Irritating antigen particles, method for producing the same, and method for using the same

Abstract

This disclosure provides cell-stimulating particles and methods for using them. It also provides populations of cells produced using the cell-stimulating particles.

Description

[Technical Field]

[0001] field The present invention generally relates to cell-stimulating particles and methods for using them. [Background technology]

[0002] Background of the Invention Various feeder cell-based methods are available for generating T lineage cells from stem cells / progenitor cells. However, the expression of cell surface proteins by supporting 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, have been shown to promote in vitro generation of precursor T cells in feeder-free and serum-free culture systems when combined with VCAM-1 (Shukla et al., 2017). Microbeads modified to present DL4 have also been shown to support in vitro differentiation of T cell lineages, although progression to mature lineages such as CD4-CD8+ cells is limited (Trotman-Grant et al., 2021). Brief stimulation of T cell precursors with CD3 antibodies in the absence of Notch signaling, followed by maturation without CD3 antibodies, has been shown to promote the generation of CD8αβ+ T cells (Iriguchi et al., 2021).

[0004] Immobilized and soluble antibodies have been previously used to stimulate and proliferate primary (i.e., donor-derived) T cells, for example, by stimulation of chimeric antigen receptors (CARs) (Philipson et al., 2020). Antigens on particles and antigens presented by antigen-presenting cells have also been previously used to stimulate and proliferate CAR+ primary T cells (Wu et al., U.S. Patent Application Publication No. 20180223255).

[0005] It is desirable to eliminate or mitigate one or more of the above drawbacks. [Overview of the project]

[0006] Summary of the Invention In one embodiment, a method for generating CD4-CD8+ T cells is provided. This method involves contacting a population of CD4+CD8+ cells expressing an antigen receptor with an antigen immobilized on a substrate. The antigen binds to the antigen receptor.

[0007] In one embodiment, CD4+CD8+ cells are induced in vitro from hematopoietic stem cells / progenitor cells.

[0008] In one embodiment, hematopoietic stem cells / progenitor cells are induced in vitro from pluripotent stem cells.

[0009] In one embodiment, CD4+CD8+ cells are induced according to a method that includes contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand.

[0010] In one embodiment, the step of contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand further includes contacting the population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand at a first ligand concentration to generate a population of progenitor T cells, 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.

[0011] 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.

[0012] In one embodiment, the method further includes the step of enriching CD4+CD8+ cells with respect to CD8α or CD8β.

[0013] In one embodiment, the immobilized Notch signaling ligand is DL4.

[0014] In one embodiment, the antigen is a CD19 polypeptide, and the antigen receptor binds to the CD19 polypeptide.

[0015] In one embodiment, the CD19 polypeptide is an engineered variant of CD19.

[0016] In one embodiment, the antigen is a polypeptide having the sequence of SEQ ID NO: 8.

[0017] In one embodiment, the substrate is a particle.

[0018] In one embodiment, the particle is composed of a material selected from the group consisting of polystyrene, iron oxide, and gold.

[0019] In one embodiment, the particle is composed of polystyrene and magnetizable iron oxide.

[0020] In one embodiment, the antigen is covalently conjugated to the particle.

[0021] In one embodiment, the step of contacting a population of CD4+CD8+ cells expressing an antigen receptor with an antigen coupled to a particle is performed at a particle:cell ratio of 1:1.

[0022] In a second aspect of the present disclosure, a population of CD4 - CD8+ T cells produced according to the method of the first aspect is provided.

[0023] In one embodiment, the population of CD4 - CD8+ T cells expresses a chimeric antigen receptor (CAR).

[0024] In one embodiment, the CAR is a CD19 CAR and the antigen is a CD19 polypeptide.

[0025] In one embodiment, a population of CD4-CD8+ T cells is induced in vitro from pluripotent stem cells.

[0026] In one embodiment, the population of CD4-CD8+ T cells is TRAC- / - and / or CD3+.

[0027] In one embodiment, more than 50% of the CD4-CD8+ cell population is CD8αα+.

[0028] A third embodiment provides a method for inducing cytotoxicity in tumor cells. This method involves exposing tumor cells to a population of CD4-CD8+ T cells. The population of CD4-CD8+ T cells is prepared according to the method of the first embodiment.

[0029] A fourth embodiment provides a method for treating a disease or condition of interest. This method comprises generating CD4-CD8+ T cells according to the method of the first embodiment and administering an effective amount of CD4-CD8+ T cells to a subject in need.

[0030] A fifth aspect provides the use of CD4-CD8+ T cells in the manufacture of a pharmaceutical product for treating a disease or condition. The CD4-CD8+ T cells are produced by the method according to the first aspect.

[0031] In a sixth embodiment, a cell-stimulating particle is provided. The cell-stimulating particle comprises a particle and a modified antigen immobilized on the surface of the particle. The modified antigen comprises one or more amino acid substitutions from the natural antigen. The modified antigen has greater solubility and / or stability in vitro compared to the natural antigen.

[0032] In one embodiment, the modified antigen includes the extracellular domain of the antigen.

[0033] In one embodiment, the modified antigen is covalently conjugated to the particle.

[0034] In one embodiment, the particles are composed of a material selected from the group consisting of polystyrene, iron oxide, and gold.

[0035] In one embodiment, the particles are composed of polystyrene and magnetizable iron oxide.

[0036] In one embodiment, the modified antigen is the CD19 antigen.

[0037] In one embodiment, the modified antigen is a polypeptide having the sequence of SEQ ID NO: 8. [Brief explanation of the drawing]

[0038] Brief explanation of the drawing To facilitate understanding of the subject matter, embodiments are shown in the accompanying drawings as non-limiting examples.

[0039] [Figure 1A] Annotated images of CD19 protein mutants on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under both non-reducing (-) and reducing (+) conditions.

[0040] [Figure 1B] This graph quantifies the binding of the CD19 antibody FMC63 to 2D-coated CD19-Fc and CD19.1-Fc as measured by direct enzyme-linked immunosorbent assay (ELISA).

[0041] [Figure 1C] The graph above shows the quantified thermal fusion curve of the CD19 protein mutant, and the graph below shows the first derivative plot of the fusion curve.

[0042] [Figure 2] This graph quantifies the binding of FITC-labeled CD19.1 to primary anti-CD19 CAR-T cells. MFI: mean fluorescence intensity, conc.: concentration.

[0043] [Figure 3] This graph quantifies the density of CD19.1-Fc and CD19.1-His proteins on beads, as measured by the bicinchoninate assay (BCA).

[0044] [Figure 4] This graph shows the quantification of CD69 expression levels in primary unmodified T cells (top) and primary CD19 CAR-T cells (bottom) using flow cytometry after using CD19-coated beads or other activation methods.

[0045] [Figure 5A] These graphs quantify cell proliferation (top) and viability (bottom) in primary CD19 CAR-T cells when using cytokines and CD19-coated beads ("IL-2 CD19"; "IL7 / IL-15 CD19") or other activation methods.

[0046] [Figure 5B] Flow cytometry plots of marker expression in primary CD19 CAR-T cells with no stimulation (left) or with CD19-coated bead stimulation (right).

[0047] [Figure 5C] This graph quantifies the proliferation rate of primary CD19 CAR-T cells after 6 days of culture using cytokines, CD19-coated beads, or other activation methods.

[0048] [Figure 6A] This graph quantifies CD19 CAR-Jurkat cell activation by soluble OKT3 or CD19.1-Fc.

[0049] [Figure 6B] This graph quantifies CD19 CAR-Jurkat cell activation by 2D-coated CD19.1-Fc compared to other stimulation conditions.

[0050] [Figure 6C] This graph quantifies CD19 CAR-Jurkat cell activation by 2D-coated CD19.1-His compared to other stimulation conditions.

[0051] [Figure 6D] This graph quantifies CD19 CAR-Jurkat cell activation by 2D-coated FMC63 compared to 2D-coated CD19.1-His and other stimulation conditions.

[0052] [Figure 7] This graph quantifies the luminescence response of 41BB-NFAT-Jurkat cells to stimulation with CD19.1-coated beads, compared to other stimulation conditions.

[0053] [Figure 8] This graph quantifies the luminescence response of 41BB-NFAT-Jurkat cells to stimulation with CD19.1-coated beads, compared to other stimulation conditions.

[0054] [Figure 9] This graph quantifies the maximum half-volume effective concentration (EC50) of FMC63 anti-CD19 antibody binding to the following: (A) CD19.1-Fc coated beads, (B) CD19.1-His coated beads, and (C) wild-type (wt) CD19-His coated beads.

[0055] [Figure 10A] This graph quantifies the viability of iPSC-derived CAR+ T lineage cells after culturing them using commercially available DL4 coating (StemSpan® Lymphocyte Differentiation Coating Material, STEMCELL Technologies) or engineered thymic niches (2D ETN) with immobilized Notch ligand and CD19-coated beads in various ratios.

[0056] [Figure 10B]This graph quantifies the proliferation rate of iPSC-derived CAR+ T lineage cells after culturing them with commercially available DL4-coated or 2D ETN and CD19-coated beads in various ratios.

[0057] [Figure 10C] This graph quantifies the viability of iPSC-derived CAR+CD4-CD8+(CD8SP) cells after culturing them using commercially available DL4-coated or 2D ETN and CD19-coated beads in various ratios.

[0058] [Figure 10D] This graph quantifies the percentage of iPSC-derived CAR+CD4-CD8+(CD8SP) cells generated after culturing with commercially available DL4-coated or 2D ETN and CD19-coated beads in various ratios.

[0059] [Figure 11A] This graph quantifies the percentage of iPSC-derived CAR+ cells that are CD8αα+ (CD8aa, dark bars) and CD8αβ+ (CD8ab, light bars) after culturing with commercially available DL4-coated and CD19-coated beads in various ratios.

[0060] [Figure 11B] This graph quantifies the percentage of iPSC-derived CAR+ cells that are CD8αα+ (CD8aa, dark bars) and CD8αβ+ (CD8ab, light bars) after culturing with various ratios of 2D ETN and CD19-coated beads.

[0061] [Figure 12A] This graph quantifies the cytotoxicity of iPSC-derived T cell lineage cells generated by various methods, as evaluated by co-culture assays with CD19+ Raji cells (WT Raji) at various effector-to-target (E:T) ratios.

[0062] [Figure 12B] This graph quantifies the cytotoxicity of iPSC-derived T cell lineage cells generated by various methods, as evaluated by co-culture assays with CD19+ Raji cells (WT Raji) at various effector-to-target (E:T) ratios.

[0063] [Figure 13A] This graph quantifies the cytotoxicity of a bulk population of iPSC-derived CAR+ T cells compared to primary untransduced T cells (primary Untxd T) and primary CD19 CAR-transduced cells (primary CAR T) with CD19+Raji cells (WT Raji, dark bars) or CD19+Raji cells (WT Raji, light bars).

[0064] [Figure 13B] This is a flow cytometry plot showing marker expression and selection strategies for iPSC-derived CAR+ T cell lineages. The outlines indicate CD4-CD8α- (double negative, DN, lower left quadrant) and CD4-CD8α+ (lower right quadrant) cell populations.

[0065] [Figure 13C] This graph quantifies the cytotoxicity of selected iPSC-derived DN (CD8-CD4-iPSC CAR DN) and CD4-CD8+ (CD8+CD4-iPSC CAR SP) cell populations against CD19+Raji cells (WT Raji, dark bars) or CD19+Raji cells (WT Raji, light bars), compared to primary CD19 CAR transduced (primary CD8+CD4-CAR T) cells.

[0066] [Figure 14A] This is a flow cytometry plot showing marker expression in CAR-modified iPSC-derived T cell lineage cells cultured on 2D ETN for 14 days without CD19-coated beads.

[0067] [Figure 14B]This graph quantifies the survival rate of CAR-modified iPSC-derived T-cells cultured for 14 days on a 2D ETN without CD19-coated beads.

[0068] [Figure 14C] This graph quantifies CAR expression in CAR-modified iPSC-derived T-cells cultured on 2D ETN for 14 days without CD19-coated beads.

[0069] [Figure 15A] Figure 14 shows flow cytometry plots illustrating marker expression in CAR-modified iPSC-derived T cell lines after long-term culture under conditions of no stimulation (top) or CD19-coated bead stimulation (bottom).

[0070] [Figure 15B] Figure 14 shows a graph quantifying the survival rate of CAR-modified iPSC-derived T cell lines after long-term culture under conditions of no stimulation (top) or with CD19-coated bead stimulation (bottom).

[0071] [Figure 16A] This graph quantifies the survival rate of iPSC-derived T cell lineage cells cultured with CD19-coated beads.

[0072] [Figure 16B] This graph quantifies the time-dependent proliferation rate of iPSC-derived T-cells cultured with CD19-coated beads.

[0073] [Figure 17] This is a flow cytometry plot showing marker expression in iPSC-derived T cell lineage cells cultured with CD19-coated beads.

[0074] [Figure 18A] This is a flow cytometry plot showing marker expression in iPSC-derived T lineage cells cultured with CD19-coated beads.

[0075] [Figure 18B] This graph quantifies the cytotoxicity of iPSC-derived T cell lineages cultured with CD19-coated beads, compared to primary T cells and target cell controls, as evaluated by target cell expression of luciferase from both wild-type (WT) and CD19 knockout (KO) Raji cells.

[0076] [Figure 19] This graph quantifies the cytotoxicity of iPSC-derived T cell lineage cells cultured with CD19-coated beads, compared to primary T cells and target cell controls, when evaluated by co-culturing with A549 CD19+ cells (left) or A549 CD9- cells (right) at various effector-to-target (E:T) ratios.

[0077] [Figure 20A] This graph quantifies the cytotoxicity of iPSC-derived T cell lineages cultured with CD19-coated beads compared to primary T cells and target cell controls at various effector-to-target (E:T) ratios. It shows the first step of a serial restorative assay ("Stim#1 Count") using target cells.

[0078] [Figure 20B] This graph quantifies the viability of iPSC-derived T cell lineage cells cultured with CD19-coated beads, compared to primary T cells and target cell controls at various effector-to-target (E:T) ratios. It shows the first step of a serial restimulation assay ("Stim#1 viability") using target cells.

[0079] [Figure 20C] This graph quantifies the cytotoxicity of iPSC-derived T cell lineages cultured with CD19-coated beads compared to primary T cells and target cell controls at various effector-to-target (E:T) ratios. The results of a second serial restimulation assay using target cells ("Stim#2 Count") are also shown.

[0080] [Figure 21] This graph quantifies the cytotoxicity of iPSC-derived T cell lineages cultured with CD19-coated beads compared to primary T cells and target cell controls at various effector-to-target (E:T) ratios in a serial restorative assay.

[0081] [Figure 22] Figure 21 shows a graph quantifying the expression of exhaustion markers (TIGIT, TIM3, LAG3, PD-1) in iPSC-derived T cell lineages cultured with CD19-coated beads, compared to primary T cells after the first cycle of the serial restimulation assay. The expression levels were 4, 3, 2, 1, or none (0).

[0082] [Figure 23A] This graph quantifies the survival rate of iPSC-derived T cell lineage cells 24 hours (day 19) after CD19 bead stimulation with 2D or 3D ETN.

[0083] [Figure 23B] This graph quantifies the expression of cellular markers (CD4-CD8+, CD8 SP; CD4+CD8+, DP; CD4+CD8-, CD4 ISP; CD4-CD8-, DN) in iPSC-derived T cell lineage cells 24 hours (day 19) after CD19 bead stimulation with 2D or 3D ETN.

[0084] [Figure 23C] This graph quantifies the percentage of CD8αα+ (CD8 aa) cells or CD8αβ+ (CD8 ab) cells among iPSC-derived T lineage cells 24 hours (day 19) after CD19 bead stimulation with 2D or 3D ETN.

[0085] [Figure 23D] This graph quantifies the survival rate of iPSC-derived T cell lineage cells 48 hours after CD19 bead stimulation with 2D or 3D ETN (day 21) followed by recovery.

[0086] [Figure 23E] This graph quantifies the expression of cellular markers (CD4-CD8+, CD8 SP; CD4+CD8+, DP; CD4+CD8-, CD4 ISP; CD4-CD8-, DN) in iPSC-derived T cell lineage cells 48 hours after CD19 bead stimulation with 2D or 3D ETN (day 21) and subsequent recovery.

[0087] [Figure 23F] This graph quantifies the percentage of CD8αα+ (CD8 aa) cells or CD8αβ+ (CD8 ab) cells among iPSC-derived T lineage cells after 48 hours of recovery following CD19 bead stimulation with 2D or 3D ETN (day 21).

[0088] [Figure 24A] This graph quantifies CAR expression in iPSC-derived T cell lineage cells 24 hours after CD19 bead stimulation with 2D or 3D ETN.

[0089] [Figure 24B] This graph quantifies CAR expression in iPSC-derived T cell lineage cells 48 hours after CD19 bead stimulation with 2D or 3D ETN (day 21) and subsequent recovery.

[0090] [Figure 25] 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.

[0091] [Figure 26A] This graph quantifies the cell viability of cells from iPSC-derived HSPCs over 28 days of differentiation under 2D ("NTX4B3-4 2D", solid line) or 3D ("NTX4B3-4 3D", dashed line) differentiation processes.

[0092] [Figure 26B]This graph quantifies the cumulative proliferation rate of cells throughout 28 days of differentiation from iPSC-derived HSPCs under 2D ("NTX4B3-4 2D", solid line) or 3D ("NTX4B3-4 3D", dashed line) differentiation processes.

[0093] [Figure 27] This is a flow cytometry plot of marker expression in NTX4B3 cells cultured for 17 or 24 days under 2D differentiation conditions.

[0094] [Figure 28] These are flow cytometry plots of marker expression in NTX4B3 cells cultured under 3D differentiation conditions for 17, 24, or 28 days.

[0095] [Figure 29A] This graph quantifies the cell viability after culturing iPSC-derived DP with antigen beads.

[0096] [Figure 29B] This graph quantifies the cumulative growth rate after culturing iPSC-derived DP with antigen beads.

[0097] [Figure 30] This is a flow cytometry plot of marker expression in NTX4B3 cells cultured with antigen beads on day 35.

[0098] [Figure 31] 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 using antigen-coated beads. The normalized green area (vertical axis) represents the number of CD19+ / + or CD19- / - target cells for each culture condition.

[0099] [Figure 32]This is a flow cytometry plot of marker expression in CD8+ cells prepared from iPSCs generated using antigen-coated beads.

[0100] [Figure 33] This graph quantifies the cumulative cytotoxicity and proliferation rate during an in vitro serial restorative assay in primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads.

[0101] [Figure 34] Flow cytometry plots of marker expression in iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads, before (top) and after (bottom) an in vitro serial restimulation assay.

[0102] [Figure 35A] This graph quantifies memory cell marker expression before ("baseline") and after ("Stim 4") an in vitro serial restorative assay in primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads. Cells were classified into various 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+; and terminally differentiated effector memory cells (TEMRA), CD62L-CD45RA+CD45RO+ that reexpress CD45RA. The percentage of cells within each memory subset is shown.

[0103] [Figure 35B]This graph quantifies the expression of exhaustion markers in primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads, before ("baseline") and after ("Stim 4") an in vitro serial restimulation assay. It shows the percentage of cells expressing various exhaustion markers.

[0104] [Figure 36A] This graph quantifies the secretion of the effector cytokine interferon-γ (IFN-γ) during an in vitro serial restorative assay in primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads.

[0105] [Figure 36B] This graph quantifies the secretion of the effector cytokine Granzyme B during an in vitro serial restorative assay in primary and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads.

[0106] [Figure 36C] This graph quantifies the secretion of the effector cytokine TNF-α during an in vitro serial restorative assay in primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads.

[0107] [Figure 37] This graph quantifies the cumulative proliferation rate during an in vitro serial restorative assay in primary CD8+ CAR-T cells and iPSC-derived CD8+ CAR-T cells generated using antigen-coated beads. [Modes for carrying out the invention]

[0108] 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.

[0109] Generally, this disclosure provides cell-stimulating particles, methods for generating CD4-CD8+ cell populations from stem cells / progenitor cells using cell-stimulating particles, CD4-CD8+ cell populations generated by the methods disclosed herein, and the use of CD4-CD8+ cell populations in the manufacture of pharmaceuticals for treating diseases or conditions.

[0110] 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.

[0111] 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) 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 have the ability to produce more limited or specific types of blood cells and are pluripotent or lineage-restricted cells derived from HSCs. Hematopoietic stem cell / progenitor cells (HSPCs) typically exist as a heterogeneous population in vivo and are used as such in this specification. HPCs and HSPCs may be characterized by the expression of one or more CD34, CD43, CD31, and CD45.

[0112] 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 hematopoietic 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.

[0113] 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 differentiate from progenitor T cells through the stages of CD4-CD8-(double negative, DN), CD4+CD8-(CD4 immature single positive, CD4ISP), early CD4+CD8+(double positive, DP), late DP, and CD4-CD8+(CD8 single positive, CD8SP) and CD4 single positive (CD4SP). Late DP can be characterized by the presence of CD4+ / CD8A+ / CD8B+ / CD3+ and TCRαβ+. At the late DP stage, the differentiation fate to the TCRγδ lineage is not open, the cell size is reduced, 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 can also be characterized by the cell surface expression of CD3 and either TCRγδ (γδ T cells) or TCRαβ (αβ T cells).

[0114] 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).

[0115] As used herein, “Notch signaling ligand” refers to any ligand that has the ability to interact with the Notch protein receptor to control differentiation and fate determination of T cells. Examples of Notch signaling ligands include delta-like 4 (DL4), delta-like 1 (DL1), delta-like 3 (DL3), Jagged1, and Jagged2.

[0116] 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 signal peptide portion of DL4. For example, a commercially available product (Sino Biologicals) containing the extracellular domain (Met1-Pro524) of human DL4 (accession number NP061947.1 of full-length DL4; SEQ ID NO: 1) fused at the C-terminus to the Fc region of human IgG1 is a DL4 protein suitable for use in the methods provided herein.

[0117] 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. The definition also includes variants of polypeptides, oligopeptides, peptides, and proteins that have amino acid sequence identity with a given polypeptide, oligopeptide, peptide, or protein. The percentage of identity may be, for example, over a certain length, for example, over the entire length of the polypeptide, 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.

[0118] 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 Ig 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 signal peptide portion of VCAM-1 (QIDSPL (SEQ ID NO: 2) or TQIDSPLN (SEQ ID NO: 3)). For example, the commercially available mouse VCAM-1-Fc chimeric protein (R&D Inc.) is a fusion of the Phe25-Glu698 region of mouse VCAM-1 (accession number CAA47989 of full-length mouse VCAM-1; SEQ ID NO: 4) and the Fc region of human IgG1, and is an example of a VCAM-1 protein available herein. The use of at least a portion of human VCAM-1 (accession numbers P19320, NP001069, EAW72950; SEQ ID NO: 5 of full-length human VCAM-1) may also be suitable for use in the manner provided herein. In this specification, references to VCAM-1 also include mutants 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. The definition also includes mutants such as polypeptides, oligopeptides, peptides, and proteins that have amino acid sequence identity with a given polypeptide, oligopeptide, peptide, or protein. 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).

[0119] As used herein, “two-dimensional engineered 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.

[0120] As used herein, “three-dimensional engineered 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.

[0121] As used herein, “antigen-presenting particles,” “antigen-coated beads,” or “antigen beads” 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”).

[0122] The three-dimensional (3D) substrate 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. Several approaches may be used individually or in combination to produce protein-coated particles, such as physicoadsorption driven by protein affinity to the particle material, chemical conjugation by reaction with amines, carboxyls, thiols, epoxy, azide reactive groups, or 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 be composed of, for example, polystyrene, iron oxide, polystyrene and magnetizable iron oxide (magnetic polystyrene), gold, or other suitable materials known in the art. ETNs and antigen beads may be used to culture cells on tissue culture plates, flasks, or other containers used for culturing cells.

[0123] As used herein, “immobilized” or “surface-bound” refers to ligands such as Notch signaling ligands or integrin ligands, antigens, peptides, or proteins that bind to a substrate via covalent or non-covalent interactions, affinity-based interactions, or other appropriate forms of interaction.

[0124] As used herein, a “richly containing” cell population means a cell population containing one or more cell phenotypes (e.g., CD4-CD8+(CD8SP), CD4+CD8+(DP), CD4+CD8-(CD4 ISP), CD4-CD8-(DN)) that exhibits a higher absolute number or ratio of the cell phenotype (e.g., CD4-CD8+(CD8SP)) compared to other cell phenotypes, and at least 25% of the cell population consists of single-cell phenotypes.

[0125] As used herein, the term “subjects” means vertebrates, preferably mammals (e.g., non-human mammals), more preferably primates, and even more preferably humans. Mammals include, but are not limited to, primates, humans, livestock, sports animals, and pets.

[0126] As used herein, the terms “treatment,” “to treat,” or “to treat” refer to an approach to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, an increase in the immune response, an increase in the T-cell response, a reduction in the degree of damage from a disease, condition, or disorder, a reduction in the duration of a disease, condition, or disorder, and / or a reduction in the number, severity, or duration of symptoms associated with a disease, condition, or disorder. The terms include administering any of the compounds, agents, drugs, or pharmaceutical compositions of this disclosure to prevent or delay the onset of one or more symptoms, complications, or biochemical signs of a disease or condition; reducing or improving one or more symptoms; shortening or reducing the duration of symptoms; or preventing or inhibiting further onset of a disease, condition, or disorder. Treatment may 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 mitigation after the onset of a disease, condition, or disorder. Beneficial or desired clinical outcomes may be an increase or decrease (as appropriate) 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% compared to an appropriate control, e.g., a subject that received no treatment.

[0127] As used herein, the terms “administer” or “dosage” refer to the placement of a drug, medicine, compound, or pharmaceutical composition disclosed herein onto 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 may be administered by any suitable route that results in effective treatment on the subject. Routes of administration of the compounds and pharmaceutical compositions disclosed herein include, but are not limited to, intravenous or intraperitoneal administration routes, or combinations thereof.

[0128] 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. For 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. For 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 drug; 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.

[0129] For the purposes of this disclosure, an effective dose of a cell population or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, 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 agents, drugs, compounds, or pharmaceutical compositions. Thus, “effective dose” may be considered in relation to the administration of one or more therapeutic agents, and a monotherapy agent may be considered effective if, in combination with one or more other agents, the desired outcome is 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.

[0130] 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, all standard pharmaceutical carriers such as phosphate-buffered saline, water, 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).

[0131] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0132] The phrase "and / or" should be understood to mean "either or both" of the elements thus combined, i.e., 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 to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements); and so on.

[0133] As used herein, the phrase “one or more” in relation to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of every element specifically enumerated in the list of elements, nor excludes 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.

[0134] 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 the 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 the numbers above and below a given value with a variance of 10%. In certain embodiments, the term "approximately" is used to modify the numbers above and below a given value with a variance of 5%. In certain embodiments, the term "approximately" is used to modify the numbers above and below a given value with a variance of 1%.

[0135] 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.

[0136] 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.

[0137] 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

[0138] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings that are generally understood by those skilled in the art. In general, 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.

[0139] 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 (J. M. Miller and M. Cales, eds., 1987); Current Protocols in Molecular Biology (F. M. Asubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Oligan et al., eds., 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Vol. 3 版This is described in detail in *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 *Limmunobiology* (CA Janeway and P. Travers, 1997).

[0140] Hematopoietic stem cell / progenitor cell population Generally, the in vitro methods provided herein for generating CD4-CD8+ cell populations involve culturing hematopoietic stem cells and / or progenitor cells under conditions and for a period of time suitable for differentiation into T cell lineage populations in the presence of Notch signaling ligands and antigen stimulation.

[0141] Hematopoietic stem cells / progenitor cells (HSPCs) typically exist as heterogeneous populations in vivo and are used as such heterogeneous populations as described herein. HPCs and HSPCs may be characterized by the expression of one or more CD34, CD43, CD31, and CD45.

[0142] 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).

[0143] 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 as disclosed herein.

[0144] Cell culture system Cells can 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 can be carried out under static conditions, dynamic or agitated conditions, or a combination of static and dynamic conditions. The bioreactor can be any type known in the art, and any type of processing / culture conditions and methods can be used, such as batch processes, fed-batch processes, and perfusion culture methods and conditions.

[0145] Notch Ligand Substrate In one embodiment, HSPC 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.

[0146] In one embodiment, cells are cultured in a three-dimensional culture system utilizing a suitable three-dimensional substrate that 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 activate Notch signaling.

[0147] For example, Notch signaling ligands, such as 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. 2019157597.

[0148] In another example, 3D ETN beads may be prepared by affinity capturing DL4 and VCAM-1, which are supported with 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 times protein molar excess) at room temperature with continuous stirring for 60 minutes. At the end of the incubation period, excess free protein is removed by magnetic separation, followed by buffer exchange. This procedure is repeated four more times, after which the 3D ETN beads are concentrated 10-fold for storage.

[0149] The quantification of protein immobilization can be carried out according to methods known in the art, such as a colorimetric bicinchoninic acid (BCA) assay, an immunofluorescence assay, or other known detection methods.

[0150] 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 stage or the 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).

[0151] Manipulated 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 other 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] TIFF2026521509000003.tif254170TIFF2026521509000004.tif251170TIFF2026521509000005.tif254170TIFF2026521509000006.tif224170

[0152] 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] TIFF2026521509000008.tif18170

[0153] 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 region (TRAC) gene (TRAC- / -).

[0154] Cell populations derived from culturing stem cells / progenitor cells using the methods provided herein are intended to be included in pharmaceutical compositions.

[0155] Cell populations derived from culturing stem cells / progenitor cells using the methods provided herein are further intended to be used 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.

[0156] For example, subjects suffering from oncological or autoimmune diseases, conditions, or disorders may benefit from administration of CD4-CD8+ cell populations as described herein.

[0157] The pharmaceutical compositions provided herein may be administered to subjects to treat a target cancer or autoimmune disorder.

[0158] The pharmaceutical compositions provided herein may be administered to a subject in an effective or therapeutically effective dose. Those skilled in the art will be able to determine such a 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, and the specific composition or route of administration selected. Those skilled in the art will also know how to select an appropriate route of administration and how to administer the compounds and compositions described herein.

[0159] 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 and then adjusted as necessary in response to the clinical response.

[0160] 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 the administration of the pharmaceutical compositions for the therapeutic treatment described above. In some embodiments, kits for preparing single-dose units are provided.

[0161] 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 recorded on a magnetic or optical storage disk) are also acceptable.

[0162] Antigen bead composition The base beads can be made of any material suitable for covalent or non-covalent coating with functional proteins, including polystyrene (PS particles) and styrene copolymers such as maleic acid (SMA particles), acrylic acid (SAA particles), and divinylbenzene (PS-DVB particles), silica (silica particles) including acrylonitrile (PAN particles), butadiene (PBR particles), and vinyl acetate (PVA particles), polymethyl methacrylate (PMMA particles), silica crosslinked with tetraethyl orthosilicate (TEOS) and / or doped with metal ions, and particles made from gold (gold particles).

[0163] Bead size The beads may be sized for easy handling in an aqueous suspension.

[0164] Protein coating In addition to the methods described above, the antigen can be directly conjugated to the carboxyl functional group on the bead. Furthermore, it is conceivable that heterobifunctional PEGs of various lengths can be conjugated to the beads, and then conjugated to the terminal carboxyl groups of the PEGs. This approach may improve the shelf life of CD19 beads and allow for control of their potency.

[0165] antigen In addition to the antigens listed in Tables 1 and 2 above, it is intended that other antigens known in the art may be used.

[0166] The antigen-presenting particles described herein are intended to include, for example, signaling regions of the antigen, such as the extracellular domain or external domain of the antigen. The antigen may be an engineered sequence or a mutant sequence derived from a natural antigen. The antigen may also be an antigen-mimicking peptide.

[0167] Example 1: Preparation of ETN and CD19.1 coated beads 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]

[0168] 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) (- / -). Appropriate coating amounts of DL4 and VCAM-1 diluted in DPBS per well were added to the tissue culture plates, as shown in Table 4. [Table 4]

[0169] 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]

[0170] Preparation of 3D ETN The dosage of 3D ETN can be calculated according to the bead diameter and can be 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]

[0171] 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 ) can vary. Table 7 provides the calculation of the Notch signaling ligand concentration for the range of bead dosages and the Notch signaling ligand density for 3.05 μm polystyrene beads.

Table 7

[0172] 3D ETN can also include surface-bound VCAM-1. VCAM-1 can be immobilized on 3D ETN at an input molar ratio in the range of 1:6 to 10:1 of DL4:VCAM-1. In one embodiment, 3D ETN is prepared at an input molar ratio of 2.5:1 of DL4:VCAM-1. In one embodiment, the final density of surface-bound VCAM-1 on the beads is equivalent to the density of the Notch ligand. For example, the surface area of VCAM-1 per unit volume can be equivalent to the surface area of the Notch ligand per unit volume shown in Table 7 above.

[0173] Cells can be cultured at a density suitable for the culture scale and format. In microplate culture, cells can be cultured, for example, at 2.5×10 5 ~2×10 6 cells / mL. In STR culture, cells can be cultured, for example, at 5×10 4 ~6×10 6 cells / mL.

[0174] Preparation of CD19 Antigen First, it was evaluated whether recombinant CD19 proteins, including wild-type and selected mutants, meet the criteria for use in preparing cell stimulation / proliferation reagents, specifically whether the recombinant proteins can be functionally produced and functionally immobilized.

[0175] Artificial antigen-presenting cells expressing the wild-type sequence of the CD19 external domain (e.g., CD19-transduced Raji cells) have been used for stimulating and proliferating anti-CD19 CAR-T cells, but the use of recombinant wtCD19 (external domain) in defined systems such as protein-functionalized beads has not been reported. Beads functionalized with anti-FMC63 scFv antibody have been shown to induce small to moderate proliferation on FMC63-derived anti-CD19 CAR T cells (Philipson, 2020). In addition to low efficiency, the use of anti-idiotype antibodies limits the applicability to specific strains of CAR T cells (i.e., in this case, FMC63-derived anti-CD19 CAR cells). Instead, antigen-coated beads may be applicable to all anti-CD19 CAR cells, regardless of CAR design.

[0176] The exodomain of wild-type human CD19 was produced in various designs using the HEK cell line according to standard recombinant protein batch production conditions. The protein designs tested included: (1) full-length wtCD19 exodomain (etCD19, Pro20-Lys291, AcroBiosystems) fused to a human IgG1a Fc tag for improved solubility and dimer presentation; (2) etCD19 fused to human serum albumin domain 2 (AD2) for increased solubility; (3) cleaved etCD19 (21-271) fused to a human IgG1a Fc tag for improved solubility and dimer presentation; and (4) etCD19 exodomain fused to a human IgG1a Fc tag containing an artificial secretion signal peptide. Lobner et al. reported the homogeneous recombinant preparation of etCD19-AD2 using a stably transfected CHO-K1 cell line and an optimized cell culture process (specifically, the supplementation of chemical chaperones and the use of a semi-continuous perfusion culture process) (Lobner 2020). The authors also demonstrated that recombinant wtCD19-AD2, when immobilized on a glass-supported bilayer lipid with the adhesion molecule ICAM1 and the co-stimulatory molecule B7-1, activates CD19 CAR-T cells, as evidenced by the induction of intracellular calcium flow and the formation of supramolecular activation clusters upon contact (Lobner, 2020). Nevertheless, recombinant production of the four designs described above, including etCD19-AD2, results in low titers, and the one-step purified protein forms higher-order oligomers.

[0177] The preparation of soluble functional proteins is crucial for the successful development of antigen-functionalized T cell proliferation reagents. Klesmith et al. created several engineered variants of etCD19 with improved solubility using directed evolution (Klesmith, 2019). It has also been previously demonstrated that engineered etCD19 variants, when fused to scFvs targeting cancer biomarkers such as CD20, can redirect anti-CD19 CAR T cells and kill CD20-expressing cells (Klesmith, 2019). In this disclosure, we confirm that these engineered variants of etCD19 with improved solubility can be used to prepare functional reagents for CAR cell proliferation. As an example, we selected CD19.1, one of the engineered variants reported by Klesmith, and created His-tagged and hFc-tagged versions in HEK cell lines using conventional batch culture conditions. Good titers were achieved in both versions. The sequences of wtCD19, CD19.1, and CD19.1-Fc are shown in Table 8 below. [Table 8]

[0178] The solubility of etCD19 mutants was compared using SDS-PAGE under non-reducing conditions. Both His-tagged CD19.1 and hFc-tagged CD19.1 migrated as single bands consistent with their theoretical molecular weights under non-reducing conditions (Figure 1A), while commercially available wild-type etCD19 protein (AcroBiosystems) and custom-produced wild-type etCD19 protein (lanes 3, 5, 7) formed higher-order oligomers (Figure 1A). Direct ELISA of the CD19 antibody FMC63 against 2D-coated CD19-Fc and CD19.1-Fc demonstrated that CD19.1 was superior to wtCD19 in binding to FMC63 (Figure 1B). Furthermore, the thermal stability of etCD19 mutants was characterized using a protein thermal shift assay (GloMelt, Biotium). CD19.1-His and CD19.1-Fc in PBS showed normal thermal denaturation profiles with denaturation midpoints of 69.9°C and 68.3°C, respectively (Figure 1C). In contrast, the thermal denaturation profiles of wild-type ecCD19 purchased from a reliable vendor (AcroBiosystems) or custom-manufactured did not show any detectable denaturation transitions (Figure 1C). Furthermore, WT ecCD19 exhibited high fluorescence at room temperature, suggesting that the protein was partially unfolded and / or highly hydrophobic (Figure 1C).

[0179] The binding affinity of CD19 mutants to immobilized FMC63 was also characterized using biolayer interferometry. The equilibrium dissociation constants (K) of CD19.1-Fc and CD19.1-His were determined. D The K values ​​were 0.103 nM and 3.18 nM, respectively. The custom-produced WT etCD19-Fc showed strong nonspecific binding to the blank sensor surface and weak binding to the FMC63 surface, suggesting that the protein formed inactive soluble aggregates. In comparison, the K values ​​of purchased WT etCD19-His and WT etCD19-His (Acrobiosystems) were... D The values ​​are 3.88 nM and 7.18 nM, respectively. However, stronger nonspecific binding is observed for these proteins.

[0180] The specific binding of CD19.1 (both -His-tagged and -hFc-tagged versions) to anti-CD19 CAR cells was evaluated by titrating FMC63-derived CAR cells with FITC-labeled CD19.1. EC50 was determined by fitting the binding curve to a four-parameter dose-response function. When titrated with anti-CD19 28z primary T cells, the EC50 values ​​for CD19.1-Fc and CD19.1-His were 1.9 nM and 15.8 nM, respectively (Figure 2). In comparison, the binding curve for commercially available FITC-CD19 (AcroBiosystems) to the same CAR cells shifted to much higher concentrations (Figure 2). Thus, engineered CD19.1 showed a much stronger binding strength to anti-CD19 CAR cells than wtCD19.

[0181] Preparation of CD19 antigen beads CD19.1 protein was conjugated using 4.5 μm smooth carboxyl-functionalized magnetic polystyrene beads by EDC / NHC conjugation as described below.

[0182] The beads were resuspended in the vial by gentle vortexing. 200 μL of beads were transferred to a low-protein-conjugated Eppendorf tube. 200 μL of activation buffer was added, mixed thoroughly, and the supernatant was decanted. These steps were repeated twice. After the final wash, the beads were resuspended in 200 μL of activation buffer. Freshly prepared EDC and NHS were quickly added to 200 μL of washed beads and incubated at room temperature for 30 minutes by mixing on a rotary mixer. The tube was placed on a magnet for 1 minute, and the supernatant was discarded. The tube was removed from the magnet, and the washed beads were resuspended in the same volume of conjugation buffer as the initial volume of beads (i.e., 200 μL) and mixed thoroughly. This washing procedure was repeated twice. The Eppendorf tube was placed on a magnet, and a volume equal to the amount of protein to be added (i.e., 200 μL minus the volume of protein) was carefully removed. Based on the total moles of carboxyl groups available for protein conjugation on the beads, 0.5 × moles, 1.0 × moles, 2.0 × moles, and 5.0 × moles of CD19.1 protein were added to the reaction mixture, respectively. The total conjugation reaction volume was 200 μL. The reaction was allowed to proceed at room temperature for 2.5 hours with gentle tilting and rotation. The reaction was quenched by adding glycine and incubated at room temperature for 30 minutes with gentle mixing. A magnet was applied for 1 minute, and the supernatant was carefully transferred to a clean Eppendorf tube. The beads were washed three times with blocking buffer. The beads were resuspended in 200 μL of blocking buffer and mixed for at least 2 hours. A magnet was applied for 1 minute, the supernatant was discarded, and replaced with an equal volume of storage buffer. 200 μL of storage buffer was added, and the coated beads were stored at 2°C to 8°C until further use.

[0183] The density of CD19.1 protein on the beads was determined by performing a bicinchoninate assay (BCA) directly on the CD19.1 coated beads. The coating efficiency, expressed as a percentage of the theoretical maximum coverage for each condition, is shown in Table 9 below. [Table 9]

[0184] As expected, the maximum CD19.1 protein load on the beads was observed when the protein concentration was highest in the reaction mixture, i.e., at 5.0 × moles (Figure 3).

[0185] The binding capacity of CD19.1 antigen-coated beads was determined by titrating the beads with an excess amount of anti-CD19-FITC (FMC63 clone). The amount of anti-CD19-FITC bound to the beads was then calculated by interpolation from a standard curve created with anti-CD19-FITC. Briefly, 20 μL of uncoated or coated beads were incubated with anti-CD19-FITC (FMC63) at room temperature for 60 minutes while gently mixing. A magnet was applied, and the supernatant was carefully removed. The beads were washed three times with buffer. The fluorescence intensity of the supernatant and washes was analyzed. The washed beads were resuspended in 500 μL of buffer, and 200 μL was transferred to the wells of a black 96-well plate, where the fluorescence intensity was measured. The data indicate that higher anti-CD19 binding can be achieved by increasing antigen density (Table 10 below). This demonstrates the ability to control antigen loading and modulate cell activation. [Table 10]

[0186] Example 2: Culture of CAR T cells using CD19-coated beads CAR T cells were co-cultured with CD19.1Fc coated beads in a 1:1 ratio, and T cell activation was evaluated by measuring CD69 expression over time using flow cytometry. Human primary CAR T cells were seeded on U-bottom plates, and an equal volume of medium containing either an activation reagent (TransAct® (Miltenyi), CD3 / CD28 beads, CD19.1-FC beads) or a control (uncoated beads, Raji target cells) was added. The cells were cultured over time, and at predetermined time points, CAR T cells were stained for viability evaluation, fixed, and stained with a T cell activation flow panel. CD19.1-Fc coated beads showed specific activation of CAR+ primary T cells compared to unmodified primary T cells (Figure 4).

[0187] For proliferation, CAR T cells were cultured in X-VIVO® 15 medium for approximately one week with (1) IL-2 (200 U / mL) or (2) IL-7 (10 ng / mL) + IL-15 (10 ng / mL) in tissue culture plates. CAR-T cells were incubated at 37°C with 5% CO2, and the medium and cytokines were changed every two days. Via 2 cassettes on a Nucleocounter were used to measure cell viability and cell number. To assess the enrichment of CAR T cells, cells were stained for viability evaluation, and CAR was detected using a CAR-specific monoclonal antibody. CAR-T cells proliferated over time and showed improved viability when CD19-coated beads were used in the presence of IL-2 or IL-7 and IL-15 (Figures 5A, 5B). Stimulation with CD19 beads enhanced CAR expression (Figure 5C).

[0188] To investigate the activity of CD19.1 on CAR T cell activation, a luciferase reporter cell line was created by transducing Jurkat-Lucia NFAT cells (Invivogen) with the FMC63-scFv-41BBz CAR. Upon stimulation with PMA and ionomycin, Jurkat-Lucia NFAT cells initiated Lucia luciferase expression (data not shown). The modified CAR Jurkat-Lucia NFAT cell line responded to conventional T cell stimulants such as TransAct (Miltenyi) and anti-CD3 / anti-CD28 Dynabeads (ThermoFisher) (Figure 6A). When CD19.1 (either the -His-tagged or -hFc-tagged version) was added in soluble form, neither luciferase activity nor T cell activation was observed (Figure 6A). CD19.1 was then immobilized on 96-well tissue culture plates by passive adsorption. Specifically, the protein was added to a 96-well plate in 100 μL of PBS and incubated at 37°C for 2 hours. When CAR Jurkat NFAT cells were seeded on CD19-coated plates, a dramatic increase in luciferase activity was observed (Figure 6B, Figure 6C). The activation induced by both proteins was dose-dependent (Figure 6B, Figure 6C). CD19.1-Fc appeared to have a lower EC50 and therefore be more potent than CD19.1-His, but the maximum stimulation was comparable between the two. The lower EC50 observed for CD19.1-His may be due to lower coating efficiency, given its smaller size. Soluble CD19.1 binds to anti-CD19 CAR T cells (see Example 1.1B), but it was clearly demonstrated that immobilization is required for its T cell activation (Figure 6A vs. Figure 6B, 6C). Immobilized CD19.1 was very potent in CAR T cell activation.

[0189] For comparison, a commercially available anti-FMC63 scFv (clone FM3-Y45, AcroBiosystems) was also immobilized, and its ability to activate T cells was investigated. Clone FM3-Y45 also dose-dependently activated reporter cells (Figure 6D). However, it was not potent, and the activation intensity was more than 30 times lower than that of CD19.1 when coated at the same mass density (Figure 6D). It should be noted that the maximum stimulation caused by immobilized CD19.1 was 3.4 times stronger than stimulation by TransAct® added at the manufacturer's recommended dose (i.e., 1:100 dilution). Stronger activation is not necessarily advantageous in terms of T cell proliferation or cytotoxic activity, but it means a wider dynamic range of activation, thereby increasing the freedom of adjustment.

[0190] To evaluate the functional activity of CD19-coated beads, the 41BB-NFAT-Jurkat reporter cell line was treated with CD19-coated beads. 5E4 cells per well were seeded in 50 μL of medium in a U-bottom 96-well plate. Then, 50 μL of medium containing CD19-coated beads, BSA-coated beads, or conventional T-cell activating beads (CD3 / CD28 Dynabeads or TransAct® human T-cell activator) was added. At each time point, 20 μL of supernatant was removed, and 50 μL of reconstituted QUANTI-Luc Gold (luciferase substrate) reagent was added to each well. Luminescence was then measured using a spectrophotometer.

[0191] Cell activation by CD19.1-coated beads was higher than that by conventional CD3 / CD28 Dynabeads and soluble T cell activator TransAct® (Figure 7).

[0192] Next, we evaluated whether CD19.1-coated beads exhibited specificity for CD19 CAR T cells. Transduced NFAT-Jurkat cells were incubated with CD19.1-coated beads. No cell activation was observed with CD19.1-coated beads, but nonspecific stimulation was observed with CD3 / CD28 Dynabeads® and the soluble T cell activator TransAct® (Figure 8).

[0193] ELISA was performed to determine the maximum half-volume effective concentration (EC50) of CD19-coated beads (CD19.1-Fc, CD19.1-His, and wtCD19-His). PBS containing BSA (0.1% w / v) buffer (pH=7.4) was added to all wells and incubated at room temperature for 5 minutes. CD19.1 coated bead solutions were added to the wells of a low-binding, flat-bottomed 96-well plate, along with uncoated beads (+ / - antibody) as control and a blank. A DynaMag® side-skirt magnet was used to attract the beads to the side of the plate, and the supernatant was removed. Next, 100 μL of HRP-conjugated anti-human CD19 (FMC63) antibody was added to each well. The plate was sealed and incubated at room temperature for 1 hour with mixing. A DynaMag® side-skirt magnet was used to attract the beads to the side of the plate, and the supernatant was removed. The plate was washed three times with PBS containing 0.05% v / v Tween-20, and the supernatant was removed using a side-skirt magnet. 100 μL of 1-step Ultra TMB-ELISA (ThermoFisher) solution was added, the plate was covered, and incubated at room temperature in the dark for 30 minutes or until the desired color developed. The reaction was stopped by adding 100 μL of 2 M sulfuric acid. Using a DynaMag® side-skirt magnet, the beads were attracted to the side of the plate, and 100 μL was pipettered from each well and transferred to another 96-well plate for absorption measurement. The plate was evaluated within 30 minutes of stopping the reaction. Absorbance from each well was read at 450 nm and 550 nm, and the 550 nm value was subtracted from the 450 nm value. The curve was fitted using a 4-parameter logistic regression model, and the EC50 was calculated. CD19.1-His coated beads were observed to have the lowest EC50 compared to CD19.1-Fc and wtCD19-His (Figures 9A, 9B, and 9C).

[0194] Both wtCD19 and CD19.1Fc were coated onto beads by adding equal amounts of protein to the reaction mixture. The total protein density on the wtCD19-coated beads and the CD19.1Fc-coated beads was similar (Table 11). As described above, an anti-CD19 binding assay was performed on the beads, and it was confirmed that the binding capacity (functional antigen density) of the CD19.1Fc-coated beads was approximately 3.5 times that of the wtCD19-coated beads (Table 11). [Table 11]

[0195] Example 3: Culture of iPSC-derived T cell precursors and T cells using CD19-coated beads In this example, iPSC-derived polyclonal TRAC+ / +CAR-28z proT cells (Banks A-E) were matured by culturing them for 3 days with CD19 antigen beads (commercial DL4 coating or 2D DL4+VCAM ETN maturation days 18-21). Cell viability, proliferation rate, and marker expression were analyzed. Bulk viability was maintained at approximately 50% under all conditions (Figure 10A), and proliferation rates were consistent across conditions (Figure 10B). CAR stimulation with CD19 coated beads increased the viability of the CD4-CD8+ (CD8SP) population (Figure 10C) and increased %CD8SP (Figure 10D). Culturing with and without CD19 beads using commercially available DL4 coating (StemSpan® Lymphocyte Differentiation Coating Material, STEMCELL Technologies) mainly produced CD8αα+ cells (Figure 11A). Culture on 2D ETN (DL4&VCAM) generated a mixed population of CD8αα+ and CD8αβ+ cells, and the percentage of CD8αβ+ cells decreased with CD19 bead stimulation (Figure 11B).

[0196] In co-culture assays, iPSC-derived cells showed cytotoxicity against WT Raji cells comparable to that of primary T cells (Figure 12A). Nonspecific killing was also observed against CD19 KO Raji cells (Figure 12B). This may be due to the innate immunity-like properties of the generated CD8 αα+ cells. In co-culture assays, the overall cytolytic activity of iPSC-derived CAR T cells and primary CAR T cells against WT CD19-positive Raji cells was similar (Figure 13A). To investigate the contribution of subpopulations of iPSC-derived cells, CD4-CD8α-(DN) and CD4-CD8α+ cells were selected (Figure 13B) and co-cultured with target cells. Both DN and CD4-CD8α+ cells contributed to the killing of WT and CD19 KO Raji cells (Figure 13C).

[0197] In further experiments, CAR-introduced iPSC-derived progenitor T cells (18 days after differentiation from HSPCs) were cultured on 2D ETN with frequent medium changes, and cell surface marker expression was evaluated after 14 days (Figure 14A). Cell viability and CAR expression were evaluated throughout the culture period (Figures 14B, 14C).

[0198] Fourteen days later, cells were stimulated with CD19 antigen beads in a bead-to-cell ratio of 1:2 for 24 hours. After bead removal, cells were evaluated two days later. Extended maturation time and antigen-dependent stimulation improved the purity of CAR+CD4-CD8αβ+CD8 cells (Figure 15A). Cell viability was also evaluated before and after stimulation with CD19 antigen-coated beads (Figure 15B).

[0199] The culture protocol described above was repeated in three independent experiments using iPSC-derived progenitor T cells. Similar viability and proliferation rates were observed across the experiments (Figures 16A and 16B). Table 12 provides output results demonstrating that culture with CD19 antigen-coated beads resulted in the generation of CD8α+CAR+ cells (Figure 17). [Table 12]

[0200] Example 4: In vitro function of iPSC-derived CD8αα cells The in vitro function of CAR-modified iPSC-derived cells generated as described above was evaluated. The generated cells included CD4-CD8-DN and CD8αα+ phenotypes, as determined by flow cytometry (Figure 18A). CAR-modified iPSC-derived cells showed effective killing of CD19+ Raji wild-type cells and nonspecific killing of CD19-KO Raji cells (Figure 18B, "iPS NTX1A2").

[0201] In the serial restimulation assay, primary CD8 CAR T cells (donor 3215) or iPSC-derived DN / CD8αα+ cells were co-cultured with CD19-overexpressing A549 cells or CD19-negative A549 parental cell line. The A549 cell line expressed GFP. Each well was imaged for GFP-positive regions for 3 days, and four replication wells were used for each E:T condition. iPSC-derived DN / CD8αα cells showed specific and nonspecific cytotoxic activity during the first stimulation (Figure 19). Primary CAR T cells maintained function after the third stimulation, but despite similar viability, iPSC-derived CAR T cells could not maintain a sufficient number of cells to continue stimulation beyond the third (Figures 20A-20C). In two co-cultures with altered E:T ratios, iPSC-derived DN / CD8αα cells showed specific and nonspecific cytotoxic activity during the first stimulation, but this effect was lost during the second stimulation (Figure 21).

[0202] The expression of fatigue markers TIGIT, TIM3, LAG3, and PD-1 was evaluated in primary cells and iPSC-derived cells after initial stimulation with target cells. Unlike primary CAR-T cells, classical fatigue markers were not expressed in iPSC-derived CD8αα+ cells after initial stimulation (Figure 22).

[0203] Example 5: Culture of T cell precursors using CD19-coated beads and ETN beads In this example, PSC-derived T cell precursors were cultured in combination with genetically modified Thymic Niche (ETN) beads in the presence of CD19 antigen-coated beads. On day 18, the precursor cells were cultured on plate-bound 2D ETN or with ETN beads at relative bead doses of 0.5x and 0.05x. All conditions were cultured for 24 hours, both in and out of the presence of CD19 beads. After 24 hours, the conditions cultured with CD19 beads were debeaded and reseeded on either fresh 2D ETN or fresh ETN beads. Cell viability and phenotype were evaluated by flow cytometry at seeding (D18), 24 hours after CD19 antigen bead stimulation (D19), and 2 days after reseeding (D21).

[0204] After 24 hours of bead stimulation (day 19), the cell phenotype was similar for both 2D ETN and 0.5×3D ETN doses (Figure 23B, Table 13). Survival rate was slightly higher with 2D ETN compared with 3D ETN on day 19 (Figure 23A). After 48 hours of recovery (day 21), survival rates were similar between 2D ETN and 0.5×ETN bead doses, but the 0.5×ETN bead dose showed a higher proportion of CD4-CD8+ (CD8 SP) cells (Figure 23D, E, Table 13).

[0205] CAR expression was also higher in ETN bead culture compared to 2D ETN on day 21 (Figure 24, Table 13). [Table 13]

[0206] Example 6: Generation of iPSC-derived CD4-CD8+ cells using a multi-step differentiation protocol In this example, CAR-modified iPSC-derived CD34+ HPCs were differentiated into CD4-CD8+ T cells using a five-step protocol (protocol schematic, Figure 25). In Stage 1, CD34-positive HPCs were seeded in culture vessels coated with DL4 and VCAM-1 ("2D"), or in 0.5 times the amount (2.7 × 10⁶). 7CAR-modified cells were mixed with 3D ETN beads (beads / mL, "3D") and cultured in progenitor cell growth medium (SFEM II + LEM, STEMCELL Technologies) for 10 days to induce CD34-CD7+CD5+ ProT cells. At this point, the cells moved to stage 2, and the ProT cells were differentiated into early DP (CD4+CD8A+) cells over 7 days. 2 × 10⁶ CAR-modified cells were then processed. 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 a protein density of 0.1× and a bead dose of 0.5×. At the end of Stage 2, the early DP cells had insufficient expression of surface CD3 / TCR required for stimulation and required further maturation. In Stage 3, the early DP cells were reseeded at 6×10⁶ 6 The cells were re-seed at a high density of cells / mL, mixed with 3D ETN beads at 0.1x protein density and 0.25x bead volume, and cultured for a further 8–13 days in SFEM II+LMM medium (timing may be cell line-dependent) to promote maturation to late DP (CD4+CD8A+CD8B+CD3+TCRαβ+ / -TCRγδ+ / -) cells. Stage 4 was initiated by enriching late DP cells with a CD8-positive selection kit (e.g., STEMCELL Technologies), followed by re-seeding in a medium containing IL-21, and cultured for 7 days with the addition of CD19 antigen-coated beads (Example 1) to promote differentiation from DP to CD8SP.

[0207] The viability and cumulative proliferation rate were evaluated during stages 1-3 of the differentiation protocol. During the first 10 days of the process, emerging proT cells maintained high viability and the highest cumulative proliferation rate (between 200 and 600 times) (Figure 10). As proT cells progressed to stage 2 (days 10-17), viability decreased significantly, and the increase in proliferation rate was minimal (Figure 26). In stage 3 (from day 17 onward), viability continued to decline slightly as DP cells matured and their phenotypes progressed, but cells maintained a viability rate of over 10% for about a week, and the decrease in proliferation rate was minimal (Figures 26A, 26B), which was an improvement over the previous protocol.

[0208] For 2D culture, 10-day ProT cells generated from CAR-modified HPCs at stage 1 were cultured 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) in a 1 × 10⁶ culture. 6 Cells were seeded in precursor maturation medium (SFEM II+LMM) at a concentration of cells / mL. On day 17, sacrificial wells were collected and flow cytometry analysis was performed. On day 22, the cultures were collected and analyzed in SFEM II+LMM 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) at a concentration of 3 × 10⁶ cells / mL. 6 Cells were reseeded at a rate of cells / mL. On day 24, sacrificial wells were collected and flow cytometry analysis was performed. The decrease in 2D ETN coating and the increase in seeding density from day 10 to day 24 led to successful generation of CD4+CD8α+ early DP by day 17, and effective maturation of this population, as evidenced by >70% CD4+CD8α / β+, >20% surface CD3, and >70% CAR+ expression on day 24 (Figure 27).

[0209] For 3D culture, 10-day ProT cells generated from CAR-modified HPCs in Stage 1 were cultured in 2 × 10⁶ cells. 6 Cells were seeded in SFEM II+LMM at a concentration of cells / mL, incubated at 37°C for 2-4 hours, mixed with 0.5× bead doses of 0.1× protein density 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. 6Cells were re-seed in SFEM II+LMM at a concentration of cells / mL, incubated at 37°C for 2–4 hours, mixed with 0.25× bead doses of 0.1× 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. Compared to the 2D process, the 3D protocol improved the conversion from CD4+ISP to CD4+CD8A+DP, and CD4+CD8α / β+ late DP appeared earlier by day 17 (Figure 28, top panel). The culture was mainly CD4+CD8α / β+DP on day 24, but these late DPs were more immature than those produced by the 2D protocol due to lower CD1a+ expression at this point (Figure 28, middle panel). Further 4 days of culture improved the expression of surface CD3, CD1a, and CAR (Figure 28, bottom panel).

[0210] Furthermore, late-stage DP was generated using either 2D or 3D ETN beads. The enriched late-stage DP from the CAR modified strain was subjected to stage 4 conditions at a rate of 1 × 10⁻⁶. 6 Cells were seeded at a concentration of cells / mL and stimulated with CD19 antigen-coated beads (Example 1) at a bead:cell ratio of 1:1 for 7 days. Enriched DP produced using the 3D protocol had a higher viability than that produced using the 2D protocol (Figure 29A). Increases in both cell number and viability were observed over time in CAR-modified strains using the CD19 bead transfer protocol (Figure 29B).

[0211] Concentrated late-stage DP from CAR modified strains under stage 4 conditions: 1 × 10 6Seeded at cells / mL and stimulated with CD19 antigen-coated beads at a bead:cell ratio of 1:1 for 7 days. At the end of this stage (day 35), the cultures were harvested and stained for select markers of T cell maturation, activation, and surface CD3 expression. Conversion with CD19 antigen-coated beads resulted in the effective generation of CAR+CD8SP with high mean fluorescence intensity (MFI) (Figure 30, top panel). For the sub-gated CD8+ population (Figure 30, top panel, gray overlay), these cells had intermediate levels of CD8α / β (43%), moderate CD3 expression (56%), as well as important integrins such as LFA-1 and CD49c (VLA-3) (Figure 30, bottom panel, sub-gated against CD8+). The expression of the activation marker CD25 was low (56%, lower right panel).

[0212] At the end of the CD8SP stage (day 35), the cultures were harvested and co-cultured with target cells regardless of CD19 expression. To measure cytotoxic activity, a continuous restimulation assay was performed using an Incucyte® (Sartorius) live cell imaging assay with GFP-expressing CD19+ cells as target cells (n = 3 technical replicates). T cells were co-cultured with target cells at an E:T ratio of 2:1 every 5 days under conditions with exogenous cytokine added. Target clearance was measured by the decrease in GFP surface area. CD8SP cells generated using CD19 beads had some non-specific activity at the first stimulation (Figure 31, dark gray inverted triangles), which may be due to activation during the DP-SP differentiation stage. iPSC-derived CD8SP was able to continuously engage with target cells over 4 stimulations (Figure 31).

[0213] The phenotype of CD8SP generated using CD19 antigen-coated beads was evaluated using flow cytometry. CD8SP was a mixture of CD8αα+ and CD8αβ+ cells with high CAR expression (Figure 32). Some CD4 expression still existed after SP migration (Figure 32). Despite the knockout of TRAC in the iPSC cell bank, the generated DP and CD8SP expressed surface CD3 but did not express TCRαβ (Figure 32).

[0214] The in vitro function of iPSC-derived CD4-CD8+ cells generated using CD19 antigen beads in the above continuous restimulation assay was compared with primary CAR-T cells by plotting cumulative cytotoxicity against cumulative proliferation fold change. Compared with primary CAR-T cells, equivalent or higher cytotoxicity was observed for iPSC-derived cells, and primary cells showed a greater cumulative proliferation fold change (Figure 33). For iPSC-derived cells, CD8αβ expression was retained even after 4 stimulations (Figure 34).

[0215] The expression of various memory markers was evaluated using flow cytometry at baseline and at the end of the 4th stimulation. Cells were classified into various 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+; and terminally differentiated effector memory cells that re-express CD45RA (TEMRA), CD62L-CD45RA+CD45RO+. At baseline, the majority of primary CAR-T cells were TEMRA cells, while iPSC-derived cells included the TCM phenotype, TSCM phenotype, TEM phenotype, and TEMRA phenotype (Figure 35A). After 4 stimulations, a greater proportion of iPSC-derived cells showed the TEM phenotype, and both iPSC-derived cells and primary cells included the TSCM and TCM phenotypes (Figure 35A).

[0216] Furthermore, the expression of exhaustion markers TIM3 and LAG3 before and after four stimulations by target cells was evaluated in iPSC-derived CD4-CD8+ cells and primary CAR-T cells generated using CD19 antigen beads. More than 60% of primary CAR-T cells were TIM3+ at baseline, and many showed TIM3+LAG3+ after exposure to target cells (Figure 35B). In contrast, iPSC-derived cells showed low expression of exhaustion markers at baseline, and TIM3 expression increased moderately after exposure to target cells (Figure 35B).

[0217] Furthermore, cytokine secretion from iPSC-derived cells was evaluated. Supernatants were collected 24–36 hours after the start of each stimulus in a long-term serial killing assay in the presence of CD19+ / + or CD19- / - target cells, and cytokines IFNγ (Figure 36A), granzyme B (Figure 36B), and TNFα (Figure 36C) were quantified using a 1300 MESO QuickPlex SQ 120MM. Cytokine secretion was found to be antigen-dependent (Figure 36). The proliferation of iPSC-derived cells generated using CD19 antigen beads also showed proliferation over four stimulations by target cells, similar to primary CD8+ CAR-T cells (Figure 37).

[0218] 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.Bauluら.TCR-engineered T cell therapy in solid tumors:State of the art and perspectives.Science Advances eadf3700(2023).Doi:10.1126 / sciadv.adf3700 2.Drougkasら.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 3.Guhaら.Assessing the Future of Solid Tumor Immunotherapy.Biomedicines 10:655(2022).Doi:10.3390 / biomedicines10030655 4.Iriguchiら.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 5.Klesmithら.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 6.Lobnerら.Getting CD19 Into Shape:Expression of Natively Folded“Difficult-to-Express”CD19 for Staining and Stimulation of CAR-T Cells.Front.Bioeng.Biotechnol.8(49):1-13(2020).Doi:10.3389 / fbioe.2020.00049 7.Philipsonら.4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling.Sci.Signal.2020 13:eaay8248.Doi:10.1126 / scisignal.aay8248 8.Quら.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ら.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.Sunら.Evolution of CD8+T Cell Receptor(TCR)Engineered Therapies for the Treatment of Cancer.Cells 10:2379(2021)Doi:10.3390 / cells10092379 11.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 12. Want et al. T Cell Based Immunotherapy for Cancer: Approaches and Strategies. Vaccines 11:835(2023). Doi:10.3390 / vaccines11040835 13. Weber et al. The Emerging Landscape of Immune Cell Therapies. Cell.2020 181(1):46-62.Doi:10.1016 / j.cell.2020.03.001 14. Wu et al., U.S. Patent Application Publication No. 20180223255 15. Zuniga-Pflucker et al. International Publication No. 2019 / 157597

Claims

1. A method for generating CD4-CD8+ T cells, - Contacting a population of CD4+CD8+ cells expressing an antigen receptor with an antigen immobilized on a substrate, wherein the antigen binds to the antigen receptor. Methods that include...

2. The method according to claim 1, wherein the CD4+CD8+ cells are induced in vitro from hematopoietic stem cells / progenitor cells.

3. The method according to claim 2, wherein the hematopoietic stem cells / progenitor cells are induced in vitro from pluripotent stem cells.

4. The aforementioned CD4+CD8+ cells, - Contacting a population of hematopoietic stem cells / progenitor cells with an immobilized Notch signaling ligand. The method according to claim 2 or 3, which is derived by a method including the following.

5. The step of contacting the population of hematopoietic stem cells / progenitor cells with the immobilized Notch signaling ligand is - The population of hematopoietic stem cells / progenitor cells is brought into contact with the 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. The method according to claim 4, including the method described in claim 4.

6. 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 claim 5, wherein the molecule / mL is

7. The method according to claim 5 or claim 6, further comprising the step of concentrating the CD4+CD8+ cells with respect to CD8α or CD8β.

8. The method according to any one of claims 4 to 7, wherein the immobilized Notch signaling ligand is DL4.

9. The method according to any one of claims 1 to 8, wherein the antigen is a CD19 polypeptide and the antigen receptor binds to the CD19 polypeptide.

10. The method according to claim 9, wherein the CD19 polypeptide is an engineered variant of CD19.

11. The method according to claim 1, wherein the antigen is a polypeptide having the sequence of SEQ ID NO:

8.

12. The method according to any one of claims 1 to 11, wherein the substrate is particles.

13. The method according to claim 12, wherein the particles are composed of a material selected from the group consisting of polystyrene, iron oxide, and gold.

14. The method according to claim 13, wherein the particles are composed of polystyrene and magnetizable iron oxide.

15. The method according to any one of claims 12 to 14, wherein the antigen is covalently conjugated to the particle.

16. The method according to any one of claims 1 to 15, wherein the step of contacting a population of CD4+CD8+ cells expressing the antigen receptor with an antigen coupled to a particle is performed in a particle:cell ratio of 1:

1.

17. A population of CD4-CD8+ T cells prepared according to the method described in any one of claims 1 to 16.

18. The population of CD4-CD8+ T cells according to claim 17, wherein the population expresses a chimeric antigen receptor (CAR).

19. The population according to claim 18, wherein the CAR is a CD19 CAR and the antigen is a CD19 polypeptide.

20. The population of CD4-CD8+ T cells according to any one of claims 17 to 19, wherein the population of CD4-CD8+ T cells is induced in vitro from pluripotent stem cells.

21. The population according to any one of claims 17 to 20, wherein the population of CD4-CD8+ T cells is TRAC- / - and / or CD3+.

22. The population according to any one of claims 17 to 21, wherein more than 50% of the population of CD4-CD8+ T cells are CD8αα+.

23. A method for inducing cytotoxicity in tumor cells, comprising exposing the tumor cells to a population of CD4-CD8+ T cells, wherein the population of CD4-CD8+ T cells is prepared according to the method described in any one of claims 1 to 16.

24. A method for treating the target disease or condition, a) To generate CD4-CD8+ T cells according to any one of claims 1 to 16, b) Administering an effective amount of the CD4-CD8+ T cells to a subject in need of treatment for a disease or condition. Methods that include...

25. Use of CD4-CD8+ T cells in the manufacture of a pharmaceutical product for treating a disease or condition, wherein the CD4-CD8+ T cells are produced by the method described in any one of claims 1 to 16.

26. A cell-stimulating particle comprising a particle and a modified antigen immobilized on the surface of the particle, wherein the modified antigen comprises one or more amino acid substitutions from a natural antigen, and the modified antigen has greater solubility and / or stability in vitro compared to the natural antigen.

27. The cell stimulating particle according to claim 26, wherein the modified antigen includes the extracellular domain of the antigen.

28. The cell-stimulating particle according to claim 26 or 27, wherein the modified antigen is covalently conjugated to the particle.

29. The cell stimulating particle according to any one of claims 26 to 28, wherein the particle is composed of a material selected from the group consisting of polystyrene, iron oxide, and gold.

30. The cell stimulating particles according to claim 29, wherein the particles are composed of polystyrene and magnetizable iron oxide.

31. The cell-stimulating particle according to any one of claims 26 to 30, wherein the modified antigen is a CD19 antigen.

32. The cell-stimulating particle according to claim 31, wherein the modified antigen is a polypeptide having the sequence of SEQ ID NO: 8.

Images