-Grant et al., 2017). Recombinant VCAM-1-FC fusion proteins were purchased from R&D Systems. Table 3 shows DL4 and VCAM-1 suitable for the preparation of 2D and 3D ETNs, as further described below. [Table 3]

[0235] Preparation of 2D ETN Six-well, twelve-well, twenty-four-well, forty-eight-well, or ninety-six-well tissue culture plates were coated with Notch signaling ligands, DL4 and VCAM-1, overnight at 4°C or for three hours at 37°C. The tissue culture plates could be stored at 4°C for up to two weeks after coating. For coating, solutions of 20 μg / mL DL4 and 10 μg / mL VCAM-1 were prepared in Dulbecco's phosphate-buffered saline (DPBS) (- / -). As shown in Table 4, appropriate coating volumes of DL4 and VCAM-1 diluted in DPBS were added per well to the tissue culture plates. [Table 4]

[0236] The tissue culture plates were gently tapped to ensure that the coating solution containing Notch signaling ligands DL4 and VCAM-1 was evenly distributed across the well surface. After sealing the tissue culture plates with Parafilm®, they were stored overnight at 4°C or at 37°C for 3 hours. The tissue culture plates coated with Notch signaling ligands DL4 and VCAM-1 overnight at 4°C were placed in a 37°C incubator for 3 hours for equilibration, after which cells were seeded. After equilibration, or after coating the tissue culture plates with Notch signaling ligands DL4 and VCAM-1 at 37°C for 3 hours, the coating solution was aspirated from the wells. The wells were washed with DPBS(- / -) using the volumes shown in Table 5, and the cell suspension was immediately added to the tissue culture plates. [Table 5]

[0237] Preparation of 3D ETN The dosage of 3D ETN can be calculated according to the bead diameter and expressed as a dosage proportional to the surface area of ​​the culture plate or flask (e.g., as shown in Table 6), the number of beads per unit volume of culture, or the bead surface area per unit volume of culture. As calculated based on unit volume, the number of beads per 1 mL concentration does not change with different containers. The "1×" bead dosage represents complete coverage of the plate surface by one layer of beads. Since the beads are nearly spherical, the total surface area of ​​the beads is four times the surface area of ​​the plate or culture vessel. Table 6 shows the range of bead concentrations for polystyrene beads with a diameter of 3.05 μm. [Table 6]

[0238] The density of Notch signaling ligands on beads (e.g., the density of surface-bound DL4) is, for example, 100 molecules / square micrometer (100 molecules / μm). 2 ) ~3000 molecules / square micrometer (3000 molecules / μm2 ) may vary. Table 7 shows the Notch signaling ligand concentrations for various bead doses and the calculated Notch signaling ligand density for 3.05 μm polystyrene beads. [Table 7]

[0239] Table 8 provides the surface area of ​​Notch signaling ligands (e.g., DL4) per unit volume for a range of bead doses, and the Notch signaling ligand densities for both 3.05 μm and 3.29 μm diameter polystyrene beads. [Table 8]

[0240] 3D ETN may also contain surface-bound VCAM-1. VCAM-1 can be immobilized on 3D ETN in input molar ratios ranging from 1:6 to 10:1 for DL4:VCAM-1. In one embodiment, 3D ETN is prepared with an input molar ratio of 2.5:1 for DL4:VCAM-1. In one embodiment, the final density of surface-bound VCAM-1 on the beads is equivalent to the density of Notch ligand. For example, the surface area of ​​VCAM-1 per unit volume may be equivalent to the surface area of ​​Notch ligand per unit volume shown in Table 8 above.

[0241] Cells can be cultured at densities suitable for the culture scale and format. In microplate culture, for example, cells are cultured at 2.5 × 10⁶ densities. 5 ~2×10 6 It can be cultured at cells / mL. In STR culture, for example, 5 × 10⁶ 4 ~6×10 6 It can be cultured at a cell / mL concentration.

[0242] Preparation of antigen beads CD19 wild-type antigen (AcroBiosystems) or the engineered variant CD19.1 (Klesmith et al., 2019) was functionalized with polyhistidine (-His) or human IgG1a Fc (-Fc) tags. The CD19 antigen was then immobilized on carboxyl-functionalized magnetic polystyrene beads (4.5 μm) using carbodiimide crosslinking agent conjugations with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide HCl (NHC) and N-hydrosuccinimide (NHS). Based on the total moles of carboxyl groups available for protein conjugation on the beads, 0.5X moles, 1.0X moles, 2.0X moles, or 5.0X moles of CD19.1 protein were added to the reaction mixture, respectively. The total conjugation reaction volume was 200 μL. The maximum CD19.1 protein loading on the beads, representing 69.4% of the theoretical maximum coverage, was observed when the protein concentration was highest in the reaction mixture, i.e., at 5.0 × moles. The sequences of wild-type CD19 and CD19.1 are shown in Table 9 below. [Table 9]

[0243] Generation and characterization of cloned iPSC strains possessing chimeric antigen receptors integrated into the TRAC gene locus. Induced pluripotent stem cell lines were genetically modified to incorporate a chimeric antigen receptor (CAR) into the T cell receptor α constant (TRAC) locus. Chimeric antigen receptor expression was evaluated at the end of clonal production from one parental iPSC line (Figure 1A shows the percentage of chimeric antigen receptor-positive cells (bar graph) and median fluorescence intensity (MFI, dots).). Parental lines (unedited) and cloned iPSC lines expressing CD19-CAR by random LVV integration (LVV CAR) were used as controls. Genetic characterization of copy number integration at the TRAC locus was evaluated by digital droplet polymerase chain reaction (ddPCR) copy number variation (CNV) assay (Biorad) by amplifying the genomic TRAC locus-CAR transgene junction. Genomic stability analysis of selected clones was evaluated by G-band karyotype analysis performed by WiCell, demonstrating a normal karyotype (Figure 1B). The iCS digital assay also indicated the expected copy number in 24 genomic regions of recurrent iPSC abnormalities (data not shown).

[0244] Next, pluripotent lymphocyte-competent CD34+ hematopoietic progenitor cells were generated from engineered iPSCs. Prior to enrichment, high purity and high yield of CD34+ cells and CD34+CD43+HPCs were obtained from iPSC lines derived from two independent donors and gene-edited clone iPSC lines (Figure 2A, B).

[0245] Prior to differentiation, the manipulated iPSCs were positive for the undifferentiated cell markers OCT4, SOX2, SSEA4, and TRA-160. As described above, when a seed bank of iPSC clones with the chimeric antigen receptor incorporated into the TRAC locus was thawed and grown, 100% chimeric antigen receptor expression was detected in the iPSCs (Figure 3A, "Stage 1"). The iPSCs were differentiated into CD34+ cells in a scalably agitated suspension culture, and the enriched HPCs were also positive for CD43 and CD45 (Figure 3B, "Stage 2"). Expression of the erythrocyte precursor marker CD235a was negative, indicating a lack of early lineage constraint (Figure 3B). Chimeric antigen receptor expression decreased during CD34 differentiation (Figure 3B). Subsequently, the CD34+ HPCs were cultured with ETN beads displaying DLL4 and VCAM-1 under conditions identified as providing high levels of Notch signaling, and the ProT phenotype was evaluated by measuring CD5 and CD7. T cell markers (CD4 and CD8) were low at this stage, typical of ProT cells (Figure 3C, "Stage 3"). The gradual decrease in Notch signaling after the ProT stage led to the acquisition of the T cell lineage fate between weeks 4 and 5, primarily producing CD4+CD8+DP cells (Figure 3D, "Stage 4"). Chimeric antigen receptor expression gradually increased as cells became bound to the T cell lineage fate (Figure 3D). The involvement of the chimeric antigen receptor promoted the maturation of DP cells to CD8 SP T cells, as further described in Example 3. iPSC-derived CD8 SP cells were a mixture of CD8ααT cells and CD8αβT cells (Figure 3E). CD8SP cells had high levels of expression of stem cell memory markers CD45RA, CD62L, and CD95 (data not shown). Chimeric antigen receptor-stimulated cells expressed the T cell activation / NK cell marker CD56 but lacked standard NK markers such as NKP44 and NKP46. Chimeric antigen receptor expression was 100% after SP transition.

[0246] Example 2: Generation of CD4+CD8+ cells by regulation of Notch signaling In this example, 5 × 10⁶ iPSC-derived CD34+ cells were placed in LEM. 4 Seed cells at a density of cells / mL, and after 24 hours, ETN beads were added in a relative bead dose of 0.5 × (2.7 × 10⁻¹⁰). 7 The cells were added in beads / mL. Cells were harvested and 5 × 10 in LEM on day 10. 5 Reseed at a density of cells / ml, and after a 4-hour rest, administer a dose of 0.5 × ETN beads (2.7 × 10⁻¹⁰). 7 Beads / mL were added. Cells were collected again on day 15 and measured 0.1 × (5.4 × 10⁻⁶). 6 Beads / mL), 0.5 × (2.7 × 10 7 beads / mL) and 1×(5.4×10 7 In a matrix having a bead dose of beads / mL, 1 × 10 6 , 2×10 6 and 4×10 6 Cells were reseeded at a density of cells / mL. After 21 days, the cell phenotype was assessed by flow cytometry. The proportions of double-negative (DN) cells, CD4 immature single-positive (CD4 ISP) cells, double-positive (DP) cells, and CD8 single-positive (CD8 SP) cells varied as a function of both cell density and bead dose, with lower bead doses (0.1×) and higher cell densities (4×10⁻¹⁶). 6 Cells / mL yielded a larger proportion of CD4 ISP and DP, but higher bead doses (1×) and lower cell densities (1×10) resulted in a larger proportion of CD4 ISP and DP. 6 The number of cells / mL generated more CD8 SP cells (Figure 4A, 4C). The percentage of DP cells was inversely proportional to the bead-to-cell ratio (Figure 4B). Modulation of Notch signaling via ETN bead dose and cell density was shown to control the trajectory of cell fate.

[0247] In further experiments, the cells were differentiated as described above until day 15. On day 15, the cells were divided into 2 × 10⁻⁶ cells. 6 Cell density of cells / mL and ETN bead dose of 0.5× (2.7×10) 7 Beads / mL) compared to standard ETN beads (800 molecules / μm 2(Figure 5, top row) or beads with lower protein density (0.1x density or 100 molecules / μm 2 The cells were reseeded with either of the two types of beads (Figure 5, bottom row). Flow cytometry analysis revealed that the beads with lower protein density produced a higher proportion of CD4 ISP and DP cells, as well as fewer DN and CD8 SP cells, than the standard ETN beads (Figure 5). Total CD8β expression was similar between the two types of beads, but a lower proportion of CD8αα+ cells was observed in the low-density beads.

[0248] The cells were differentiated as described above until day 21. High bead dose (1×, 5.4×10 7 beads / mL) and low cell density (1 × 10⁻⁶) 6 CD56 expression varied with the bead-to-cell ratio, as the highest percentage of CD56+ cells was produced at lower bead doses (0.5× and 0.1× beads / mL) and higher cell densities (2×10⁶). 6 and 4×10 6 The cell dose (0.1 × beads / mL) resulted in fewer CD56+ cells (Figure 6A). On day 21, CD3 showed the opposite trend to CD56, with a lower bead dose (0.1 × beads / mL) and a higher cell density (4 × 10⁶). 6 The highest CD3 expression was obtained under the cell / mL condition; however, CD3 expression was very low under all conditions (<3%) (Figure 6B).

[0249] Cell differentiation was further extended beyond day 21 to day 23. Similar to day 21, the phenotype on day 23 was a function of both cell density and bead dose, with the lowest bead dose and highest cell density producing the most double-positive cells, and CD8 SP cells at high bead doses (1×, 5.4×10⁶). 7 beads / mL) and low cell density (1 × 10⁻⁶) 6The richest bead-to-cell ratio was observed at cell-to-mL (Figure 7A). Notably, more DP and less DN and CD4 ISP were observed on day 23 under lower bead-to-cell ratio conditions compared to day 21 (Figures 7A, 7C). The percentage of DP was inversely correlated with the bead-to-cell ratio, with a slightly steeper slope compared to day 21 (Figure 7B).

[0250] The cells were differentiated up to day 23 as shown in Figure 7. High bead dose (1×, 5.4×10) 7 beads / mL) and low cell density (1 × 10⁻⁶) 6 CD56 expression varied with the bead-to-cell ratio, as the highest percentage of CD56+ cells was produced at lower bead doses (0.5× and 0.1× beads / mL) and higher cell densities (2×10⁶). 6 and 4×10 6 The higher the bead dose (0.1x, 5.4x10⁶), the lower the CD56+ cells (Figure 8A). On day 23, CD3 showed the opposite trend to CD56, with lower bead doses (0.1x, 5.4x10⁶). 6 Beads / mL), high cell density (4 × 10 6 CD3 expression was highest under the cell / mL condition (Figure 8B). CD3 expression on day 23 was higher than that observed on day 21, but was still below 6% under all conditions (Figure 8B).

[0251] The differentiation conditions on days 21 and 23 and the resulting cell phenotypes are summarized in Table 10 below. [Table 10]

[0252] Example 3: Generation of CD8+ cells by temporal control of Notch signaling iPSC-derived CD34+ HPCs were differentiated into CD8SP T cells using a 5-stage protocol (protocol schematic, Figure 9). During Stage #1, CD34 HPCs were seeded on DL4 and VCAM-1 coated culture vessels ("2D") or mixed with 3D ETN beads at a 0.5x bead dose (2.7 × 10⁷ beads / mL, "3D") in precursor growth medium (SFEM II + LEM) and cultured for 10 days to generate CD34-CD7+CD5+ ProT cells.

[0253] At this point, the cells moved to stage #2, where ProT cells differentiated into early DP (CD4+CD8A+) over 7 days using two methods. In one method, cells (unmodified or TCR-modified cell lines) were divided into 2 × 10⁶ cells. 6 The cells were reseeded at an intermediate seeding density of cells / mL and mixed with 3D ETN beads at a 0.25-fold dose in precursor maturation medium (SFEM II + LMM). Alternatively, the cells (CAR modified) were seeded at 2 × 10⁶ 6 The cells were reseeded at an intermediate seeding density of cells / mL and mixed with 3D ETN in precursor maturation medium (SFEM II + LMM) at 0.1x protein density and 0.5x dose. At the end of Stage #2, the early DPs required further maturation because they did not have sufficient surface CD3 / TCR expression for stimulation.

[0254] During Stage #3, in one method, the initial DP was mixed with 3D ETN beads at a 0.25x bead dose (TCR modified cell line) to 6 × 10 6 Late DP (CD4+CD8A+CD8B+CD3+TCRαβ+ / -TCRγδ+ / -) was matured by reseeding at a higher seeding density of cells / mL. Alternatively, early DP was reseeded with 3D ETN beads at 0.1x protein density and 0.25x bead dose in SFEM II+LMM (unmodified or CAR-modified cell lines) and cultured for a further 8–13 days (timing may be cell line-dependent).

[0255] Stage #4 was initiated by enriching late DP via, for example, a CD8-positive selection kit (STEMCELL Technologies), followed by re-seeding in DP-SP medium containing IL-21, addition of an appropriate T cell activator, and culture for 7 days to induce conversion from DP to CD8SP. In one method, the T cell activator is an anti-CD3 / CD28 / CD2 or anti-CD3 / CD28 CD3-stimulating factor such as Immunocult®, combined with an integrin ligand such as retronectin®, fibronectin, laminin, or ICOS-L, which are fibronectin fragments. The CD3-stimulating factor may also be ICAM-1. In another method, for example in CAR-modified cell lines, the T cell activator is an antigen-coated bead, such as a CD19-coated bead.

[0256] Finally, the newly migrated CD8SP cells were moved to Stage #5, where they were activated and proliferated (for either unmodified or TCR-modified cell lines). The CD8SP cells were then placed in 5 × 10¹⁶ fluorinated anti-CD28 antibody-containing activation medium on non-TC-treated vessels coated with plate-bound CD3 antibody (OKT3) and fibronectin fragment (Retronectin®). 5 Seed cells at a concentration of 1 / mL for 3 days (activation step), then 1 × 10¹⁶ cells were placed on a non-TC treated container in growth medium. 5 Re-seed cells at 2 / mL for 2 days, then place 3 × 10 units on a non-TC treated container in growth medium. 5 The cells were re-seed at a rate of 2 / mL for an additional 2 days (growth step). It is also intended that CD8SP may be activated / growthed upon contact with ICAM-1.

[0257] Survival rates and cumulative proliferation rate dynamics were evaluated during stages 1–3 of the 3D differentiation protocol. During the first 10 days of the process, emerging proT cells remained highly viable, with cumulative proliferation rates at their peak (between 200–600x) (Figure 10). As proT cells entered stage #2 (days 10–17), a substantial decrease in survival rates was observed, with minimal increases in proliferation rates (Figure 10). During stage #3 (from day 17 onward), survival rates continued to decrease slightly due to improvements in DP maturity and phenotype, but cells were able to maintain a survival rate of over 10% for approximately one week, with minimal decreases in proliferation rates, representing an improvement from previous protocols.

[0258] One method involves transferring 10-day ProT cells (unmodified strain) generated at Stage #1 to precursor maturation medium (SFEM II+LMM) in non-tissue culture (TC) treated plates pre-coated with 1.37 μg / ml human DL4 and 1 μg / ml human VCAM-1 (intermediate 2D ETN) at a rate of 1 × 10⁶ cells. 6 Cells were seeded at a concentration of 1 / mL. On day 17, sacrificial wells were collected and flow cytometry analysis was performed. On day 22, the cultures were collected and 3 × 10¹⁶ cells were sampled in non-TC treated plates pre-coated with 0.68 μg / mL human DL4 and 0.5 μg / mL human VCAM-1 (lower 2D ETN). 6 Cells / mL were replated with SFEM II+LMM. On day 24, sacrificial wells were collected and flow cytometry analysis was performed. The decrease in 2D ETN coating and increase in seeding density from day 10 to day 24 led to successful generation of CD4+CD8α+ early DP by day 17, resulting in effective maturation of this population, as evidenced by >70% CD4+CD8α / β+, 20-30% surface CD3, and 4-8% TCRα / β+ expression on day 24 (Figure 11). Cells were 23.37% and 15.32% CD1a+ on day 24 (NTX4A1 and NTX6A1 strains, data not shown).

[0259] Alternatively, 2 × 10⁶ ProT cells (unmodified cell line) generated in Stage #1 are used.6 Cells were seeded at SFEM II+LMM at a concentration of cells / mL, incubated at 37°C for 2-4 hours, mixed with 0.25 times the dose of 3D ETN beads, and cultured for 7 days. On day 17, cells were harvested and flow cytometry analysis was performed, and the remaining cells were divided into 5 × 10⁶ cells. 6 Cells were re-seed in SFEM II+LMM at a concentration of cells / mL, incubated at 37°C for 2–4 hours, mixed with 0.25x dose of 0.1x protein density 3D ETN beads (Example 2), and cultured for a further 11–13 days. On day 24, sacrificial wells were collected and flow cytometry analysis was performed. In contrast to the 2D process, the 3D protocol improved the conversion from CD4+ISP to CD4+CD8A+DP, resulting in earlier appearance of CD4+CD8α / β+ late DP by day 17 for both the unmodified cell lines NTX4A1 (Figure 12) and NTX6A1 (Figure 13). The cultures were predominantly CD4+CD8α / β+DP on day 24, but these late DPs were less mature than those produced by the 2D protocol due to lower endogenous TCRα / β surface expression at this point (Figures 12, 13). Further culturing for 4–6 days improved surface CD3 and TCRα / β in both cell lines (Figures 12 and 13). CD1a expression also increased from 4.04% to 12.17% in the NTX4A1 cell line from day 24 to day 28, and from 4.73% to 15.36% in the NTX6A1 cell line.

[0260] In 2D culture, 10-day ProT cells generated from CAR-modified HPCs in Stage 1 were placed in non-TC treated plates pre-coated with 1.37 μg / ml human DL4 and 1 μg / ml human VCAM-1 (intermediate 2D ETN) in 1x10⁶ cells of progenitor cell maturation medium (SFEM II + LMM). 6 Cells were seeded at a concentration of 1 / mL. On day 17, sacrificial wells were collected and flow cytometry analysis was performed. On day 22, the cultures were collected and 3 × 10¹⁶ cells were sampled in non-TC treated plates pre-coated with 0.68 μg / mL human DL4 and 0.5 μg / mL human VCAM-1 (lower 2D ETN). 6Cells / mL were replated with SFEM II+LMM. On day 24, sacrificial wells were collected and flow cytometry analysis was performed. The decrease in 2D ETN coating and increase in seeding density from day 10 to day 24 led to successful generation of CD4+CD8α+ early DP by day 17, resulting in effective maturation of this population, as evidenced by >70% CD4+CD8α / β+, >20% surface CD3 and >70% CAR+ expression on day 24 (Figure 14). CD1a expression on day 24 was 23.25%.

[0261] In 3D culture, 10-day-old ProT cells generated at stage 1 from CAR-modified HPCs were seeded at 2 x 10⁶ cells / mL in SFEM II + LMM, incubated at 37°C for 2-4 hours, mixed with 0.5 times the amount of 3D ETN beads with 0.1 times the protein density, and cultured for 7 days. On day 17, the cells were harvested and flow cytometry analysis was performed, and the remaining cells were divided into 5 x 10⁶ cells. 5 Cells were re-seed in SFEM II+LMM at a concentration of cells / mL, incubated at 37°C for 2–4 hours, mixed with 0.25x dose of 0.1x protein density 3D ETN beads, and cultured for a further 11 days. On day 24, sacrificial wells were collected and flow cytometry analysis was performed. In contrast to the 2D protocol, the 3D protocol improved the conversion of CD4+ISP to CD4+CD8α+DP, and CD4+CD8α / β+ late DP appeared earlier by day 17 (Figure 15, top row). The cultures were mainly CD4+CD8α / β+DP on day 24 (Figure 15, middle row), but these late DPs were less mature than those produced by the 2D protocol due to lower CD1a+ surface expression at this point (2.61%). Further 4 days of culture improved surface CD3 and CAR expression (bottom row of Figure 15) and increased CD1a expression (8.97%).

[0262] Next, late DP was generated using either 2D ETN or 3D ETN beads. Concentrated late DP from two unmodified strains, NTX4A1 and NTX6A1, was collected in a medium containing IL-21 and 1.25% ImmunoCult® activator anti-CD3 / CD28 / CD2, at a rate of 1 × 10⁶. 6Cells / mL were seeded for 7 days under stage #4 conditions. Concentrated late DP from CAR modified strains was seeded at 1 × 10⁶ under stage #4 conditions. 6 Cells were seeded at a concentration of cells / mL and stimulated for 7 days with either CD3 stimulation using 1.25% ImmunoCult® activator (anti-CD3 / CD28 / CD2 beads) or antigen stimulation using CD19 antigen-coated beads in a 1:1 bead:cell ratio (Example 1). The enriched DP produced using the 3D protocol had higher viability than that produced using the 2D protocol. When using the anti-CD3 / CD28 / CD2 transfer protocol (Figure 16A, B, left), a decrease in cell viability (Figure 16A, left) and cell number (Figure 16B, left) was observed during the 7-day DP-SP transfer, regardless of the cell line or protocol used to generate the input DP. In contrast, when the CD19 bead transfer protocol was used with CAR-modified cell lines (Figure 16A, B, right), an increase in both cell number and viability was observed.

[0263] Next, concentrated late-stage DP from two unmodified strains, NTX4A1 and NTX6A1, was added to DP-SP medium containing IL-21 and 1.25% ImmunoCult® activator anti-CD3 / CD28 / CD2, at a rate of 1 × 10⁶. 6 Cells were seeded at a concentration of cells / mL for 7 days under Stage #4 conditions. At the end of this stage (Day 35 for NTX4A1 - Figure 17, upper panel, and Day 37 for NTX6A1 - Figure 17, lower panel), the cultures were harvested and stained for selected markers of T cell maturation, activation, and TCR expression. Partial DP-SP migration was observed in both cell lines, which was evidenced by the presence of residual DP in the culture (Figure 17, left panel, upper right quadrant). Looking at the CD8SP population (Figure 17, gray overlay) in more detail, these cells were highly positive for CD8α / β (82–85%), expressed surface CD3 and TCRα / β (30–58%), as well as key integrins such as LFA-1 and CD49c (VLA-3) and the activation marker CD25.

[0264] Concentrated late-stage DP from CAR modified strains under stage #4 conditions: 1 × 10 6 Cells were seeded at 1 / mL and stimulated for 7 days with either CD3 stimulation using 1.25% ImmunoCult® activator (anti-CD3 / CD28 / CD2, upper panel of Figure 18) or antigen stimulation using CD19 antigen beads in a 1:1 bead:cell ratio (lower panel of Figure 18). At the end of this stage (day 35), the cultures were harvested and stained for selected markers of T cell maturation, activation, and surface CD3 expression. Both conversion methods resulted in the effective generation of high MFI CAR+CD8SP (60-63%). Anti-CD3 / CD2-transitioned cells had more residual DP after transition than those under the CD19 bead condition (Figure 18A, B). A more detailed examination of the CD8SP population (gray overlay) revealed that these cells possessed intermediate levels of CD8α / β (43%), moderate to high surface CD3 expression (86% for anti-CD3 / CD28 / CD2 and 56% for CD19 beads), as well as important integrins, such as LFA-1 and CD49c (VLA-3). The activation marker CD25 was highly expressed in anti-CD3 / CD28 / CD2-transplanted CD8SP (78.57%, Figure 18, upper panel) and lower under CD19 bead conditions (56%, Figure 18, lower panel). Both CD3 activation via anti-CD3 / CD28 / CD2 and CAR activation via CD19 beads were effective conversion methods for successfully translocating CAR+DP to CD8SP. Differentiation of unmodified cell lines yielded CD4-CD8α+CD8β+CD25+CD69+LFA-1+ / CD49c+CD3+TCRαβ+TCRγδ+CD56lo cells. Differentiation of CAR-modified TRAC- / - cell lines yielded CD4-CD8a+CD8B+CD25+CD69+LFA-1+ / CD49c+CD3+CAR+TCRγδ+CD56lo cells.

[0265] Tables 11-14 below summarize the differentiation conditions and resulting cell phenotypes for the unmodified cell lines NTX4A1 and NTX6A1 (Tables 11 and 12), the CAR-modified cell line NTX4B3 (Table 13), and the TCR-modified cell line TCR-174 (Table 14). Cell seeding density is before the addition of beads (ETN or CD19 antigen-coated beads). [Table 11] [Table 12] [Table 13] [Table 14]

[0266] Example 4: In vitro function of CAR+CD8+SP cells At the end of the CD8SP stage (day 35 from HPC), iPSC-derived cell cultures that had transitioned to CD8SP containing ImmunoCult® activator anti-CD3 / CD28 / CD2 or antigen-coated beads (Example 3) were harvested and cultured with target cells, with or without CD19 expression. A serial restimulation assay was performed using an Incucyte®-based assay (Sartorius) to measure cytotoxic activity with GFP-expressing CD19+ cells as target cells (n=3 technical replicates). T cells were co-cultured with target cells and exogenous cytokine support in a 2:1=E:T ratio every 5 days. Target clearance was measured by the decrease in GFP surface area. CD8SP cells generated by both methods exhibited some nonspecific activity during the initial stimulation (Figure 19A, Figure 19B, dark gray inverted triangles) potentially to be activated during the DP-SP stage of differentiation. iPSC-derived CD8SP was able to sequentially engage with target cells over four stimulations (Figure 19A, B).

[0267] Cell proliferation was calculated by counting at the end of each round of target exposure. iPSC-derived CAR-T cells proliferated 28,000-fold after 4 rounds of antigen exposure (Figure 20A). Equivalent effector cytokine production 24 hours after stimulation 1 was measured by MSD in iPSCs, and primary CD8+ CAR-Ts co-cultured with CD19+ / + and CD19- / - target cells were measured (Figure 20B, C).

[0268] Cumulative cytotoxicity and growth rate were evaluated for primary cells and iPSC-derived cells over four exposures to the target (IL-2, IL-7, and IL-21, or IL-15, IL-7, and IL-21 supplemented, Figure 21). Growth rate and cytotoxicity were calculated after each stimulus. Cumulative growth rate was calculated by productting the cumulative growth rate from previous stimuli with the growth rate of the current stimulus. Cytotoxicity was calculated through the relative decrease in area under the curve (AUC) from the target-only control: cytotoxicity = 1 - AUC / AUC control (cytotoxicity of 0 corresponds to no tumor control, and cytotoxicity of 1 corresponds to immediate total tumor control). Cumulative cytotoxicity was calculated by summing the cumulative cytotoxicity from previous stimuli with the cytotoxicity of the current stimulus. Compared to primary CAR-T cells, iPSC-derived cells showed comparable or higher cytotoxicity, while primary cells exhibited greater cumulative proliferation (Figure 21, horizontal dashed line indicates the maximum cumulative cytotoxicity achievable with each stimulus).

[0269] The expression of T cell memory markers was assessed using flow cytometry at baseline and at the end of the fourth stimulation. Cells were assigned to T cell memory subsets based on the following expression profiles: T stem cell memory (TSCM), CD62L+CD45RA+CD95+; T central memory (TCM), CD62L+CD45RA-CD45RO+; T effector memory (TEM), CD62L-CD45RA-CD45RO+; terminally differentiated effector memory cells reexpressing CD45RA (TEMRA), CD62L-CD45RA+CD45RO+. Anti-CD3 / CD28 / CD2-transferring CD8SP and CD19 bead-transferring CD8SP had increased memory phenotypes at baseline and post-stimulation 4 compared to primary T cells (Figure 22A). The expression of various exhaustion markers was also assessed using flow cytometry at baseline and at the end of stimulation 4. Anti-CD3 / CD28 / CD2-transferred CD8SP and CD19 bead-transferred CD8SP showed reduced expression of exhaustion markers at baseline and post-stimulation compared to primary T cells (Figure 22B).

[0270] Example 5: Characterization of CD8+ SP cells Deep characterization of iPSC-derived cells in the ProT stage (day 10), DP stage (day 22), and iPSC CD8-T (process-completed cells; Figure 23, showing y-axis labels for Example 3) was performed based on CITEseq data (single-cell mRNA + protein) using a publicly available dataset for thymocytes as a reference (Park et al., 2020). Gene sets corresponding to DN (initial), DN(P), DN(Q), DP(P), DP(Q), CD8-T, CD8αα, and CD8-Tmem (23A, x-axis labels) were obtained from the reference dataset (Park et al., 2020). Gene signature enrichment was calculated using single-sample gene set enrichment analysis (ssGSEA) at the single-cell level. The overall enrichment pattern was summarized as a bubble plot, where the area of ​​each circle represents the percentage of cells expressing the signature, and the shading represents the mean expression level (Figure 23A). This analysis revealed that iPSC-derived ProT(D10) cells were equivalent to DN(early) cells in human thymus (Figure 23A). Similarly, iPSC-derived cells at day 22 corresponded to the in vivo DP cell stage, and process-termination cells corresponded to in vivo T cells. Further analysis was performed by creating homogeneous manifold approximation plots (UMAPs) based on single-cell mRNA and protein expression using iPSC-derived cells from D10(ProT), D22(DP), process-termination (iPSC CD8-T), and in vivo primary CD8 T cells (Figure 23B). The overlapping cells, shaded in bright blue (process-termination) and purple (primary T), demonstrate that iPSC-derived process-termination cells were equivalent to in vivo primary T cells based on single-cell mRNA and protein expression. Single-cell protein expression profiles of different T cell markers were created for iPSC-derived ProT (D10), DP (D22), CD8-T (process terminated), and primary CD8-T cells (Figure 23C).

[0271] Next, iPSC-derived termination cells were characterized by flow cytometry (Figure 23), and their gene expression profiles were compared to those of primary T cells. Gene expression profiles of cell markers were filtered using a data-driven analysis strategy with Seurat (Stuart et al., 2019) (in the heatmap shown in Figure 25A, columns represent cells and rows represent filtered markers). Cell populations were annotated with SingleR (Aran et al., 2019) with reference to the thymus atlas dataset (Park et al., 2020) (Figure 25B). The majority of primary T cells (over 90%) were assigned as CD8 T cells (Figure 25B).

[0272] The above approach was also applied to iPSC-derived termination cells (Figures 26A and 26B). The majority of iPSC-derived termination cells (over 80%) were assigned as DP cells, and 20% of the cells were assigned as CD8 T cells (Figure 26B).

[0273] Example 6: Generation of CD8+ SP cells with exogenous TCR Successful generation of late DP and CD8SP in TCR-modified cell lines was achieved using a 3D protocol (n=2 clones). A TCR-modified CD34+ bank was differentiated to late DP (stage #3) using the 3D protocol as described above (Example 3). Cultures were harvested on day 25 and enriched for CD8α using the EasySep® CD8-positive selection kit, followed by staining with CD4, CD8A, CD8B, CD3, TCRVβ1 antibodies and APC conjugate MAGE-A4 tetramer to evaluate DP% and surface TCR expression (Figures 27A, 28A). The enriched late DP was cultured in 1x10⁶ cells in a medium containing IL-21 and 1.25% ImmunoCult® activator anti-CD3 / CD28 / CD2. 6Cells / mL were seeded for 7 days under stage #4 conditions (Example 3). At the end of this stage (day 32), flow cytometry immunophenotyping of the cultures was performed to evaluate the expression of selected markers for T cell maturation, activation, and TCR expression. Late DP derived from TCR-modified strains successfully transitioned to CD8α / β+SP T cells with higher CD3 and TCR surface expression compared to the DP stage (89% for clone 172, Figure 27B, and 78% for clone 174, Figure 28B). Detection of surface TCRs was most accurate over time when using MAGE-A4 tetramer staining.

[0274] TCR-modified CD8SPs (n=2 clones) generated in Stage #4 were activated using the anti-CD3 antibody OKT3 and the fibronectin fragment RetroNectin® (both coated on the culture plate) and soluble CD28 costimulation, and proliferated twice for a total of 14 days. At the end of this stage, immunophenotyping of the proliferated CD8SPs was performed by flow cytometry for selected markers of T cell memory and exhaustion. The proliferated TCR-modified CD8SPs had a low exhaustion profile (14–24% LAG3+) and retained CD8α (74–84%), surface CD3 (85–90%), and MAGE-A4 TCR expression (84–90% CD3+ TCRVβ1+, >96% tetramer+) over the two proliferation cycles (Figures 29, 30). Of the CD3+ cells, 81–87% of the population were T SCM The composition is (CD95+CD62L+CD45RA+e), and 10-14% is T EFF The cell type was (CD95+CD62L-CD45RA+) (Figures 29 and 30). The cells had a viability rate of over 80% and proliferated 26-fold and 126-fold after two cycles (data not shown).

[0275] At the end of this stage, cells were evaluated for in vitro function using a flow cytometry-based cytotoxicity assay and compared with unmodified iPSC-derived CD8SP and primary CD8SP manipulated to express MAGE-A4 TCR. Target-specific cytotoxicity was evaluated using GFP-expressing A375 wild-type (WT) cells as the target line, and nonspecific killing was evaluated using GFP-expressing A375 β-2-microglobulin (B2M) KO cells. A375 B2M KO cells were labeled with CellTrace® Blue reagent and mixed with A375 WT target cells in a 1:1 ratio. Target cells were seeded, and effector cells were added in six different E:T ratios between 0.03125:1 and 1:1 based on the number of A375 WT target cells. No effector cells were included in the control well. Cells were cultured for 24 hours before harvest, stained with survival dyes, and analyzed by flow cytometry. The number of viable A375 WT cells (GFP+CellTrace-) and A375 B2M KO cells (GFP+CellTrace+) was quantified using counting beads for normalization. The viability of A375 WT and A375 B2M KO cells was calculated separately in each well by normalizing to a control well without effector cells (average WT or B2M KO cells / 0 = E:T). The normalized specific killing percentage was calculated by [1 - (percentage of viable WT / percentage of viable B2M KO)] × 100. iPSC-derived TCR SSI CD8SP exhibited similar or better normalized specific killing compared to primary TCR-T cells (Figure 31), but also had higher levels of background nonspecific killing (not shown). iPSC-derived CD8SP lacking MAGE-A4 TCR did not show target-specific killing.

[0276] Example 7: Combination of Notch inhibition and Notch stimulation for control of cellular phenotype Regulation of Notch signaling using gamma-secretase inhibition (GSI) was found to alter the cell differentiation trajectory. iPSC-derived CD34 cells (TRAC- / - cell line, "NTX4B1") were subjected to 0.5 × (2.7 × 10⁻⁶)7 Cells were differentiated in LEM for 10 days using an ETN bead dose of (beads / mL). On day 10, cells were harvested and divided into 2 × (1.08 × 10⁶)⁶ cells. 8 ETN dose of beads / mL: 1 x 10 6 Cells were reseeded at a cell density of cells / mL. On day 11, the gamma-secretase inhibitor DAPT was added at doses of 0.1, 0.3, 0.5, and 1 μM. DAPT was added only on day 11 (Figure 32 Panel A; 1 GSI dose) or on day 11, as well as during medium changes on days 13 and 15 (Figure 32 Panel B; 3 GSI doses). In the absence of GSI, cells were predominantly CD8 SP (approximately 40%) or DN (approximately 50%). Addition of a single GSI dose on day 11 resulted in a dose-dependent decrease in CD8 SP and an increase in both the CD4 ISP and DP populations (Figure 32A). With three GSI doses (Figure 32B), a more abrupt decrease in the CD8 SP percentage was observed at a 0.1 μM dose. The DP percentage increased with GSI dose, but CD4 ISP peaked at 0.3–0.5 μM doses, and the percentage decreased beyond that point.

[0277] Cell viability, proliferation, and DP differentiation responded to GSI dose. As shown in Figure 32, cells were differentiated in the presence of various doses of GSI from day 11 to day 17. The percentage of DP cells generated depended on both GSI dose and number of treatments, as higher concentrations and more frequent treatments resulted in more DP cells (Figure 33A). Cell viability was also responsive to GSI dose, with lower viability observed at doses greater than 0.3 μM with a single GSI treatment or greater than 0.1 μM with three GSI additions (Figure 33B). Cell proliferation (compared to the day 10 introduction) decreased at doses greater than 0 or 0.1 μM with three or single treatments, respectively (Figure 33C).

[0278] Between day 10 and day 17, cells were differentiated using 0.1×ETN bead dose (Figure 34, top row), 2× bead dose + 1 μM GSI (single addition, Figure 34, middle row), or 2× bead dose + 0.3 μM GSI (triple addition, Figure 34, bottom row). All three conditions produced similar proportions of CD5+ / CD7+, CD4 ISP, DP, and CD8ab+ cells, suggesting that different orthogonal methods—ETN bead dose, GSI concentration, and frequency of GSI addition—to control Notch signaling levels can produce comparable cell populations.

[0279] The GSI dose-response was performed as shown in Figure 32. As a control, cells seeded on 2D ETN were also treated with GSI (single GSI addition only). For both CAR and WT cells, 2D ETN had more DP cells at 0 GSI dose than 3D ETN (Figures 35A, 35B). Cells differentiated on 2D ETN had peak DP levels at a GSI dose of 0.3 μM, while DP was richest at 1.0 μM and 0.75 μM with one (3D-1T) and three (3D-3T) GSI additions with 3D ETN, respectively. The difference in dose-response curves between 2D and 3D ETN is likely related to the higher levels of Notch signaling conferred by 3D ETN. Both CAR and WT cells differentiated on 2D and 3D ETN showed a GSI dose-dependent increase in the DP cell population.

[0280] Cells were differentiated as shown in Figure 35. The percentages of CD8αSP, DP, DN, and CD4ISP as a function of GSI dose are shown for CAR (Figure 36, left) cells and unmodified (Figure 36, right) cells. The distribution of cells in each quadrant is shown for 3D ETN with 3 GSI doses (Figure 36, top row), 3D ETN with a single GSI dose (Figure 36, middle row), and 2D ETN with a single GSI dose (Figure 36, bottom row). CAR and WT cells differentiated with 2D and 3D ETN showed a GSI dose-dependent increase in the DP cell population.

[0281] Example 8: TCR activation The viability and cumulative proliferation rate dynamics of iPSC-derived TCR-modified cells were evaluated during stages 1–3 of the 3D differentiation protocol (Example 3). During the first 10 days of the process, the emerging TCR-modified proT cells maintained high viability and reached peak proliferation (200–340x, Figure 37A, B). When the proT cells entered stage #2 (days 10–17), a substantial decrease in viability was observed, and the increase in proliferation rate was minimal (Figure 37A, B). During stage #3 (from day 17 onward), viability gradually decreased due to DP constraint and maturation (Figure 37A, B). Despite constitutive TCR surface expression, the TCR-modified strains had similar viability and proliferation rates to the unmodified NTX6A1 parent strain.

[0282] TCR-modified iPSC-derived CD34+ banks were differentiated to late DP (stage #3) using the 3D protocol as described above (Example 3). On day 28, the cultures were harvested, stained for selected maturation markers (Figure 38, upper panel), and the remaining cells were used for the CD8α enrichment step using the EasySep® CD8 Positive Selection Kit. To confirm the purity of the enrichment, flow cytometry analysis of CD4 / CD8A / CD8B was performed (Figure 38, lower panel). At this point, the TCR-modified CD4+CD8α / β+DP expressed high levels of important maturation markers such as CD28 and CD2, suggesting that the cells were at the optimal time to transition to stage #4 (DP-SP transition). Furthermore, no expression of CD137 (41BB) was detected at this stage. CD8α enrichment resulted in effective depletion of CD4-CD8α / β-DN from the cultures and increased the purity of the late DP population (n=2 clones, only one shown).

[0283] Concentrated late-stage DP was added to DP-SP medium containing IL-21, 1 × 10⁶ 6Cells were seeded at cell / mL under stage #4 conditions for 7 days. Multiple activation conditions were tested to determine the optimal method of transition for TCR-modified strains. Two main methods were tested: two types of ImmunoCult® activators (i.e., CD3-based activation) and pHC tetramer activation (MAGE-A4 and an unrelated HIV gag tetramer as a control) using different co-stimulatory reagents (i.e., antigen-based activation). At the end of stage #4, ImmunoCult® activator CD3 / CD28 was the best method of conversion, with viability similar to the input and a growth fold exceeding 0.7x (Figure 39, n=2 clones). In addition to improved viability and growth fold, ImmunoCult® activator CD3 / CD28 resulted in highly efficient DP-SP conversion, with virtually all late DPs transitioning to >90% CD8α / β+SP (Figure 40, conditions 1 and 3). These CD8SPs also expressed important activation markers such as CD25 and CD69, which are expected to be expressed after conversion. However, MAGE-A4 tetramer-based translocation was not very effective in converting DP to CD8SP, as evidenced by the high percentage of residual CD4+CD8α / β+DP in the culture, as well as the low expression of CD25 and CD69 (Figure 40, conditions 2, 5, and 4). It should be noted that the CD8α / β+ population remained high under these conditions (>90%), likely because both DP and CD8SP expressed these markers. As expected, unrelated tetramer HIV-gag was ineffective in converting DP to SP, as virtually all cells were still DP cells (Figure 40, conditions 6-8).

[0284] The concentrated late-stage DP was seeded under stage #4 conditions as described above (Figures 39 and 40). At the end of this stage, the cell phenotype was evaluated by flow cytometry for selected memory and co-stimulatory markers of interest (Figures 41 and 42). CD8SP generated using ImmunoCult® activators (conditions 1 and 3) highly expressed key maturation markers such as CD95 (>98%), CD45RA (>80%), CD62L (>66%), and CD27 (>92%) (Figure 41). CD2 was also expressed, and was higher under condition 3 (ImmunoCult® activator without CD2) (Figure 42). CD45RO expression was lower compared to the tetramerization condition. Other co-stimulatory molecules such as CD137, ICOS, and CD28 were expressed at low levels (Figure 42). MAGE-A4 tetramer transfer samples (conditions 2, 5, and 4) showed higher expression of CD45RO (>87%) and lower expression of CD45RA (15–27%) and CD95 (69–79%) compared to the ImmunoCult® activator condition (Figure 41), suggesting that this mixed population was phenotypically more immature than the ImmunoCult® transfer condition. CD2 (>67%), as well as CD27 (>80%) and CD28 (>57%) under some conditions, were expressed. The latter may be due to the presence of DP, which also expressed this marker. Similarly, CD137 and ICOS were underexpressed, as in the ImmunoCult® activator condition (Figure 42). Finally, the addition of different co-stimulatory reagents during stage #4 showed virtually no phenotypic or viability and growth rate changes after transfer (data not shown).

[0285] TCR-modified CD8SP cells generated in Stage #4 using ImmunoCult® activator anti-CD3 / CD28 / CD2 (Condition #1) or MAGE-A4 tetramer and soluble CD28 (Condition #2) were activated and proliferated using the following four different methods: OKT3 and retronectin® coating and soluble anti-CD28 co-stimulation ("A"); OKT3 and retronectin® coating and soluble 41BB co-stimulation ("B"); OKT3 and retronectin® coating and soluble ICOS-L Fc co-stimulation ("C"); and T Cell TransAct® CD3 / CD28 activator ("D"). Cell counts were measured on days 3 and 7 to evaluate viability and proliferation rate during this stage. The addition of different co-stimulatory reagents did not result in differences in viability or proliferation rate (Figure 43). T Cell TransAct® CD3 / CD28 stimulation was the least successful condition tested. Cells generated under condition #1 showed more consistent improvements in viability and proliferation rate over time, as demonstrated by the good performance observed in both TCR-modified cell lines used. In contrast, cells generated under condition #2 had a clone-dependent response to the activation conditions tested. TCR-modified clone 172 showed lower overall performance compared to clone 174.

[0286] TCR-modified CD8SP cells, generated in Stage #4 using ImmunoCult® activator CD3 / CD28 / CD2 (Condition #1) or MAGE-A4 tetramer and soluble anti-CD28 (Condition #2), were activated and proliferated using the four different methods described above (see Figure 43). At the end of this stage, the cell phenotype was evaluated by flow cytometry for the selected marker of interest. The addition of different co-stimulatory reagents did not result in any difference in the CD8SP phenotype (Figures 44A, B, C, D). CD8α / β+ surface expression was retained under all conditions tested, but a more abrupt decrease in this population was observed in cells derived from Condition #2 (Figures 44C, D). Anti-CD3 / CD28 / CD2-transitioned CD8SP cells showed lower surface expression of CD25 and CD69 compared to CD8SP cells derived from Condition #2, suggesting that these cells were less activated at the end of one round of proliferation. Interestingly, the T Cell TransAct® CD3 / CD28 activation condition ("D") generated cells with a more activated profile compared to conditions A, B, and C using plate-coated OKT3 and retronectin®. Regarding memory markers, anti-CD3 / CD28 / CD2-transitioning CD8SP in activation conditions A, B, and C expressed higher percentages of CD45RA (>95%) and CD62L (>88%), as well as lower percentages of CD45RO (47-70%), compared to CD8SP derived from condition #2 (Figure 45A, B). T Cell TransAct® (condition D) had a higher percentage of CD45RO (87.57%) compared to conditions based on OKT3 and retronectin®. Almost all cells were CD95+. Anti-CD3 / CD28 / CD2-transitioning CD8SP cells showed fewer activation phenotypes, higher expression of CD45RA and CD62L, and lower expression of CD45RO after one proliferation compared to MAGE-A4 tetramer-transitioning cells (Figure 45A, B).

[0287] Example 9: Generation, characterization, and function of iPSC-derived TCR-T cells Clonal iPSC lines were generated using the MAGEA4 T cell receptor (TCR) integrated into the TRAC locus (Schematic, Figure 46A). Clones were generated using the Namocell® single-cell deposition system, and targeted biallelic insertions at the TRAC locus were screened using digital droplet polymerase chain reaction (ddPCR). TCR expression was characterized at the end of clonal production for four selected biallelic iPSC clones (Figure 46B). Vector copy number (VCN) values ​​were determined using a ddPCR copy number variation (CNV) assay (Biorad) by amplifying the genomic TRAC locus-TCR transgene junction, and the copy number integration at the TRAC locus was genetically characterized (Figure 46B). Representative scatter plots of the selected clones (174) and unedited controls are shown in Figure 46C. Genomic stability analysis was performed on the 174 selected clones (Figures 46D, E). G-band karyotype analysis performed by WiCell showed a normal karyotype (Figure 46D), and a summary of the iCS-digital® assay (Stem Genomics) indicated the expected copy numbers in 24 genomic regions of recurrent iPSC abnormalities (Figure 46E).

[0288] iPSC-derived MAGE-A4 TCR-expressing CD34+ hematopoietic progenitor cells (HPCs) were differentiated into CD8SP T cells using a four-stage protocol (Schematic Diagram, Figure 47A). During Stage 1, CD34 HPCs were seeded in 2D ETN-coated vessels in precursor growth medium (SFEM II+LEM) (Example 1) and cultured for 10 days to generate CD34-CD7+CD5+ ProT cells (Stage 2). At this point, the cells were transferred to Stage 3, and the ProT cells were cultured for 18 days with varying doses of DL4 and VCAM-1 paramagnetic beads (Example 3) to allow for the emergence of late-stage DP (CD4+CD8A+CD8B+CD3+TCRαβ+). Finally, Stage 4 was initiated by enrichment of late-stage DP via a CD8-positive selection kit (STEMCELL Technologies) and activation using ImmunoCult® activator anti-CD3 / CD28 for 7 days to induce conversion from DP to SP.

[0289] Stage 1 CD34+ HPCs expressed high levels of CD43 and CD45 hematopoietic markers, and intermediate TCR expression was detected using TCRVβ1 antibody (Figure 47B). Stage 2 ProT cells were predominantly CD7+ (93.41%), co-expressing CD34 (13.88%), CD5 (21.19%), and CD56 (24.78%) (Figure 47C). Stage 3 late DPs were predominantly CD4+CD8A+CD8B+ (approximately 86%) (Figures 47D, E), with high levels of CD5+CD7+ and CD28+CD2+ costimulatory molecules (Figure 47E). Stage 4 CD8SPs were CD4-CD8A+ (73.14%), co-expressing high levels of CD8B (Figure 47F). The resulting CD8SP also expressed important activation markers (CD25 and CD69) (Figure 47F), a low fatigue profile (12.47% LAG3+) (Figure 47F), a mixture of TSCM, TCM, and TEM memory phenotypes (Figure 47G), and was predominantly CD2+ and CD27+ (Figure 47H).

[0290] MAGE-A4 TCR expression dynamics were analyzed during T cell differentiation. Cells were stained with APC-conjugated MAGE-A4 tetramer reagent at day 10 (ProT), day 28 (Late DP), and day 35 (CD8SP) to detect surface TCR expression levels during T cell differentiation. MAGE-A4 TCR expression was lower at the CD34 HPC stage (26.07%), but as cells were confined to more T lineages, TCR surface expression increased as a result (Figure 48).

[0291] TCR+CD8 SP iPSC-T cells were suitable for downstream in vitro proliferation while maintaining key true T cell phenotypic markers. A biphasic iPSC-T proliferation culture protocol consisting of an activation stage and a maintenance stage was developed (Figure 49A). Viability and proliferation rate (against day 0) were analyzed over a 7-day culture period (Figure 49B). Key true T cell phenotypic markers were maintained until the end of the proliferation protocol, as assessed by flow cytometry (Figure 49C). CD8SP cells still predominantly retained the stem cell memory phenotype but largely lacked common exhaustion markers (Figure 49D).

[0292] Next, the cytotoxicity and specificity of TCR+CD8 SP iPSC-T cells were evaluated using an in vitro serial restimulation assay (Schematic, Figure 50A). Effector cells, either TCR+CD8 SP iPSC-T cells or primary CD8+ cells transduced with an exogenous TCR using adeno-associated virus (AAV), were seeded with Nuclight® green-labeled tumor targets in multiple effector-to-target (E:T) ratios and co-cultured in Incucyte® for 5 days to monitor cytotoxicity (Stim 1). After harvesting on day 5, effector cells from a 2:1 E:T ratio were counted and re-seeded at a 2:1 E:T ratio for subsequent stimulation with the new tumor target. This process was repeated for a total of 4 rounds of activity (total 19 days).

[0293] T cell phenotype was monitored by flow cytometry through a restorative assay. The E:T ratio and assay duration (activation rounds) affected the co-expression of inhibitory receptors PD-1, TIGIT, LAG3, TIM3, and CD39 on effector cells as measures of CD8 T cell exhaustion (Figure 50B).

[0294] To monitor target cell death, cells were gradually exposed to one round of activation at lower E:T ratios using Incucyte®. The difference in cytotoxicity between iPSC-derived CD8+ TCR+ cells and primary CD8+ TCR+ cells was most pronounced at lower E:T ratios, showing a 3-fold difference in activity (Figure 50C). Using the optimal E:T ratio (2:1) for iPSC-derived CD8+ TCR+ cells, iPSC-derived CD8+ TCR+ cells demonstrated repetitive killing of tumor targets comparable to primary CD8+ TCR+ cells (Figure 50D).

[0295] To evaluate the specificity in the Incucyte® cytotoxicity assay, iPSC-derived CD8+ TCR+ cells or primary CD8+ TCR+ cells were seeded with or without β2M expression (antigen-positive) tumor target cells (antigen-negative). Both iPSC-derived CD8+ TCR+ and primary CD8 TCR cells showed similar specificity for antigen-positive cells (Figure 50E).

[0296] We used CITESeq to characterize the iPSC-derived cell populations and primary cell populations in detail. Homogeneous manifold approximation projection (UMAP) plots were created based on CITESeq data (mRNA and surface protein expression at single-cell resolution) for peripheral blood-derived CD8+ T cells (PBMC-CD8T): peripheral blood-derived CD4+ T cells (PBMC-CD4-T), peripheral blood-derived T cells activated with anti-CD3 / anti-CD28 beads (PBMC-T-activated), peripheral blood-derived natural killer cells (PBMC-NK), and iPSC-derived TCR+CD8+ cells (iPS-TCR) (Figure 51A). Furthermore, we referred to the thymus atlas dataset (Park et al., 2020) and annotated the cell populations "iPS-TCR" and "PBMC-T-Activated" with SingleR (Aran et al., 2019) (Figure 51B). Cellular markers were screened based on statistical expression thresholds and shown as bubble plots using Seurat (Stuart et al., 2019) (Figure 51C).

[0297] While this disclosure has been described with reference to specific embodiments, various modifications thereto will be apparent to those skilled in the art. Any examples provided herein are included solely for illustrative purposes and are not intended to limit the disclosure. Any drawings provided herein are for illustrative purposes only to illustrate various aspects of the disclosure and are not intended to depict the disclosure to scale or to limit it. The claims attached herein should not be limited by the preferred embodiments described above, and the broadest interpretation consistent with this specification as a whole should be given. All technical disclosures enumerated herein are incorporated herein by reference in their entirety. References 1.Aran et al.Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage.Nat.Immunol.20(2)163-172(2019).Doi:10.1038 / s41590-018-0276-y 2.Baulu et al.TCR-engineered T cell therapy in solid tumors:State of the art and perspectives.Science Advances eadf3700(2023).Doi:10.1126 / sciadv.adf3700 3.Drougkas et al.Comprehensive clinical evaluation of CAR-T cell immunotherapy for solid tumors: a path moving forward or a dead end? J Cancer Res Clin Oncol 149:2709-2734(2023).Doi:10.1007 / s00432-022-04547-4 4.Guha et al.Assessing the Future of Solid Tumor Immunotherapy.Biomedicines 10:655(2022).Doi:10.3390 / biomedicines10030655 5.Iriguchi et al.A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.Nat.Commun.12,430(2021).Doi:10.1038 / s41467-020-20658-3 6.Klesmith et al.Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins.Mol.Pharmaceutics 16(8)3544-3558(2019).Doi:10.1021 / acs.molpharmaceut.9b00418 7.Park et al.A cell atlas of human thymic development defines T cell repertoire formation.Science 2020 367(6480):1-11.Doi:10.1126 / science.aay3224 8.Qu et al.Tumor buster-where will the CAR-T cell therapy ‘missile’ go? Molecular Cancer 21:201(2022).Doi:10.1186 / s12943-022-01669-8 9.Shukla et al.Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1.Nat.Methods.2017 14(5):531-538.Doi:10.1038 / nmeth.4258 10.Stuart et al.Comprehensive Integration of Single-Cell Data.Cell 177(7):1888-1902(2019).Doi:10.1016 / j.cell.2019.05.031 11.Sun et al.Evolution of CD8+T Cell Receptor(TCR)Engineered Therapies for the Treatment of Cancer.Cells 10:2379(2021)Doi:10.3390 / cells10092379 12.Trotman-Grant et al.DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell free system.Nat.Commun.2021 12(5023)1-11.Doi:10.1038 / s41467 13.Want et al.T Cell Based Immunotherapy for Cancer:Approaches and Strategies.Vaccines 11:835(2023).Doi:10.3390 / vaccines11040835 14.Weber et al.The Emerging Landscape of Immune Cell Therapies.Cell.2020 181(1):46-62.doi:10.1016 / j.cell.2020.03.001 15.Zuniga-Pflucker et al.WO 2019 / 157597

Claims

1. A method for generating a population of CD4-CD8+ T cells, —A population of hematopoietic stem cells / progenitor cells is brought into contact with an immobilized Notch signaling ligand at a first ligand concentration, thereby generating a population of progenitor T cells. —The method involves contacting the population of precursor T cells with the immobilized Notch signaling ligand at a second ligand concentration to generate a population of CD4+CD8+ cells, wherein the concentration of the second ligand is less than the concentration of the first ligand. —In the absence of the Notch signaling ligand, the population of CD4+CD8+ cells is brought into contact with a T cell activator, thereby generating a population of CD4-CD8+ T cells. Methods that include...

2. The method according to claim 1, wherein the Notch signaling ligand is DL4.

3. The method according to claim 2, wherein the step of contacting the population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand at the first ligand concentration further comprises contacting the population of hematopoietic stem cells / progenitor cells with immobilized VCAM-1.

4. The first ligand concentration is 3.15 × 10 １１ ~1.26 x 10 １２ The concentration is molecules / mL, and the second ligand concentration is 3.94 × 10⁻⁶. １０ ~6.31 x 10 １１ The method according to any one of claims 1 to 3, wherein the amount is molecules / mL.

5. The method according to any one of claims 1 to 4, further comprising: - Before contacting the population of CD4+CD8+ cells with the T cell activator, the population of CD4+CD8+ cells is contacted with the immobilized Notch signaling ligand at a third ligand concentration, wherein the concentration of the third ligand is less than the concentration of the second ligand.

6. The third ligand concentration is 3.94 × 10 １０ ~6.31 x 10 １１ The method according to claim 5, wherein the molecule / mL is

7. The method according to any one of claims 1 to 6, further comprising enriching CD4+CD8+ cells with respect to CD8α or CD8β.

8. The method according to any one of claims 1 to 7, wherein the T cell activator is a CD3 stimulating factor and an integrin ligand.

9. The method according to claim 8, wherein the CD3 stimulating factor is an anti-CD3 / anti-CD28 / anti-CD2 reagent, an anti-CD3 reagent, or a major histocompatibility complex (pMHC) tetramer, and the integrin ligand is laminin, ICAM, fibronectin, fibronectin fragment, VCAM-1, or ICOS-L.

10. The method according to claim 9, wherein the CD3 stimulating factor is an anti-CD3 / anti-CD28 / anti-CD2 reagent and the integrin ligand is a fibronectin fragment.

11. The method according to any one of claims 1 to 7, wherein the T cell activator is a chimeric antigen receptor (CAR) activator.

12. The method according to claim 11, wherein the CAR activator is an antigen immobilized on a substrate.

13. The method according to claim 12, wherein the antigen is a CD19 antigen and the substrate is a particle.

14. The method according to any one of claims 1 to 13, wherein the step of contacting the population of CD4+CD8+ cells with the T cell activator in the absence of the Notch signaling ligand is performed in a cell culture medium containing IL-7, IL-15, and IL-21.

15. The method according to any one of claims 1 to 14, wherein the step of contacting the population of CD4+CD8+ cells with the T cell activator in the absence of the Notch signaling ligand is performed in the absence of IL-2.

16. The method according to any one of claims 1 to 15, further comprising: - Contacting the aforementioned population of CD4-CD8+ T cells with T cell activators, as well as IL-7, IL-15, and IL-21.

17. The method according to claim 16, wherein the T cell activating factor is a CD3 stimulating factor.

18. The method according to claim 17, wherein the CD3 stimulating factor is an anti-CD3 / anti-CD28 / anti-CD2 reagent, an anti-CD3 reagent, or a peptide major histocompatibility complex (pMHC) tetramer.

19. The method according to any one of claims 1 to 18, further comprising increasing the cell density of the population of precursor T cells by contacting the immobilized Notch signaling ligand at the second ligand concentration, and / or increasing the cell density of the population of CD4+CD8+ cells by contacting the T cell activator and integrin ligand in the absence of the Notch signaling ligand.

20. The method according to any one of claims 1 to 19, wherein the hematopoietic stem cells / progenitor cells are obtained from pluripotent stem cells.

21. The method according to any one of claims 1 to 20, wherein the population of CD4-CD8+ T cells comprises a nucleic acid sequence encoding a CAR or an exogenous T cell receptor (TCR).

22. The method according to any one of claims 1 to 21, wherein the population of CD4-CD8+ T cells is CD3+.

23. The method according to any one of claims 1 to 21, wherein the population of CD4-CD8+ T cells is TRAC- / -.

24. The method according to any one of claims 1 to 22, wherein the population of CD4-CD8+ T cells is enriched with respect to CD8αβ+ cells.

25. The method according to any one of claims 1 to 22, wherein the population of CD4-CD8+ T cells comprises subpopulations of TCRγδ+, CD49c+, and / or CD31+ cells.

26. A population of CD4-CD8+ T cells prepared according to the method described in any one of claims 1 to 25.

27. The population of CD4-CD8+ T cells according to claim 26, wherein the population of CD4-CD8+ T cells includes a nucleic acid sequence encoding a CAR or exogenous TCR.

28. The population according to claim 26 or claim 27, wherein the population of CD4-CD8+ T cells is CD3+.

29. The population according to any one of claims 26 to 28, wherein the population of CD4-CD8+ T cells is TRAC- / -.

30. A population of CD4-CD8+CD3+TRAC- / - cells induced in vitro from pluripotent stem cells.

31. A population of CD4-CD8+CD3+TRAC- / - cells according to claim 30, comprising a nucleic acid sequence encoding a CAR or exogenous TCR.

32. The population of CD4-CD8+CD3+TRAC- / - cells according to claim 30 or claim 31, wherein the population of CD4-CD8+ T cells includes a subpopulation of TCRγδ+, CD49c+ and / or CD31+ cells.

33. A pharmaceutical composition comprising a population of CD4-CD8+ T cells and a pharmaceutically acceptable carrier, wherein the population of CD4-CD8+ T cells is CD3+ and TRAC- / -.

34. A method for treating the target disease or condition, a) To generate a population of CD4-CD8+ T cells as described in any one of claims 1 to 25, b) Administering an effective amount of the aforementioned population of CD4-CD8+ T cells to a subject who needs it. Methods that include...

35. The method according to claim 34, wherein the population of CD4-CD8+ T cells is CD3+ and / or TRAC- / -.

36. The method according to claim 34 or 35, wherein the disease or condition is a hematological malignancy, and the CD4-CD8+ T cells include a nucleic acid sequence encoding a CAR or exogenous T cell receptor (TCR).

37. Use of a population of CD4-CD8+ T cells in the manufacture of a pharmaceutical product for treating a disease or condition, wherein the population of CD4-CD8+ T cells is produced by the method according to any one of claims 1 to 25.

38. A method for generating a population of CD4-CD8+ cells, —A population of hematopoietic stem cells / progenitor cells is brought into contact with an immobilized Notch signaling ligand at a first ligand concentration, thereby generating a population of progenitor T cells. —The method involves contacting the population of precursor T cells with the immobilized Notch signaling ligand at a second ligand concentration to generate a population of CD4+CD8+ cells, wherein the concentration of the second ligand is less than the concentration of the first ligand. Methods that include...

39. The method according to claim 38, wherein the Notch signaling ligand is DL4.

40. The concentration of the first ligand is 3.15×10 １１ ~1.26×10 １２ molecules / mL, and the concentration of the second ligand is 3.94×10 １０ ~6.31×10 １１ molecules / mL. The method according to claim 39 or claim 40.

41. The method according to any one of claims 38 to 40, wherein the population of CD4+CD8+ cells is CD1a+CD28+ and ICOS+.

42. The method according to any one of claims 38 to 41, wherein the population of CD4+CD8+ cells comprises a nucleic acid sequence encoding a CAR or exogenous TCR.

43. A population of CD4+CD8+ cells prepared according to the method described in any one of claims 38 to 42.

44. A population of CD4+CD8+ cells induced in vitro from pluripotent stem cells, wherein the CD4+CD8+ cells are CD1a+CD28+ and ICOS+.

45. A method for generating a population of CD4-CD8+ T cells, —By contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand, a population of progenitor T cells is generated. —The population of precursor T cells is brought into contact with a Notch signaling inhibitor and the immobilized Notch signaling ligand, thereby generating a population of CD4+CD8+ cells. —In the absence of the Notch signaling ligand, the population of CD4+CD8+ cells is brought into contact with a T cell activator, thereby generating a population of CD4-CD8+ T cells. Methods that include...

46. The method according to claim 45, wherein, in the step of contacting the population of progenitor T cells with the Notch signaling inhibitor and the immobilized Notch signaling ligand, the activity of the immobilized Notch signaling ligand is reduced compared to the activity of the immobilized Notch signaling ligand in the step of contacting the population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand.

47. The method according to claim 45 or 46, wherein the Notch signaling inhibitor is a gamma secretase inhibitor.

48. The method according to claim 47, wherein the gamma-secretase inhibitor is provided at a concentration of 0.1 to 1 micromolar.

49. A method according to any one of claims 45 to 48, - In the step of contacting the population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand, the immobilized Notch signaling ligand is 3.15 × 10 １１ ~1.26 x 10 １２ Provided at a first ligand concentration of molecules / mL, and / or - In the step of contacting the population of precursor T cells with the Notch signaling inhibitor and the immobilized Notch signaling ligand, the immobilized Notch signaling ligand is 3.15 × 10 １１ ~2.52 x 10 １２ Provided at a second ligand concentration of molecules / mL, method.

50. A method for generating a population of CD4+CD8+ cells, —By contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand, a population of progenitor T cells is generated. - The population of precursor T cells is brought into contact with a Notch signaling inhibitor and the immobilized Notch signaling ligand, thereby generating a population of CD4+CD8+ cells. Methods that include...

51. The method according to claim 50, wherein, in the step of contacting the population of progenitor T cells with the Notch signaling inhibitor and the immobilized Notch signaling ligand, the activity of the immobilized Notch signaling ligand is reduced compared to the activity of the immobilized Notch signaling ligand in the step of contacting the population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand.

52. The method according to claim 50 or 51, wherein the Notch signaling inhibitor is a gamma-secretase inhibitor.

53. The method according to claim 52, wherein the gamma-secretase inhibitor is provided at a concentration of 0.1 to 1 micromolar.

54. A method according to any one of claims 50 to 53, - In the step of contacting the population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand, the immobilized Notch signaling ligand is 3.15 × 10 １１ ~1.26 x 10 １２ Provided at a first ligand concentration of molecules / mL, and / or - In the step of contacting the population of precursor T cells with the Notch signaling inhibitor and the immobilized Notch signaling ligand, the immobilized Notch signaling ligand is 3.15 × 10 １１ ~2.52 x 10 １２ Provided at a second ligand concentration of molecules / mL, method.

55. A method for treating the target disease or condition, a) To generate a population of CD4-CD8+ T cells as described in any one of claims 45 to 49, b) Administering an effective amount of the aforementioned population of CD4-CD8+ T cells to a subject who needs it. Methods that include...

56. The method according to claim 55, wherein the population of CD4-CD8+ T cells is CD3+ and / or TRAC- / -.

57. Use of a population of CD4-CD8+ T cells in the manufacture of a pharmaceutical product for treating a disease or condition, wherein the population of CD4-CD8+ T cells is produced by the method described in any one of claims 45 to 49.

Images