A method for rapidly producing engineered T cells that express chimeric antigen receptors.

Abstract

Methods and compositions for the production of engineered T cells expressing heterologous proteins, such as chimeric antigen receptors (CARs), are disclosed herein. Methods for the rapid production of engineered T cells expressing heterologous proteins, such as chimeric antigen receptors (CARs), under certain conditions are disclosed herein. Similar methods for the rapid production of engineered T cells containing heterologous genes are also contemplated, wherein the heterologous genes may or may not encode proteins. Compositions suitable for use in conjunction with the disclosed methods are further disclosed.

Description

[Technical Field]

[0001] Technical field This disclosure generally relates to methods and compositions for the production of engineered T cells that express heterologous proteins, such as chimeric antigen receptors (CARs).

[0002] Sequence List This disclosure includes an electronic amino acid and / or nucleotide sequence listing prepared in accordance with ST.26 standards, which was created on 19 July 2024 and submitted with this specification in an .xml file named "KYVA-002-01WO_Sequence_Listing.xml" with a size of 50KB, and is incorporated herein by reference. [Background technology]

[0003] background Autologous T cell-based therapies, such as T cells modified to express chimeric antigen receptors (CAR-T cells), have demonstrated significant therapeutic benefits for patients with cancer. However, numerous obstacles remain to the production of autologous CAR-T cells. [Overview of the Initiative]

[0004] Summary of Disclosure A method for the rapid production of engineered T cells expressing heterologous proteins, such as chimeric antigen receptors (CARs), under certain conditions is disclosed herein. Similar methods for the rapid production of engineered T cells containing heterologous genes are also contemplated, wherein the heterologous genes may or may not encode proteins. Compositions suitable for use in conjunction with the disclosed methods are further disclosed.

[0005] In one embodiment, the present disclosure provides a method for producing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, and culturing the T cells for 29 to 71 hours in serum-free culture medium containing interleukin-2 (IL-2) protein; and (c) harvesting the T cells; optionally, step (b) being performed at least 1 hour after the start of step (a); and the harvested T cells comprising one or more T cells engineered to express a heterologous protein.

[0006] In one embodiment, the present disclosure provides a method for producing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, and culturing the T cells for 31 to 71 hours in a culture medium containing interleukin 7 (IL7) and interleukin-15 (IL-15) proteins; and (c) harvesting the T cells; the harvested T cells comprising one or more T cells engineered to express a heterologous protein.

[0007] In one embodiment, the present disclosure provides a method for producing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein; and (c) culturing the T cells for 31 to 71 hours in a serum-free culture medium containing interleukin-7 (IL-7) and / or interleukin-15 (IL-15) proteins; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express a heterologous protein.

[0008] In some embodiments, the present disclosure provides a method for producing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28 in the absence of cytokines; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, and culturing the T cells for 24 to 72 hours in a culture medium containing at least one cytokine; and (c) harvesting the T cells; step (b) being performed about 18 hours after step (a); and the harvested T cells comprising one or more T cells engineered to express a heterologous protein. In certain embodiments, the at least one cytokine in step (c) comprises one or more of IL-2, IL-7, IL-15, and / or IL-21 (e.g., 1, 2, 3, or 4). In certain embodiments, the at least one cytokine in step (c) comprises IL-2. In certain embodiments, at least one cytokine in step (c) includes IL-21. In certain embodiments, at least one cytokine in step (c) includes IL-7 and IL-15. In certain embodiments, at least one cytokine in step (c) includes IL-2, IL-7, and IL-15. In certain embodiments, at least one cytokine in step (c) includes IL-21, IL-7, and IL-15.

[0009] In some embodiments, the present disclosure provides a method for producing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, and culturing the T cells for 31 to 71 hours in a culture medium containing the interleukin-21 (IL-21) protein; and (c) harvesting the T cells; the harvested T cells comprising one or more T cells engineered to express a heterologous protein. In certain embodiments, the culture medium further comprises IL-7 and / or IL-15.

[0010] In certain embodiments, the present disclosure provides a method for producing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein, and culturing the T cells in a culture medium for 24 to 72 hours; and (c) harvesting the T cells; wherein the culture medium is free from IL-2, IL-7, IL-15, or IL-21; and the harvested T cells comprise one or more T cells engineered to express a heterologous protein. In certain embodiments, the culture medium is free from cytokines. In certain embodiments, step (a) does not involve the use of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, step (a) is performed in the absence of cytokines. In certain embodiments, step (c) does not involve the use of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, step (c) is carried out in the absence of cytokines. In certain embodiments, the method is carried out in the absence of cytokines. In some embodiments, the culture medium is a basal culture medium.

[0011] In certain embodiments, the culture medium contains serum. In some embodiments, the culture medium further does not contain added cytokines or growth factors. In certain embodiments, the culture medium is serum-free.

[0012] In certain embodiments, the starting population of T cells is seeded into the culture at a concentration of about 1×10 6 cells per mL. In certain embodiments, the starting population of T cells is seeded into the culture at a concentration of about 2×10 6 cells per mL. In certain embodiments, the starting population of T cells is seeded into the culture at a concentration of about 5×10 6 cells per mL. In certain embodiments, (a) the starting population of T cells is seeded into the culture at a concentration of about 1×10 6 cells per mL; and (b) the culture medium contains IL-2. In certain embodiments, (a) the starting population of T cells is seeded into the culture at a concentration of about 1×10 6 cells per mL; and (b) the culture medium contains IL-7 and IL-15. In certain embodiments, (a) the starting population of T cells is seeded into the culture at a concentration of about 1×10 6 cells per mL; and (b) the culture medium contains IL-7, IL-15, and IL-21. In certain embodiments, (a) the starting population of T cells is seeded into the culture at a concentration of about 1×10 6 cells per mL; and (b) the culture medium contains IL-21. In certain embodiments, (a) the starting population of T cells is seeded into the culture at a concentration of about 1×10 6 cells per mL; and (b) the culture medium does not contain IL-2, IL-7, IL-15, or IL-21 (i.e., does not contain any one of IL-2, IL-7, IL-15, and IL-21). In certain embodiments, (a) the starting population of T cells is seeded into the culture at a concentration of about 2×10 6 cells per mL; and (b) the culture medium contains IL-2. In certain embodiments, (a) the starting population of T cells is seeded into the culture at a concentration of about 2×10 6(b) The cells are seeded into the culture at a concentration; (b) The culture medium contains IL-7 and IL-15. In a particular embodiment, (a) the starting population of T cells is about 2 × 10⁶ per mL. 6 (b) The cells are seeded into the culture at a concentration; (b) The culture medium contains IL-7, IL-15, and IL-21. In a particular embodiment, (a) the starting population of T cells is approximately 2 × 10⁶ per mL. 6 (b) The cells are seeded into the culture at a concentration; (b) The culture medium contains IL-21. In a particular embodiment, (a) the starting population of T cells is approximately 2 × 10⁶ per mL. 6 (a) The cells are seeded into the culture at a concentration; (b) The culture medium is free of IL-2, IL-7, IL-15, or IL-21 (i.e., free of any one of IL-2, IL-7, IL-15, and IL-21). In a particular embodiment, (a) the starting population of T cells is approximately 5 × 10⁶ per mL. 6 (b) The cells are seeded in the culture at a concentration; (b) The culture medium contains IL-2. In a particular embodiment, (a) the starting population of T cells is about 5 × 10⁶ per mL. 6 (b) The cells are seeded into the culture at a concentration; (b) The culture medium contains IL-7 and IL-15. In a particular embodiment, (a) the starting population of T cells is about 5 × 10⁶ per mL. 6 (b) The cells are seeded into the culture at a concentration; (b) The culture medium contains IL-7, IL-15, and IL-21. In a particular embodiment, (a) the starting population of T cells is approximately 5 × 10⁶ per mL. 6 (b) The cells are seeded in the culture at a concentration; (b) The culture medium contains IL-21. In a particular embodiment, (a) the starting population of T cells is approximately 5 × 10⁶ per mL. 6 (b) The cells are seeded into the culture at a concentration; (b) The culture medium is free of IL-2, IL-7, IL-15, or IL-21 (i.e., free of any one of IL-2, IL-7, IL-15, and IL-21).

[0013] In a particular embodiment, the method involves (d) heating the T cells collected in step (c) at a temperature of 38°C or lower (e.g., 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9°C or lower), 2-8 The further step includes maintaining the temperature between °C (for example, 2, 3, 4, 5, 6, 7, or 8 °C) or below -80 °C (for example, -80, -81, -82, -83, -84, -85, -86, -87, -88, -89, -90, -91, -92, -93, -94, -95, -96, -97, -98, -99, below -100 °C, or lower).

[0014] In certain embodiments, one or more agents that activate CD3 and / or CD28 include an anti-CD3 antibody, an anti-CD28 antibody, or both. In certain embodiments, one or more agents that activate CD3 and / or CD28 include a first agent that binds to CD3 and a second agent that binds to CD28. In certain embodiments, the first agent and the second agent are the same agent (e.g., a bispecific antibody that specifically binds to CD3 and CD28).

[0015] In certain embodiments, the method includes a step of obtaining a sample containing a T cell starting population from a subject prior to step (a). In certain embodiments, the method includes a step of obtaining a sample containing a T cell starting population from a subject prior to step (a). In certain embodiments, the sample is a whole blood sample obtained from a subject. In certain embodiments, the sample is a leukocyte apheresis sample obtained from a subject.

[0016] In a particular embodiment, the starting population of T cells consists of helper T(Th) cells and cytotoxic T(Th) cells. C ) cells, memory T(T) M ) cells, regulatory T (T reg) cells, including innate immune-like T cells. In certain embodiments, Th cells include Th1 cells, Th2 cells, Th17 cells, Th9 cells, Tfh cells, and / or Th22 cells. In certain embodiments, memory T cells include central memory T cells (T CM ) Cells, effector memory T(T EM ) Cells, tissue-resident memory T(T) RM ) cells, and virtual memory T(T) VM ) cells are included. In certain embodiments, innate immune-like T cells are natural killer T (NKT) cells, mucosa-associated invariant T (MAIT) cells, and γδ T cells.

[0017] In certain embodiments, the polynucleotide is contained in the delivery vehicle. In certain embodiments, the delivery vehicle is lipid nanoparticles. In certain embodiments, the delivery vehicle is a nucleic acid vector. In certain embodiments, the nucleic acid vector is a viral vector. In certain embodiments, the viral vector is a lentiviral vector.

[0018] In certain embodiments, the method results in an expansion of the T cell starting population of 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.9-fold, 1.8-fold, 1.7-fold, 1.6-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold or less, or lower, after step (c). In certain embodiments, after step (c), at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 75% of the cells in the T cell starting population are manipulated to express a heterologous protein.

[0019] In certain embodiments, the heterologous protein comprises a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises (a) an antigen-binding fragment of an anti-CD19 antibody; (b) a transmembrane domain; (c) an intracellular T cell signaling domain derived from human CD3ζ; and (d) an intracellular T cell signaling domain derived from human CD28.

[0020] In a particular embodiment, one or more T cells collected in step (c) are compared to T cells that have not been in contact with target cells with increased amounts of IFNγ, granzyme B, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP-10, MC It secretes one or more proteins selected from the group consisting of P1, MCP4, TNFα, TNFβ, TGFβ, GM-CSF, MIP1α, MIP1β, CCL11, perforin, RANTES, sCD137, and VEGF (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32). In certain embodiments, the secretion of one or more proteins after contact with target cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or higher compared to the secretion of one or more proteins in the absence of target cells. In certain embodiments, one or more T cells collected in step (c) show increased expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 after contact with target cells expressing CD19, compared to the expression of one or more T cell activation markers in the absence of target cells. In certain embodiments, the expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more, compared to the expression of one or more T cell activation markers in the absence of target cells.In certain embodiments, one or more T cells collected in step (c) exhibit increased cytotoxicity against target cells expressing CD19 compared to cytotoxicity against cells that do not express CD19. In certain embodiments, cytotoxicity increases by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more compared to cytotoxicity against cells that do not express CD19. In certain embodiments, one or more T cells collected in step (c) exhibit increased proliferation after contact with target cells expressing CD19 compared to proliferation in the absence of target cells. In certain embodiments, proliferation increases by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, or more, compared to proliferation in the absence of target cells. In certain embodiments, proliferation is measured 0 to 240 hours after contact with target cells. In certain embodiments, proliferation is measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, 48, 72, 96, 120, 144, 168, 192, 216, and / or 240 hours after contact with target cells. In certain embodiments, proliferation is measured as a multiple change in the number of CD3+ cells in the T cells of step (c) compared to the number of CD3-positive (CD3+) cells in the starting population of T cells. In a particular embodiment, proliferation is measured as a multiplicative change in the number of T cells in step (c) expressing the heterologous protein after contact with target cells expressing CD19, compared to the number of T cells in step (c) expressing the heterologous protein in the absence of target cells.In a particular embodiment, the T cells collected in step (c) have an increased amount of naive and stem cell memory T(T) cells compared to the starting population of T cells. NSCM ) cells (for example, T SCM (shows cells). In certain embodiments, the T cells collected in step (c) are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% more than the starting population of T cells. NSCM Cells (for example, T SCM Includes cells. In certain embodiments, the T cells collected in step (c) are substantially naive and stem cell memory T cells compared to the starting population of T cells. NSCM ) cells (for example, T SCM (For example, T cells in step (c) show the same percentage of cells) NSCM The percentage of cells is the T cells in the starting population of T cells. NSCM Compared to the number of cells, 4%, 3%, 2%, 1% or less (T cells in T cells) NSCM The absolute difference between the percentages of cells (increase or decrease). In a particular embodiment, the T cells collected in step (c) have a reduced amount of T effector memory (T) compared to the starting population of T cells. EM ) cells are included. In certain embodiments, the T cells collected in step (c) are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% fewer than the starting population of T cells. EM Includes cells. In certain embodiments, the T cells collected in step (c) are substantially T compared to the starting population of T cells. EM Showing the same percentage of cells (for example, T cells in step (c)) EM The percentage of cells is the T cells in the starting population of T cells. EMCompared to the number of cells, 4%, 3%, 2%, 1% or less (T cells in T cells) EM The absolute difference between the percentages of cells (increase or decrease).

[0021] In certain embodiments, the method is performed ex vivo. [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1 is a bar graph showing T cell expansion and proliferation, represented by a multiple increase in the number of CD3+ cells 72 hours after thawing compared to the number of CD3+ cells at thawing.

[0023] [Figure 2] Figure 2 is a graph showing the percentage of CAR expression 72 hours after thawing.

[0024] [Figure 3A-B] Figures 3A-3B show the percentage of TNSCMs (naive and stem cell memory T cells (CD45RO- / CCR7+)) after thawing. "UNTD" represents the untransduced control.

[0025] [Figure 4A-B] Figures 4A–4D are bar graphs representing the memory phenotype of CAR+ cells at thawing (Figures 4A and 4C) or 72 hours after thawing (Figures 4B and 4D). The "enriched" condition did not involve transduction. [Figure 4C-D] Figures 4A–4D are bar graphs representing the memory phenotype of CAR+ cells at thawing (Figures 4A and 4C) or 72 hours after thawing (Figures 4B and 4D). The "enriched" condition did not involve transduction.

[0026] [Figure 5]Figure 5 shows the percentage of cell lysis of NALM6 target cells when incubated with CAR-T cells generated under conditions D and E. "D" represents the KYV-101 6-day condition, "E" represents the KYV-101 9-day condition, and "UNTD" represents the untransduced condition.

[0027] [Figure 6A] Figures 6A–6D are bar graphs showing the secretion frequencies of 32 cytokines from cells produced using the KYV 3-day v2 protocol ("C") (Figures 6A and 6C) or the KYV-101 9-day protocol ("E") (Figures 6B and 6D) after overnight incubation in the presence of CD19+ NALM6 target cells. "ut" corresponds to untransduced control cells provided in different ways at each step of the 3-day or 9-day protocol. [Figure 6B] Figures 6A–6D are bar graphs showing the secretion frequencies of 32 cytokines from cells produced using the KYV 3-day v2 protocol ("C") (Figures 6A and 6C) or the KYV-101 9-day protocol ("E") (Figures 6B and 6D) after overnight incubation in the presence of CD19+ NALM6 target cells. "ut" corresponds to untransduced control cells provided in different ways at each step of the 3-day or 9-day protocol. [Figure 6C] Figures 6A–6D are bar graphs showing the secretion frequencies of 32 cytokines from cells produced using the KYV 3-day v2 protocol ("C") (Figures 6A and 6C) or the KYV-101 9-day protocol ("E") (Figures 6B and 6D) after overnight incubation in the presence of CD19+ NALM6 target cells. "ut" corresponds to untransduced control cells provided in different ways at each step of the 3-day or 9-day protocol. [Figure 6D]Figures 6A–6D are bar graphs showing the secretion frequencies of 32 cytokines from cells produced using the KYV 3-day v2 protocol ("C") (Figures 6A and 6C) or the KYV-101 9-day protocol ("E") (Figures 6B and 6D) after overnight incubation in the presence of CD19+ NALM6 target cells. "ut" corresponds to untransduced control cells provided in different ways at each step of the 3-day or 9-day protocol.

[0028] [Figure 7] Figure 7 is a bar graph showing the multifunctionality of CAR-T cells generated by process C or E.

[0029] [Figure 8] Figure 8 is a bar graph showing the percentage of cells generated by process C or E from all pluripotent cells that secreted the indicated cytokines.

[0030] [Figure 9] Figure 9 is a flowchart illustrating the experimental design and conditions for six CAR-T cell production processes.

[0031] [Figure 10] Figure 10 provides data demonstrating the target-specific cytotoxicity of CAR T cells generated using the method of this disclosure.

[0032] [Figure 11] Figure 11 provides results regarding target-dependent cytokine release by CAR T cells derived from fresh starting material of healthy donors (HD) 24 hours after in vitro co-culture with CD19+ NALM6 target cells at the indicated E:T (effector:target) ratio.

[0033] [Figure 12]Figure 12 shows data for CD19-targeted CAR-T cells produced using the method of this disclosure and CD19+ NALM6-targeted cells co-cultured at the indicated E:T ratio (CAR-T effector: NALM6 target), where the % of target cell toxicity was measured by flow cytometry at each indicated time point. At each time point, a fresh series of target cells were added to the co-culture to evaluate the successive recurrent cytotoxicity of CAR-T cells over time.

[0034] [Figure 13] Figure 13 provides target-specific growth data for CAR T cells produced using the method of this disclosure.

[0035] [Figure 14] Figure 14 shows the TBNK / memory phenotypes of pre-enrichment and post-enrichment materials for cells generated starting from T cells isolated from whole blood.

[0036] [Figure 15] Figure 15 shows the TBNK / memory phenotype of the final CAR T cell product (e.g., after expanded proliferation) generated using the 8-day process.

[0037] [Figure 16] Figure 16 shows the TBNK / memory phenotypes of pre-enrichment and post-enrichment materials for cells generated starting from T cells isolated from whole blood.

[0038] [Figure 17] Figure 17 shows the TBNK / memory phenotypes of pre-enrichment and post-enrichment materials for cells generated starting with T cells isolated from whole blood.

[0039] [Figure 18] Figure 18 outlines the steps of the 3-day method of this disclosure, starting with whole blood (WB) starting material (SM).

[0040] [Figure 19A] Figure 19A shows the T cell memory phenotype of cells generated using the 3-day method of this disclosure.

[0041] [Figure 19B] Figure 19B shows the cytolytic activity of cells generated using the 3-day method of this disclosure.

[0042] [Figure 19C] Figure 19C shows the results of the serial rechallenge assay.

[0043] [Figure 19D] Figure 19D shows that 3-day Ingenui-T cells successfully killed autologous primary B cells in a dose-dependent manner. Ingenui-T cells or untransduced T cells were co-cultured with autologous (donor-matched) PBMCs at the shown effector-to-target (E:T) ratio, representing the ratio of CAR+ T cells (effectors) to total PBMCs (targets). B cell viability, defined by the expression of CD19 or CD20 surface markers, was measured at 48 hours by flow cytometry. Data are presented as mean ± SD from 3 technical replicates per condition from one donor representing N=2 healthy donors. ****p<0.0001, compared matched E:T ratio, by two-way ANOVA (GraphPad Prism).

[0044] [Figure 20A] Figure 20A shows the expansion multiplier, viability, and T cell purity of CAR-T cells produced in the KYV 3-day process starting from leukocyte apheresis material. Conditions "A", "B", "C", and "D" for KYV 3-day indicate the different cultured cytokine(s) used. "NT" = untransduced. N=4 healthy donors per condition.

[0045] [Figure 20B]Figure 20B shows the yield and T cell purity of CAR-T cells produced in the KYV 3-day process starting from freshly collected whole blood from n=5 donors.

[0046] [Figure 21A] Figure 21A shows the CAR+ expression percentage, analyzed by flow cytometry, in CAR-T cells produced in the KYV 3-day process starting from leukocyte apheresis material. CAR expression was analyzed in total CD3+ T cells or CD4+ or CD8+ T cells at the time of collection. Conditions "A," "B," "C," and "D" of the KYV 3-day process indicate the different cultured cytokine(s) used. N=4 healthy donors per condition.

[0047] [Figure 21B] Figure 21B shows the CAR+ expression percentage, analyzed by flow cytometry, in CAR-T cells produced in the KYV 3-day process starting from newly collected whole blood. CAR expression was analyzed within total T cells. N=5 donors.

[0048] [Figure 22A] Figure 22A shows the CD4+ and CD8+ percentages analyzed within total CD3+ in CAR-T cells produced in the KYV 3-day process starting from leukocyte apheresis material. The KYV 3-day conditions "C1", "C2", "C3", and "C4" indicate the different cultured cytokine(s) used. N=4 healthy donors per condition. "NT" = non-transduction control. "Conv 9 days" refers to donor-matched CAR-T cells produced in the conventional 9-day culture process. "Aph SM" refers to the leukocyte apheresis donor starting material prior to the KYV 3-day process.

[0049] [Figure 22B]CD4+ and CD8+ percentages within total CD5+ in CAR-T cells produced in the KYV 3-day process starting from newly collected whole blood. Total of N=7 donors. "WB SM" refers to the donor whole blood starting material prior to the KYV 3-day process.

[0050] [Figure 23A] Figures 23A-23B show the results of T cell memory phenotyping analyzed by flow cytometry in CD4+ or CD8+ T cells in CAR-T cells produced in the KYV 3-day process starting from leukocyte apheresis material. The KYV 3-day conditions "C1", "C2", "C3", and "C4" indicate the different cultured cytokine(s) used. N=4 healthy donors per condition. "NT" = non-transduction control. "Conv 9 days" refers to donor-matched CAR-T cells produced in the conventional 9-day culture process. "Aph SM" refers to leukocyte apheresis donor starting material prior to the KYV 3-day process. Tnaive = Naive (CCR7+CD45RO-CD95-); Tscm = Stem Cell Memory (CCR7+CD45RO-CD95+); Tcm = Central Memory (CCR7+CD45RO+); Temp = Effector Memory (CCR7-CD45RO+); Te = Effector (CCR7-CD45RO-CD95+). [Figure 23B]Figures 23A-23B show the results of T cell memory phenotyping analyzed by flow cytometry in CD4+ or CD8+ T cells in CAR-T cells produced in the KYV 3-day process starting from leukocyte apheresis material. The KYV 3-day conditions "C1", "C2", "C3", and "C4" indicate the different cultured cytokine(s) used. N=4 healthy donors per condition. "NT" = non-transduction control. "Conv 9 days" refers to donor-matched CAR-T cells produced in the conventional 9-day culture process. "Aph SM" refers to leukocyte apheresis donor starting material prior to the KYV 3-day process. Tnaive = Naive (CCR7+CD45RO-CD95-); Tscm = Stem Cell Memory (CCR7+CD45RO-CD95+); Tcm = Central Memory (CCR7+CD45RO+); Temp = Effector Memory (CCR7-CD45RO+); Te = Effector (CCR7-CD45RO-CD95+).

[0051] [Figure 23C] Figure 23C shows the results of T cell memory phenotyping analysis within total CD3+ T cells in CAR-T cells produced in a KYV 3-day process starting from newly collected whole blood, compared to a conventional 9-day process starting from leukocyte apheresis material. A total of N=4 donors were involved. "WB SM" and "Aph SM" refer to the donor's whole blood or leukocyte apheresis starting material, respectively, before the culture process. T cell memory subsets were analyzed as defined in Figures 23A–23B.

[0052] [Figure 24]Figure 24 shows the target-dependent cytotoxic activity of anti-CD19 CAR-T cells produced from the KYV 3-day process against CD19+-expressing target cells, measured by the cytolysis percentage of CD19+ NALM6 target cells or CD19- CEM / C1 control cells after co-culture with anti-CD19 CAR-T cells produced from the KYV 3-day process, at the indicated E:T (effector:target) ratio. N=2 donors are shown. Cytolytic activity was measured by a luminescence assay and normalized to target cells alone (0:1).

[0053] [Figure 25A] Figure 25A shows the results of killing or outgrowth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells produced in the KYV 3-day process from leukocyte apheresis starting material, at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time=0. KYV 3-day conditions "C1", "C2", "C3", and "C4" indicate different cultured cytokines used. "NT" = untransduced control T cells. One representative donor is shown from n=4.

[0054] [Figure 25B] Figure 25B shows the results of killing or outgrowth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells produced from freshly collected whole blood in a KYV 3-day process, at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time=0. "NT" = untransduced control T cells. "Conv 9 days" refers to donor-matched CAR-T cells produced from leukocyte apheresis starting material in a conventional 9-day culture process. One representative donor is shown from n=4.

[0055] [Figure 26] Figure 26 shows the CAR-mediated cytotoxic activity of anti-CD19 CAR-T cells produced from the KYV 3-day process against CD19+ primary human B cells, using the measured cytolysis percentage of CD19+ primary human B cells in co-culture with anti-CD19 CAR-T cells produced from newly collected whole blood using the KYV 3-day process. The E:T (effector:target) ratio represents the ratio of CAR+ T cells seeded in co-culture to total PBMCs (peripheral blood mononuclear cells). "Conv 9 days" refers to donor-matched CAR-T cells produced from leukocyte apheresis starting material using the conventional 9-day culture process. One representative donor is shown in each panel from n=5.

[0056] [Figure 27] Figure 27 shows target-dependent cytokine release by anti-CD19 CAR-T cells produced from freshly collected whole blood via the KYV 3-day process in response to CD19+-expressing target cells, mediated by IFN-gamma production measured by anti-CD19 CAR-T cells produced from freshly collected whole blood via the KYV 3-day process, in co-culture with CD19+ NALM6 target cells or CD19- CEMC1 control cells at the indicated E:T (effector:target) ratio. Culture supernatant was collected and analyzed by ELLA. N=2 donors are shown.

[0057] [Figure 28A]Figure 28A shows effector dose-dependent CAR-mediated cytokine release by anti-CD19 CAR-T cells produced from leukocyte apheresis starting material via the KYV 3-day process in co-culture with CD19+ NALM6 target cells at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD. The KYV 3-day conditions "C1", "C2", "C3", and "C4" indicate different cultured cytokines used in the production process. "NT" = untransduced control T cells. N=4 healthy donors per condition.

[0058] [Figure 28B] Figure 28B shows cytokine release by anti-CD19 CAR-T cells produced from freshly collected whole blood in a KYV 3-day process in co-culture with CD19+ NALM6 target cells at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. Culture supernatant was collected and the indicated cytokines were analyzed by MSD. "Conv 9 days" refers to donor-matched CAR-T cells produced in a conventional 9-day culture process derived from leukocyte apheresis starting material. N=4 healthy donors per condition.

[0059] [Figure 28C] Figure 28C shows cytokine release by anti-CD19 CAR-T cells produced in the KYV 3-day process from freshly collected whole blood in co-culture with CD19+ NALM6 target cells at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. Culture supernatant was collected and the indicated cytokines were analyzed by MSD. "Conv 9 days" refers to donor-matched CAR-T cells produced in the conventional 9-day culture process from leukocyte apheresis starting material. N=4 healthy donors per condition.

[0060] [Figure 29] Figure 29 shows the duration of in vitro cytotoxicity by KYV 3-day or Conv 9-day anti-CD19 CAR T cells derived from healthy donors in a serial rechallenge assay against CD19+ NALM6 tumor cells. KYV 3-day CAR T cells were derived from leukocyte apheresis starting material ("APH," upper panel) or freshly collected whole blood ("WB," lower panel). CAR T cells were co-cultured with NALM6 target cells in three replicates at the indicated effector:target (E:T) ratio, and NALM6 cell viability was analyzed every 2-3 days by flow cytometry. Time to loss of CAR-mediated cytotoxic activity (days), defined as the assay time point at which >95% survival of target cells was detected, was measured for each individual replicate. Data are representative of n=4 donors.

[0061] [Figure 30] Figure 30 shows the results of in vitro expansion of anti-CD19 CAR-T cells produced from a KYV 3-day process compared to a conventional 9-day process, in response to CD19+ expressing target cells. In vitro expansion of anti-CD19 CAR T cells from KYV 3-day or conventional ("Conv") 9-day cells in response to repeated stimulation in co-culture with CD19+ expressing REH target cells. KYV 3-day CAR T cells were derived from leukocyte apheresis starting material ("APH", panel A, n=4) or other newly collected starting material ("WB", panel B, n=3) and compared to donor-matched Conv 9-day CAR T cells derived from leukocyte apheresis material. CAR T cells were co-cultured with mitomycin C-treated REH target cells in a 1:1 ratio, and cells were reseated every 3-4 days with new target cells. The total expansion proliferation of CAR+ T cells (gated by flow cytometry analysis) was measured on day 16.

[0062] [Figure 31A]Figure 31A shows the in vivo activity of anti-CD19 CAR-T cells produced from the KYV 3-day process compared to the conventional 9-day process in NSG mice with CD19+ NALM6 tumors. Mean NALM6 tumor growth in NSG mice treated with indicated doses of donor-matched anti-CD19 CAR T cells produced from either the KYV 3-day process or the conventional ("Conv") 9-day process, both derived from leukocyte apheresis ("APH") starting material. NALM6-luciferase tumor cells were intravenously injected into mice on day 7 prior to T cell transfer. On day 0, mice were given a single IV injection of indicated doses of CAR T cells. Tumor volume in each animal was measured twice weekly using IVIS bioluminescence imaging and is shown as total flux (photons / second). Data are shown as mean ± SEM for all animals per group. Data are representative of two studies using n=2 independent donors.

[0063] [Figure 31B] Figure 31B shows individual NALM6 tumor growth curves in NSG mice treated with donor-matched anti-CD19 CAR T cells at a dose of 1e6 CAR+ T cells. CAR T cells were prepared from freshly collected whole blood ("WB") using the KYV 3-day process, or from leukocyte apheresis starting material using the conventional ("Conv") 9-day process. Tumor cells were inoculated, mice treated, and analyzed as described in Figure 31A. N=5 animals per group. [Modes for carrying out the invention]

[0064] Detailed explanation A method for the rapid production of engineered T cells expressing heterologous proteins, such as chimeric antigen receptors (CARs), under certain conditions is disclosed herein. Similar methods for the rapid production of engineered T cells containing heterologous genes are also contemplated, wherein the heterologous genes may or may not encode proteins. Compositions suitable for use in conjunction with the disclosed methods are further disclosed.

[0065] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the subject matter of the claims pertains. In general, the nomenclature used in connection with the techniques described herein is well known and commonly used in the art. The above general statements and the following detailed descriptions should be understood to be merely illustrative and descriptive, and not to be limitations on any subject matter of the claims. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0066] As used herein, the singular forms "a," "and," and "the" include multiple referents unless the context explicitly indicates otherwise. For example, a reference to "an antibody" includes a plurality of antibodies, and in certain embodiments, a reference to "an antibody" includes multiple antibodies, and so on.

[0067] As used herein, all numbers or numerical ranges include all integers within or encompassing such a range, and fractions of values ​​or integers within or encompassing such a range, unless the context explicitly indicates otherwise. For example, a reference to the range 90–100% includes not only 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., but also 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, a reference to the range of 1 to 5,000 times includes not only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, but also 1.1, 1.2, 1.3, 1.4, 1.5 times, 2.1, 2.2, 2.3, 2.4, 2.5 times, and so on.

[0068] When used herein, "approximately" refers to the number itself and the range that includes the number itself and the range that includes the range from 10% below that number to 10% above that number. The "approximately" range refers to the range from 10% below the lower limit of that range to 10% above the upper limit of that range.

[0069] As used herein, terms such as “activate,” “activating,” and “activated” encompass a variety of related biological processes when used in the context of T cell activation (e.g., induction of intracellular signaling pathways associated with T cell activation, altered expression of cell surface markers, cytokine release, proliferation, etc.). Generally, T cell activation occurs as a result of the engagement of the T cell receptor complex or its functional portion (e.g., CD3) and a co-stimulatory molecule (e.g., CD28) on T cells by the major histocompatibility complex (MHC) and a co-stimulatory molecule on antigen-presenting cells, respectively. The induction of intracellular signaling cascades associated with T cell activation includes activation of the PI3K pathway, recruitment of PH domain-containing proteins (e.g., PDK1), and eventual cytokine production (e.g., IL-2). Altered expression of T cell surface markers occurs as a result of activation, leading to increased expression of one or more of the following: CD69, CD71, CD25, CD137, HLA-DR, CTLA-4, etc. The production and secretion of cytokines, chemokines, and other proteins (e.g., IFNγ, granzyme B, IL-1β, and / or IL-2) can also result from T cell activation.

[0070] As used herein, the term “basic culture medium” refers to a culture medium containing the minimum set of components essential for the survival of cells (e.g., T cells). "Basic culture medium" is typically an aqueous solution containing amino acids (e.g., glycine, arginine, asparagine, aspartic acid, cysteine, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and / or L-racemates of valine), vitamins (e.g., biotin, choline chloride, calcium D-pantothenate, folate, niacinamide, para-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and / or i-inositol), salts (e.g., calcium nitrate, iron nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium pyruvate, and / or sodium phosphate), a source of sugars (e.g., D-glucose), and optionally a reducing agent (e.g., glutathione). Typical examples of basal culture media include, but are not limited to, RPMI 1640, Eagle Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium Eagle (α-MEM), and Glasgow Minimum Essential Medium (Glasgow MEM). In most cases, the “basal culture medium” does not contain protein additives (e.g., cytokines, growth factors, and / or albumin). In some embodiments, the “basal culture medium” has a pH of 7.0 to 7.4, for example, 7.0, 7.1, 7.2, 7.3, or 7.4. In some embodiments, the “basic culture medium” has a volume osmolality of 290–320 mOsmol (e.g., 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 mOsmol). Those skilled in the art will recognize that culture media having other pH and volume osmolality values ​​can be used. In some embodiments, the “basic culture medium” is hypotonic, isotonic, or hypertonic.

[0071] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to a chimeric receptor protein comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain having antigen-binding specificity. In some cases, the extracellular domain may include an antigen-binding domain. In some cases, the transmembrane domain may include a transmembrane domain derived from a membrane-bound protein or a native polypeptide obtained from a transmembrane protein. Examples of transmembrane domains include, but are not limited to, transmembrane domains derived from T cell receptor alpha or beta chains, CD3 zeta chains, CD28 polypeptides, or CD8 polypeptides. In some cases, the intracellular domain may include a cytoplasmic signaling domain (e.g., any of the cytoplasmic signaling domains described herein) and one or more costimulatory domains (e.g., any of the exemplary costimulatory domains described herein).

[0072] As used herein, terms such as “contact,” “contacting,” and “contacted” include exposing one composition (e.g., cells or a population of cells, e.g., T cells) to another composition (e.g., polynucleotides) by any means such that they can directly interact with each other. Those skilled in the art will recognize that an exemplary method of contacting a population of cells with a drug (e.g., a heterologous protein, e.g., a nucleic acid encoding a CAR) is by mixing an aqueous suspension of cells with an aqueous solution or aqueous suspension of the drug. Even when a drug is delivered in excess of the cells, not all individual cells in a population can immediately engage in direct interaction with the drug. However, over a long period of time (e.g., 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, or longer), a substantial number of cells (e.g., at least 1%, 5%, 10%, 15%, 20%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more) will interact directly with the drug molecule, depending on the method.

[0073] As used herein, the term “cytotoxic” refers to the ability of a cell, e.g., a T cell, to express a chimeric antigen receptor (CAR) according to a method disclosed herein, to induce cell death (e.g., apoptosis or necrosis) of another cell (e.g., a target cell). For example, an engineered T cell expressing a CAR can induce a cytotoxic response against a target cell expressing a target antigen. Binding between the antigen-binding domain of the CAR and the target antigen can, in certain embodiments, result in T cell activation and target cell death. Assays for detecting CAR-T cell-induced cytotoxicity include, but are not limited to, chromium-releasing assays, bioluminescence assays (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analysis, flow cytometry (e.g., in combination with a viability dye, e.g., CTV), and CFSE / PI assays.

[0074] As used herein, the term “delivery vehicle” refers to any pharmaceutical carrier, diluent, excipient, etc., which are generally intended for use in connection with the administration of biologically active drugs, including nucleic acids. For example, a delivery vehicle may include, but is not limited to, lipid or polymer-based transfer vehicles for the delivery of nucleic acids, including lipid nanoparticles, liposomes, polymer nanoparticles (nanocapsules or nanospheres), etc. In certain embodiments, the delivery vehicle is lipid nanoparticles. In the context of nucleic acid delivery to target cells, “delivery vehicle” may also include any vector (e.g., viral vector or non-viral vector) capable of delivering nucleic acids to target cells. In certain embodiments, the delivery vehicle is a viral vector, for example, a lentiviral vector.

[0075] As used herein, the term "engineered" refers to a cell (e.g., a T cell) that is in contact with a nucleic acid encoding a heterologous protein (e.g., a CAR), meaning that the nucleic acid or fragment encoding the heterologous protein is stably integrated into the cell's genome after contact.

[0076] As used herein, terms such as “harvesting” refer to the isolation and / or collection of cells or populations of cells (e.g., T cells) after incubation of said cells under culture conditions. In certain embodiments, harvesting includes one or more conditions, such as temperature, cell culture medium, and / or changes in the availability of certain agents (e.g., one or more agents that activate CD3 and / or CD28, one or more cytokines, and / or polynucleotides encoding heterologous proteins), such that the step immediately preceding the harvesting step is terminated.

[0077] As used herein, the term “heterogeneous” refers to a nucleic acid or polypeptide sequence or domain that is not present in its native form or in the quantities present in its native environment. For example, in some embodiments, heterogeneous nucleic acids (e.g., genes) are not present between their adjacent flanking sequences, and, for example, heterogeneous sequences are not found bound to nucleic acid or polypeptide sequences present at one or both ends. In some embodiments, heterogeneous proteins are not present at all in native cells before being engineered to express the protein. In some embodiments, heterogeneous proteins are present in native cells with different post-translational modifications from the protein before being engineered to express the protein. In some embodiments, heterogeneous proteins are present in substantially lower quantities than the protein in native cells before being engineered to express the protein.

[0078] As used herein, the term “sample” refers to a biological sample obtained from a subject (e.g., a human), such as a blood sample (e.g., a whole blood sample). In certain embodiments, the sample is a blood fraction of a desired type (e.g., serum or plasma), or a blood sample processed by conventional methods to isolate one or more cell types of interest (e.g., peripheral blood mononuclear cells (PBMCs), lymphocytes, e.g., T lymphocytes). For example, “sample” may refer to a leukocyte apheresis sample obtained from the blood of a subject. In certain embodiments, “sample” refers to a whole blood sample obtained from a subject.

[0079] In certain embodiments, a method for the rapid production of engineered T cells containing heterologous genes or expressing heterologous proteins, such as chimeric antigen receptors (CARs), is disclosed herein. Compositions suitable for use in conjunction with the disclosed method are also disclosed. The method disclosed herein provides certain advantages over previous CAR-T production methods, including the production of more potent CAR-T cells compared to CAR-T cells produced using longer production protocols, thereby promoting the use of lower CAR-T cell doses for therapeutic use. Furthermore, the disclosed method also preserves the "stem cell nature" (i.e., a less differentiated phenotype) of T cells, thereby producing CAR-T cells with higher potential for proliferation. The shorter CAR-T production times obtained from the disclosed method also facilitate the scaling down of CAR-T cell production, resulting in reduced costs, reduced "needle-to-needle" time (i.e., the time from harvesting a patient's T cells to returning and delivering engineered autologous T cells to the patient), and improved patient access.

[0080] CAR-T cells Chimeric antigen receptors (CARs) are artificially constructed hybrid receptor proteins or polypeptides containing an antigen-binding domain of an antibody, e.g., an antigen-binding fragment, which can take various formats such as a single-stranded variable fragment (scFv), linked via a transmembrane domain to one or more intracellular signaling or activation domains (including, optionally, a costimulatory domain). Autologous T-cell-based therapies, e.g., T cells modified to express CARs, have demonstrated significant therapeutic benefits for patients with cancer. While we do not wish to be bound by specific theories or mechanisms, it is believed that by inducing an antigen-specific response against cells expressing a target antigen, CARs provide one or more of the following benefits: targeting and destruction of target antigen-expressing cells, reduction or elimination of target cells, promotion of immune cell infiltration into target tissues, and enhancement / expansion of the anti-cancer response. Autoimmune responses can also be reduced by targeting autoimmune-mediating cells (e.g., B cells) using CAR-T cells. While this harbinges the potential of unprecedented therapies, CAR-T cell generation faces considerable obstacles, including long manufacturing times, when relevant to the scope of this invention. Extending the CAR-T cell manufacturing process can unfavorably result in batch loss, reduced in vivo expansion and persistence of CAR-T cells, increased batch-to-batch variability of the final cell product, and increased manufacturing costs. Therefore, this disclosure provides methods and compositions for the rapid manufacture of CAR-T cells. It is understood that similar methods and compositions may be used for the rapid manufacture of T cells expressing other heterologous genes.

[0081] Method for producing manipulated T cells In certain embodiments, a method for producing a population of T cells engineered to express heterologous proteins is disclosed herein. T cells are leukocytes that have fully matured in the thymus and can identify certain foreign antigens, performing various roles in the immune system, including the activation and deactivation of other immune cells. Generally, T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or T cells derived from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from mammals. Examples of T cells, but not limited to, include naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subgroups thereof. T cells may be CD3+ cells. T cells may be CD4+, CD8+, or CD4+ and CD8+. For example, T cells can be CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 or Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral cells, for example, but not limited to, blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, γδ T cells, etc. T cells can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include central memory T cells (Tcm cells) and effector memory T cells (T EM Cells and T EMRA Examples of cells include T cells. T cells can also refer to genetically modified T cells (e.g., engineered T cells), such as T cells that have been modified to express heterologous proteins, such as chimeric antigen receptors (CARs). T cells can also be differentiated from stem cells or progenitor cells.

[0082] In certain embodiments, the disclosed method relates to the generation of engineered T cells expressing CARs having binding specificity to a target antigen. In certain embodiments, the disclosed method is used to generate a population of engineered T cells from a starting population of cells over a short process (e.g., 2, 3, or 4 days). For example, the method of the present disclosure can generate a population of engineered T cells expressing a heterologous protein (e.g., CAR) for 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, or 96 hours. In certain embodiments, the method enables the generation of engineered T cells (e.g., T cells expressing CAR) in three days. Thus, the disclosed method, in certain embodiments, includes (1) optionally a step of isolating a biological sample from a subject (e.g., a human subject) containing a T cell starting population; (2) a step of activating the T cell starting population with a CD3-binding agent and a costimulatory molecule (e.g., CD28-binding agent); (3) a step of contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein (e.g., CAR) and culturing the T cells under conditions and for a time suitable for promoting the expression of the heterologous protein by the T cells; and (4) a step of harvesting, and optionally a step of storing the engineered T cells for later use (e.g., therapeutic use or quality control testing). The following sections describe each of the aforementioned steps in more detail.

[0083] Sample preparation step In certain embodiments, a method for isolating a starting population of T cells from a biological sample, for example, a sample obtained from a subject (e.g., a human subject), is disclosed herein. Non-limiting examples of biological samples include cells, tissues (e.g., tissue obtained by biopsy), blood, serum, plasma, or any sample derived therefrom. In certain embodiments, the sample is a whole blood sample or an apheresis (e.g., leukocyte apheresis) sample obtained from a subject. In certain embodiments, the method includes the step of obtaining the sample from the subject. In certain embodiments, the method includes the step of obtaining the sample from the subject.

[0084] In certain embodiments, the method disclosed herein includes the step of obtaining a starting population of T cells from a biological sample obtained from a subject. In certain embodiments, the biological sample is a leukocyte apheresis sample. In certain embodiments, the biological sample is a whole blood sample. In certain embodiments, the starting population of T cells includes helper T(Th) cells and cytotoxic T(Th) cells. C ) cells, memory T(T) M Th cells include regulatory T (Treg) cells and innate immune-like T cells. In certain embodiments, Th cells include Th1 cells, Th2 cells, Th17 cells, Th9 cells, Tfh cells, and / or Th22 cells. In certain embodiments, T M The cells are central memory T cells (T CM ) Cells, effector memory T(T EM ) Cells, tissue-resident memory T(T) RM ) cells, and virtual memory T(T) VM ) cells are included. In certain embodiments, innate immune-like T cells include natural killer T (NKT) cells, mucosa-associated invariant T (MAIT) cells, and γδ T cells.

[0085] In certain embodiments, the method includes the step of isolating a starting population of T cells from a sample. Isolation of T cells may include the initial purification of T cells from a mixture of plasma, lymphocytes, platelets, erythrocytes, monocytes, and granulocytes. Methods for isolating T cells from biological samples, e.g., whole blood samples or leukocyte apheresis samples, are well known. Exemplary methods may include leukocyte apheresis, elutriation, density gradient centrifugation, and enrichment by selection. For example, the method may include the steps of obtaining or having obtained a biological sample, e.g., fresh, refrigerated, frozen, or cryopreserved leukocyte apheresis product, or hematopoietic tissue, e.g., a whole blood sample, an alternative source of bone marrow sample, or a biopsy or removal of a tumor or organ (e.g., thymectomy), from an entity, e.g., a laboratory, hospital, or healthcare provider, and performing the aforementioned isolation to produce an enriched population of T cells suitable for heterologous protein expression (e.g., a starting population of T cells).

[0086] Furthermore, the purity of the starting population of T cells can be increased by using one or more selection steps, such as negative selection or positive selection. Negative selection typically involves removing unwanted cell types from a mixed population of cells in a sample by using one or more agents that selectively bind to unwanted cell types, while positive selection typically involves isolating a desired cell population by using one or more agents that selectively bind to desired cell types. Enrichment of a T cell population by negative selection can be achieved, for example, by using a combination of antibodies against surface markers specific to negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunosorbent or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail could include antibodies against CD14, CD20, CDb, CD16, HLA-DR, and CD8.

[0087] On the other hand, the positive selection step can be used to specifically select for a desired cell type. In certain embodiments, positive selection of T cells may include incubation of a mixed population of cells containing T cells with a drug having a CD3 binding moiety (e.g., anti-CD3 antibody conjugate beads) for a time sufficient to positively select the desired T cells. In certain embodiments, the period is about 30 minutes. In certain embodiments, the period is in the range of 30 minutes to 36 hours or longer, and all integer values ​​in between. In certain embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In certain embodiments, the period is 10 to 24 hours, for example, 18 hours. Longer incubation times may be used to isolate T cells in any situation where fewer T cells are present compared to other cell types.

[0088] In a particular embodiment, the starting population of T cells is CD8 + T cells (e.g., CD8) + Includes cytotoxic T cells. In certain embodiments, the starting population of T cells is CD4 + T cells (e.g., CD4 + The population further includes helper T cells. In certain embodiments, the starting population of T cells is comprised of 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 20-30%, 20-40%, 20-50%, 20-60%, 30-40%, 30-50%, or 30-60% CD8 cells. + T cells (e.g., CD8) + (Cytotoxic T cells) are included. In certain embodiments, the starting population of T cells is 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 30-40%, 30-50%, 30-60%, or 30-70% CD4 +T cells (e.g., CD4 + The population of T cells further includes helper T cells. In certain embodiments, the starting population of T cells is CD8 in ratios of 1:5-5:1, 1:4-4:1, 1:3-3:1, 1:2-2:1, 1:5-2:1, 1:4-2:1, 1:3-1:1, or 1:2-1:1. + T cells (e.g., CD8) + Cytotoxic T cells and CD4 + T cells (e.g., CD4 + Includes helper T cells. In some embodiments, the cell mixture includes CD8+ T cells and CD4+ T cells in a ratio of about 1:2.

[0089] In certain embodiments, the T cell starting population is generated to achieve a desired degree of purity. For example, the T cell starting population may contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more T cells of the total number of cells in the population. The purity of the T cell starting population may be measured using routine methods, such as fluorescence-assisted cell sorting (FACS), immunopanning, or microarray-based methods. Various known T cell phenotyping methods may also be applied to further increase the purity of the T cell starting population.

[0090] Furthermore, one or more freeze-thaw cycles can be performed on a starting population of T cells to enrich them with a desired cell type. For example, freezing and thawing can improve the purity of a T cell population by further removing granulocytes and, to a certain extent, monocytes in a mixed population of cells. Routine and conventional methods for freezing and thawing T cells can be used in conjunction with the methods disclosed herein. In certain embodiments, after freezing, the frozen cells are thawed, washed, and allowed to rest at room temperature for, for example, one hour before activation using the disclosed methods.

[0091] In certain embodiments, the T cell starting population may be assayed for viability using known methods. For example, the T cell starting population may be assayed using one or more known markers of T cell identity and viability markers (e.g., dyes, antibodies, etc.), where the overlap of signals indicating both T cell identity and viability indicates the viability of the T cell starting population. In certain embodiments, the starting population includes a percentage of surviving T cells of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher than that of the total number of T cells in the T cell starting population.

[0092] In certain embodiments, the T cell starting population may be assayed for exhaustion and / or activation status. For example, the T cell starting population may be tested for exhaustion status using one or more (e.g., 1, 2, 3, or more) T cell exhaustion markers, including, but not limited to, the overexpression of one or more of LAG-3, PD-1, PD-L1, TIM-3, 2B4, CD160, TIGIT, CTLA-4, VISTA, etc. The T cell activation status in the starting population can be assessed by testing for the overexpression of one or more T cell activation markers (e.g., CD69, CD71, CD25, CD137, HLA-DR, CTLA-4, L2RA / CD25, IFNγ, TNFα, etc.). Additional indicators of T cell activation include, but not limited to, T cell proliferation and differentiation.

[0093] Following the isolation and enrichment of T cell starting populations from the original biological sample, the T cell starting population may be incubated under culture conditions suitable for maintaining T cells in a resting state before activation. The T cell starting population may be seeded at a desired density to facilitate T cell activation and / or transduction by a nucleic acid vector encoding a heterologous protein (e.g., CAR). For example, the T cell starting population may be seeded at 1 × 10⁶ 5 cells / mL~1×10 7The cells may be seeded into the culture at a concentration of cells / mL. In a particular embodiment, the starting population of T cells is approximately 1 × 10⁶ 6 The cells are seeded into the culture at a concentration of cells / mL. In a particular embodiment, the starting population of T cells is 1 × 10⁶ 6 The cells are seeded into the culture at a concentration of cells / mL. In a particular embodiment, the starting population of T cells is approximately 2 × 10⁶ 6 The cells are seeded into the culture at a concentration of cells / mL. In a particular embodiment, the starting population of T cells is 2 × 10⁶ 6 The cells are seeded into the culture at a concentration of cells / mL. In a particular embodiment, the starting population of T cells is approximately 5 × 10⁶ 6 The cells are seeded into the culture at a concentration of cells / mL. In a particular embodiment, the starting population of T cells is 5 × 10⁶. 6 The cells are seeded into the culture at a concentration of cells / mL.

[0094] In certain embodiments, prior to activation, the T cell starting population is incubated in a culture medium containing 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1% serum (e.g., human serum). In certain embodiments, prior to activation, the T cell starting population is incubated in a culture medium containing 2% serum. In certain embodiments, prior to activation, the T cell starting population is incubated in serum-free culture medium. In certain embodiments, prior to activation, the T cell starting population is incubated in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In certain embodiments, one or more cytokines are IL-2. In certain embodiments, one or more cytokines are IL-7 and IL-15. In certain embodiments, one or more cytokines are IL-2, IL-7, and IL-15. In certain embodiments, one or more cytokines are IL-21. In certain embodiments, one or more cytokines are IL-21, IL-7, and IL-15. In certain embodiments, T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL of IL-2, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are contacted with 100 ng / mL of IL-2. In certain embodiments, T cells are exposed to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-7, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are exposed to 10 ng / mL of IL-7.In certain embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-15, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are contacted with 10 ng / mL of IL-15. In certain embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, prior to activation, a starting population of T cells is incubated in the absence of any cytokine selected from IL-2, IL-7, IL-15, and IL-21. In a particular embodiment, prior to activation, the starting population of T cells is incubated in a cytokine-free culture medium.

[0095] T cell activation step In certain embodiments, a method for producing a population of engineered T cells expressing a heterologous protein (e.g., CAR) is disclosed herein, comprising a step of activating a T cell starting population. In certain embodiments, the activation of the T cell starting population comprises a step of contacting the T cell starting population with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the T cells. In certain embodiments, the costimulatory molecule is CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In certain embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody or its antigen-binding fragment (e.g., full-length IgG, Fab fragment, single-domain antibody, scFv, diabody, triabody, etc.). In certain embodiments, the agent that stimulates the CD3 / TCR complex is a small molecule or peptide ligand. In certain embodiments, the costimulatory molecule is CD28. In certain embodiments, the agent stimulating the co-stimulatory molecule is an anti-CD28 antibody or its antigen-binding fragment. In certain embodiments, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulatory molecule includes beads (e.g., magnetic beads). In certain embodiments, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulatory molecule is a solid surface (e.g., beads) containing an anti-CD3 antibody and / or anti-CD28 antibody covalently attached thereto. In certain embodiments, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulatory molecule does not include beads. In certain embodiments, the agent stimulating the CD3 / TCR complex and the agent stimulating the co-stimulatory molecule (e.g., CD28) includes a first agent stimulating CD3 and a second agent stimulating the co-stimulatory molecule. In certain embodiments, the agent stimulating the CD3 / TCR complex and the agent stimulating the co-stimulatory molecule (e.g., CD28) are the same agent. In certain embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule (e.g., CD28) are bispecific antibodies that specifically bind to CD3 and CD28.In certain embodiments, the agents that stimulate the CD3 / TCR complex and the agents that stimulate the co-stimulatory molecule (e.g., CD28) are beads (e.g., magnetic beads) containing anti-CD3 antibodies and anti-CD28 antibodies covalently attached thereto. In examples where the agents that stimulate the CD3 / TCR complex and the agents that stimulate the co-stimulatory molecule (e.g., CD28) are beads, it is understood that the beads may remain attached to the T cells at the end of the activation step (i.e., at the beginning of the transfection step), and that the T cells are detached from the beads before harvesting as a result of the spontaneous degradation of the protein portion of the agent.

[0096] In certain embodiments, the agent stimulating the CD3 / TCR complex and / or the agent stimulating the co-stimulatory molecule comprises an anti-CD3 antibody and / or an anti-co-stimulatory molecule antibody covalently attached to a colloidal polymer matrix (e.g., a nanomatrix). In certain embodiments, the matrix comprises or consists of a polymer material that is nontoxic to cells, for example, a biodegradable or biocompatible inert material. In certain embodiments, the matrix is ​​composed of hydrophilic polymer chains, which, due to the hydration of the chains, achieve maximum mobility in aqueous solution. In certain embodiments, the mobile matrix may be collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan, or an extracellular matrix composition. Examples of polysaccharides include cellulose ether, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum, or alginates. Other polymers may include polyester, polyether, polyacrylate, polyacrylamide, polyamine, polyethyleneimine, polyquaternium polymer, polyphosphazene, polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, block copolymer, or polyurethane.

[0097] In certain embodiments, the step of contacting a T cell starting population with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule is performed once at the start of the activation step. In certain embodiments, the step of contacting a T cell starting population with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule is performed once at the start of the activation step and one or more times (e.g., 1, 2, 3, or more) throughout the duration of the activation step. While we do not wish to be bound by any theory, those skilled in the art will recognize that the duration of binding of the CD3 / TCR complex stimulating drug and / or the drug that stimulates a co-stimulatory molecule to one or more cells in a T cell starting population depends, among other factors, on the specific drug(s) used, the concentration of the drug(s), and the concentration of cells seeded in the culture.

[0098] In certain embodiments, the duration of the activation step is 12–24 hours (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). In certain embodiments, the duration of the activation step is approximately 18 hours (e.g., 16, 17, 18, 19, or 20 hours). In certain embodiments, the duration of the activation step is 18 hours. In certain embodiments, after the activation step, the T cells remain associated with the agent stimulating the CD3 / TCR complex and / or the agent stimulating the co-stimulatory molecule on the surface of the T cells so that activation may continue during subsequent steps. In certain embodiments, activation continues effectively until the T cells detach from the agent (e.g., by spontaneous degradation of the protein portion of the agent).

[0099] In certain embodiments, during the activation step, the T cell starting population is incubated in a culture medium containing 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1% serum. In certain embodiments, during the activation step, the T cell starting population is incubated in a culture medium containing 2% serum. In certain embodiments, during the activation step, the T cell starting population is incubated in a culture medium containing basal culture medium and serum (e.g., 2% serum). In certain embodiments, the culture medium further contains no additional cytokines or growth factors other than serum-derived proteins. In certain embodiments, the culture medium further contains no additional proteins (e.g., soluble proteins) other than serum-derived proteins.

[0100] In certain embodiments, during the activation step, the T cell starting population is incubated in serum-free culture medium. In certain embodiments, during the activation step, the T cell starting population is incubated in basal culture medium that is not supplemented with serum. In certain embodiments, the basal culture medium does not contain any cytokines or growth factors. In certain embodiments, the basal culture medium does not contain any added proteins. It is understood that during cell culture, cells in the culture medium may secrete proteins. It is also understood that agents that stimulate the CD3 / TCR complex and / or agents that stimulate co-stimulatory molecules may degrade spontaneously over time, releasing fragments into the culture medium. These proteins are not considered “added proteins”.

[0101] In certain embodiments, during the activation step, the T cell starting population is incubated in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In certain embodiments, one or more cytokines are IL-2. In certain embodiments, one or more cytokines are IL-7 and IL-15. In certain embodiments, one or more cytokines are IL-2, IL-7, and IL-15. In certain embodiments, one or more cytokines are IL-21. In certain embodiments, one or more cytokines are IL-21, IL-7, and IL-15. In certain embodiments, T cells are exposed to 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL of IL-2, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are exposed to 100 ng / mL of IL-2. In certain embodiments, T cells are exposed to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-7, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are contacted with 10 ng / mL of IL-7. In certain embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-15, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are contacted with 10 ng / mL of IL-15. In certain embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-21, either alone or in combination with one or more other cytokines.In certain embodiments, prior to activation, a starting population of T cells is incubated in the absence of any cytokine selected from IL-2, IL-7, IL-15, and IL-21. Either serum-free culture medium (e.g., basal culture medium) or serum-supplemented culture medium may be provided in or without the cytokines and combinations thereof disclosed herein.

[0102] In certain embodiments, prior to activation, the T cell starting population is incubated in a cytokine-free culture medium or without added cytokines. In other embodiments, the step of contacting the T cell starting population with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule (e.g., CD28) is performed simultaneously with contacting the cell population with one or more cytokines. In certain embodiments, the step of contacting the T cell starting population with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule (e.g., CD28) is performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, or longer, before contacting one or more cytokines.

[0103] Transfection step: Delivery of heterologous nucleic acids to T cells In certain embodiments, a method for generating an engineered population of T cells expressing a heterologous protein (e.g., CAR) is disclosed herein, comprising an activation step followed by the step of delivering nucleic acids encoding the heterologous protein to the T cells. Polynucleotides encoding heterologous proteins (e.g., CAR) are also disclosed herein.

[0104] In certain embodiments, delivery of a heterologous protein-coding nucleic acid(s) to T cells occurs approximately 18 hours or less after the start of the activation step (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour or less, or shorter). In certain embodiments, delivery of a heterologous protein-coding nucleic acid(s) to T cells occurs approximately 18 hours after the start of the activation step (e.g., 16, 17, 18, 19, or 20 hours). In certain embodiments, delivery of a heterologous protein-coding nucleic acid(s) to T cells occurs 18 hours after the start of the activation step. If transfection is initiated after the start of the activation step, the cells at the end of the activation step (i.e., the beginning of the transfection step) are referred to herein as the “activated T cell population,” but at least a portion of the cells in the population may not be fully activated, and further activation may occur during the transfection step. In other embodiments, delivery of a nucleic acid(s) encoding a heterologous protein to a starting population of T cells is performed in conjunction with (for example, simultaneously with) the activation step.

[0105] In a particular embodiment, the disclosed method comprises the step of contacting a population of T cells with a polynucleotide encoding a heterologous protein after an activation step, thereby resulting in the cells exhibiting stable expression of the heterologous protein for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, or longer.

[0106] In certain embodiments, a polynucleotide encoding a heterogeneous protein (e.g., CAR) comprises a codon-optimized nucleic acid sequence having one or more nucleotide differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) compared to the parent (i.e., non-codon-optimized) nucleic acid. Nucleic acid sequences can be codon-optimized according to various principles, such as the principle that the frequency of synonymous codons (e.g., codons coding for the same amino acid) in coding DNA is biased across different species. Such codon degeneracy allows the same polypeptide to be coded by various nucleotide sequences. This process may be performed on any of the sequences described herein to enhance expression or stability. Codon optimization may be performed using conventional methods. Sequences around the translation start site can be converted to consensus Kozak sequences according to known methods.

[0107] nucleic acid vectors In addition to achieving high rates of transcription and translation, stable expression of exogenous genes (e.g., polynucleotides encoding heterologous polypeptides or their functional fragments) in mammalian cells can be achieved by the integration of gene-containing polynucleotides into the nuclear genome of mammalian cells. Various vectors have been developed for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Expression vectors for use in the compositions and methods described herein contain polynucleotide sequences encoding heterologous proteins (e.g., CARs), as well as additional sequence elements used, for example, for the expression of these agents and / or the integration of these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used for the expression of heterologous proteins include plasmids containing regulatory sequences, e.g., promoter and enhancer regions, that direct the transcription of the gene. Other useful vectors for the expression of heterologous proteins contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, intra-sequence ribosome entry sites (IRESs), and polyadenylation signaling sites to direct the efficient transcription of the gene supported in the expression vector. Expression vectors suitable for use by the compositions and methods described herein may also contain polynucleotides encoding markers for the selection of cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and noseoslysin.

[0108] Expression vectors for use in the compositions and methods described herein may express heterologous proteins (e.g., CARs) from monocistronic or polycistronic expression cassettes. A monocistronic expression cassette contains a polynucleotide sequence encoding a single gene. Host cells described herein may be transfected with multiple vectors, e.g., multiple vectors each containing a monocistronic expression cassette, or a single vector containing two or more monocistronic expression cassettes. A polycistronic expression cassette can be used to simultaneously express two or more proteins from a single transcript. A polycistronic expression cassette may contain dicistronic or tricistronic expression cassettes, which can be used to produce two or three proteins, respectively, from a single transcript, and may contain an IRES sequence to recruit ribosomes that initiate translation from a region of mRNA other than the 5' cap. Alternatively, foot-and-mouth disease virus 2A (FMDV 2A) polynucleotides can be used to express two or more genes (e.g., two genes, three genes, or more) and can be used in a polycistronic expression cassette to produce multiple genes at equimolar levels from the same transcript. FMDV 2A mediates a cotranslational cleavage event, which separates proteins linked by the 2A sequence, and multiple 2A sequences may be used in a single vector. Other virus-derived 2A-like sequences, including those derived from equine rhinitis A virus (E2A), porcine rhinitis virus-1 (P2A), and tosea signalavirus (T2A), can also be used in the compositions and methods described herein.

[0109] Viral vector Viral genomes provide an abundant source of viruses that can be used for the efficient delivery of exogenous genes into mammalian cells. Viral genomes are particularly useful vectors for gene delivery as polynucleotides contained within such genomes, typically integrated into the nuclear genome of mammalian cells by generalized or specific transduction. These processes occur as part of the natural viral replication cycle and do not require added proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae viral vectors, e.g., lentiviral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses, e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), as well as double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia ankara (MVA), fowlpox, and canary pox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis viruses. Examples of retroviruses include avid leukemia sarcoma, avid C virus, mammalian C, B, and D viruses, onchoretrovirus, HTLV-BLV group, alpharetrovirus, gammaretrovirus, and spumavirus.Other examples include mouse leukemia virus, mouse sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus.

[0110] Exemplary lentiviral vectors that may be used in accordance with this disclosure include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), and canine arthritis encephalitis virus (CAEV).

[0111] Retroviral vectors are typically constructed such that a large portion of the sequence encoding the structural genes of the virus is deleted and replaced by the gene of interest or the expression cassette of interest (e.g., an engineered nucleic acid as described herein). Most often, the structural genes (i.e., gag, pol, and env) are removed from the retroviral backbone using genetic engineering techniques known in the art. This may involve digestion by a suitable restriction endonuclease, or in some examples, by a Bal 31 exonuclease, such as creating a fragment containing the appropriate portion of the packaging signal. Thus, in some embodiments, the minimal retroviral vector contains, from 5' to 3', a 5' long-chain terminal repeat sequence (LTR), a packaging signal, an exogenous promoter and / or enhancer as needed, the exogenous gene of interest (or engineered nucleic acid), and a 3' LTR. In some embodiments, if an exogenous promoter is not provided, gene expression may be driven by a 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression. In many embodiments, structural genes are provided in separate vectors for the production of lentiviruses, and the resulting virions can be made replication-deficient. Specifically, with respect to lentiviruses, the packaging system may include a single packaging vector encoding the Gag, Pol, Rev, and Tat genes, as well as a third separate vector encoding the envelope protein Env (usually VSV-G due to its broad infectivity). To improve the safety of the packaging system, the packaging vector can be split to express Rev from one vector and Gag and Pol from another. Tat can also be excluded from the packaging system by using a retroviral vector containing a chimeric 5'LTR, where the U3 region of the 5'LTR is replaced with a heterologous regulatory element.

[0112] Nucleic acids (e.g., genes) packaged in retroviruses (e.g., lentiviruses) can be incorporated into the proviral backbone in several common ways. The most direct construction involves replacing the retroviral structural gene with a single gene, which is then transcribed under the control of a viral regulatory sequence within the LTR. Retroviral vectors have also been constructed that can introduce two or more genes into target cells. Typically, in such vectors, one gene is under the regulatory control of the viral LTR, while the second gene is expressed independently of the spliced ​​message or under the control of its own internal promoter.

[0113] Therefore, the nucleic acids (e.g., genes) packaged in retroviruses are flanked by 5'LTR and 3'LTR, which play a role in promoting the transcription and polyadenylation of virion RNA, respectively. The term “long-terminus repeat” or “LTR” refers to a domain of base pairs located at the end of retroviral DNA, where, in the context of their native sequences, it is a direct repeat and contains U3, R, and U5 regions. LTRs generally provide the underlying functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain numerous regulatory signals, including transcriptional regulatory elements, polyadenylation signals, and sequences necessary for the replication and integration of the viral genome. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. In certain embodiments, the R region includes a transactivation response (TAR) gene element, which interacts with a transactivator (tat) gene element to enhance viral replication. This element is not required in embodiments where the U3 region of the 5'LTR is replaced by a heterologous promoter.

[0114] In some embodiments, the retroviral vector includes a modified 5'LTR and / or 3'LTR. Modification of the 3'LTR is often done to improve the safety of the lentiviral or retroviral system by making the virus replication-deficient. In some embodiments, the retroviral vector is a self-inactivating (SIN) vector. As used herein, a SIN retroviral vector refers to a replication-deficient retroviral vector in which the 3'LTR U3 region is modified (e.g., by deletion or substitution) to prevent viral transcription after the first round of viral replication. This is because the 3'LTR U3 region is used as a template for the 5'LTR U3 region during viral replication, and therefore the viral transcript cannot be produced without the U3 enhancer-promoter. In some embodiments, the 3'LTR is modified so that the U5 region is replaced, for example, with an ideal polyadenylated sequence. Modifications to the LTR, such as modifications to the 3'LTR, 5'LTR, or both the 3' and 5'LTR, are also included in some embodiments of this disclosure.

[0115] In some embodiments, the U3 region of the 5'LTR is replaced with a heterologous promoter to drive transcription of the viral genome during viral particle generation. Examples of heterologous promoters that can be used include, for example, the promoters of simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., very early), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase). Typical promoters can drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination for generating a replicable virus because the complete U3 sequence is not present in the viral generation system.

[0116] The facilitation to the 5'LTR is a sequence required for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (Psi site). As used herein, the terms “packaging signal” or “packaging sequence” refer to a sequence located within the retroviral genome that is required for capsid formation of the retroviral RNA strand during viral particle formation (see, e.g., Clever et al., 1995 J. Virology, 69(4):2101-09). The packaging signal may also be the minimal packaging signal (also called the psi [Ψ] sequence) required for capsid formation of the viral genome.

[0117] In some embodiments, the retroviral vector (e.g., a lentiviral vector) further comprises a FLAP. As used herein, the term "FLAP" refers to a nucleic acid whose sequence comprises the central polypurine sequence and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al. (2000) Cell 101:173. During reverse transcription, the central start of the positive-strand DNA at the cPPT and the central termination at the CTS result in the formation of a triple-stranded DNA structure: the central DNA flap. While we do not wish to be bound by any theory, the DNA flap may act as a cis-activity determinant of nuclear translocation of the lentiviral genome and / or increase the viral titer. In some embodiments, the retroviral vector backbone comprises one or more FLAP elements upstream or downstream of the heterologous gene of interest in the vector. For example, in some embodiments, the transfer plasmid comprises a FLAP element. In some embodiments, the vector of this disclosure includes a FLAP element isolated from HIV-1.

[0118] In some embodiments, the retroviral vector (e.g., a lentiviral vector) further includes transport elements. In some embodiments, the retroviral vector includes one or more transport elements. The term “transport element” refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA transport elements, but not limited to, include human immunodeficiency virus (HIV) RREs (e.g., see Cullen et al., (1991) J. Virol. 65: 1053; and Cullen et al., (1991) Cell 58: 423) and hepatitis B virus post-transcriptional regulatory elements (HPREs). Generally, RNA transport elements are located within the 3' UTR of a gene and may be inserted as one or more copies.

[0119] In some embodiments, the retroviral vector (e.g., lentiviral vector) further includes post-transcriptional regulatory elements. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; see Zufferey et al., (1999) J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and optimized post-transcriptional regulatory elements (oPRE; see Schambach et al., (2006) Gene Therapy 13, 641-45) (Liu et al., (1995), Genes Dev., 9:1766). Post-transcriptional regulatory elements are generally located at the 3' end of heterologous nucleic acid sequences. This configuration results in the synthesis of an mRNA transcript in which its 5' position contains a heterologous nucleic acid coding sequence and its 3' position contains a post-transcriptional regulatory element sequence. In some embodiments, the vectors of the present disclosure lack or do not contain post-transcriptional regulatory elements such as WPRE or HPRE, because in some cases these elements do not increase the risk of cell transformation and / or substantially or significantly increase the amount of mRNA transcript or mRNA stability. Accordingly, in certain embodiments, the vectors of the present disclosure lack or do not contain WPRE or HPRE as an added safety measure.

[0120] Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. Therefore, in some embodiments, retroviral vectors (e.g., lentiviral vectors) further include polyadenylation signals. The terms “polyadenylation signal” or “polyadenylation sequence,” as used herein, refer to a DNA sequence that directs both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase H. Since transcripts lacking polyadenylation signals are unstable and rapidly degraded, efficient polyadenylation of recombinant transcripts is desirable. Examples of polyadenylation signals that can be used in the vectors of this disclosure include ideal polyadenylation sequences (e.g., AATAAA, ATTAAA AGTAAA), bovine growth hormone polyadenylation sequences (BGHpA), rabbit β-globin polyadenylation sequences (rβgpA), or other suitable heterologous or endogenous polyadenylation sequences known in the art.

[0121] In some embodiments, the retroviral vector further includes an insulator element. The insulator element may contribute to protecting the retroviral expression sequence, such as a therapeutic gene, from the effects of the integration site, which can be mediated by a cis-acting element present in the genomic DNA, resulting in the deregulation of the expression of the transferred sequence (i.e., positional effects; see, e.g., Burgess-Beusse et al., (2002) Proc. Natl. Acad. Sci., USA, 99:16433; and Zhan et al., 2001, Hum. Genet., 109:471). In some embodiments, the retroviral vector includes the insulator element in one or both of the LTRs, or somewhere in the region of the vector to be integrated into the cellular genome. Suitable insulators for use in this disclosure include, but are not limited to, chicken β-globin insulators (see Chung et al., (1993). Cell 74:505; Chung et al., (1997) Proc. Natl. Acad. Sci., USA 94:575; and Bell et al., 1999. Cell 98:387). Examples of insulator elements include, but are not limited to, insulators derived from β-globin loci, such as chicken HS4.

[0122] Non-exclusive examples of lentiviral vectors include pLVX-EF1alpha-AcGFP1-C1 (Clontech catalog #631984), pLVX-EF1alpha-IRES-mCherry (Clontech catalog #631987), pLVX-Puro (Clontech catalog #632159), pLVX-IRES-Puro (Clontech catalog #632186), pLenti6 / V5-DEST(trademark) (Thermo Fisher), and pLenti6.2 / V5-DEST(trademark) (Thermo Fisher). Fisher), pLKO.1 (Addgene plasmid #10878), pLKO.3G (Addgene plasmid #14748), pSico (Addgene plasmid #11578), pLJM1-EGFP (Addgene plasmid #19319), FUGW (Addgene plasmid #14883), pLVTHM (Addgene plasmid #12247), pLVUT-tTR-KRAB (Addgene plasmid #11651), pLL3.7 (Addgene plasmid #11795), pLB (Addgene plasmid #11619), pWPXL (Addgene plasmid #12257), pWPI (Addgene plasmid #12254), EF.CMV.RFP (Addgene plasmid #17619), pLenti CMV Puro Examples include DEST (Addgene plasmid #17452), pLenti-puro (Addgene plasmid #39481), pULTRA (Addgene plasmid #24129), pLX301 (Addgene plasmid #25895), pHIV-EGFP (Addgene plasmid #21373), pLV-mCherry (Addgene plasmid #36084), pLionII (Addgene plasmid #1730), and pInducer10-mir-RUP-PheS (Addgene plasmid #44011). These vectors can be modified to suit therapeutic use. For example, a selection marker (e.g., puro, EGFP, or mCherry) can be deleted or replaced with a second exogenous gene of interest.Further examples of lentiviral vectors include U.S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, and 6,287,814. This information is disclosed in publications No. 6,013,516, No. 6,797,512, No. 6,544,771, No. 5,834,256, No. 6,958,226, No. 6,207,455, No. 6,531,123, and No. 6,352,694, as well as in PCT Publication WO2017 / 091786.

[0123] In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR), such as a viral vector, disclosed herein, is administered to a population of cells (e.g., a T cell starting population) at an infection multiplicity (MOI) of 0 to 24 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a population of cells (e.g., a T cell starting population) at an MOI of about 4. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a population of cells (e.g., a T cell starting population) at an MOI of about 5. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a population of cells (e.g., a T cell starting population) at an MOI of about 6. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a population of cells (e.g., a T cell starting population) at an MOI of about 7. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a population of cells (e.g., a T cell starting population) at an MOI of about 8. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a population of cells (e.g., a T cell starting population) at an MOI of about 9. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a population of cells (e.g., a T cell starting population) at an MOI of about 10.

[0124] Nucleic acid transfer vehicle Nucleic acid delivery vehicles are another advantageous method for delivering polynucleotides encoding heterologous proteins of this disclosure to target cells (e.g., a starting population of T cells). In certain embodiments, the nucleic acid delivery vehicle is nanoparticles, e.g., lipid nanoparticles (e.g., LNPs), non-lipid polymer core-shell nanoparticles, or biodegradable nanoparticles. In certain embodiments, the LNPs include one or more of ionized lipids, PEGylated lipids, structural lipids (e.g., cholesterol), and / or helper lipids. While we do not wish to be bound by theory, it is believed that the delivery vehicles described herein encapsulate nucleic acids encoding heterologous proteins from degradation and provide effective delivery of nucleic acids to target cells in vivo and in vitro.

[0125] Cell culture conditions In certain embodiments, the step of contacting an activated T cell population with a polynucleotide encoding a heterologous protein (e.g., CAR) is carried out in a culture medium containing 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1% serum (e.g., human serum). In certain embodiments, the step of contacting an activated T cell population with a polynucleotide encoding a heterologous protein (e.g., CAR) is carried out in a culture medium containing 2% serum. In certain embodiments, during the transfection step, the activated T cell population is incubated in a culture medium containing basal culture medium and serum (e.g., 2% serum). In certain embodiments, the culture medium further contains no additional cytokines or growth factors other than serum-derived proteins. In certain embodiments, the culture medium further contains no additional proteins other than serum-derived proteins.

[0126] In certain embodiments, the step of contacting an activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., CAR) is performed in serum-free culture medium. In certain embodiments, the step of contacting an activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., CAR) is performed in basal culture medium that is not supplemented with serum. In certain embodiments, the basal culture medium does not contain any cytokines or growth factors. In certain embodiments, the basal culture medium does not contain any added proteins. It is understood that during cell culture (e.g., during the transfection step, or in a previous activation step if there is no medium exchange between the activation step and the transfection step), cells in the culture medium may secrete proteins. It is also understood that agents that stimulate the CD3 / TCR complex and / or agents that stimulate co-stimulatory molecules may degrade spontaneously over time, releasing fragments into the culture medium. None of these proteins are considered “added proteins”.

[0127] In certain embodiments, the step of contacting an activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., CAR) is carried out in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In certain embodiments, one or more cytokines are IL-2. In certain embodiments, one or more cytokines are IL-7 and IL-15. In certain embodiments, one or more cytokines are IL-2, IL-7, and IL-15. In certain embodiments, one or more cytokines are IL-21. In certain embodiments, one or more cytokines are IL-21, IL-7, and IL-15. In certain embodiments, T cells are exposed to 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL of IL-2, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are exposed to 100 ng / mL of IL-2. In certain embodiments, T cells are exposed to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-7, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are contacted with 10 ng / mL of IL-7. In certain embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-15, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are contacted with 10 ng / mL of IL-15.In certain embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are contacted with 20 ng / mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, T cells are cultured in the absence of any cytokine selected from IL-2, IL-7, IL-15, and IL-21. Either serum-free culture medium (e.g., basal culture medium) or serum-supplemented culture medium may be provided in the presence or absence of the cytokines and combinations disclosed herein during the transfection step.

[0128] In certain embodiments, the step of contacting an activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., CAR) is carried out in a cytokine-free culture medium or without added cytokines. In other embodiments, cytokines are added to the culture medium at the start of a previous step (the activation step). It is understood that at the beginning of the transfection step, the effective concentration of cytokines may decrease as a result of degradation or increase as a result of secretion from cells (e.g., T cells) in the culture. In certain embodiments, additional cytokines are added at the beginning of the transfection step (e.g., in the same type and amount as those added at the beginning of the activation step).

[0129] In certain embodiments, the step of contacting an activated population of T cells with a polynucleotide encoding a heterogeneous protein (e.g., CAR) is performed concurrently with the step of contacting one or more cytokines. In certain embodiments, the step of contacting an activated population of T cells with a polynucleotide encoding a heterogeneous protein (e.g., CAR) is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, or longer, after contact with one or more cytokines. In a particular embodiment, the step of contacting an activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., CAR) is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 hours or longer.

[0130] Duration of culture When T cells are brought into contact with a polynucleotide encoding a heterologous protein (e.g., CAR) of the present disclosure, the cells facilitate the integration of the nucleic acid into the T cell genome for stable expression of the heterologous protein in the T cells, and / or for a desired T cell phenotype (e.g., T NSCMT cells can be cultured in a culture medium under conditions and for a period of time sufficient to realize the cellular phenotype. For example, T cells contacted with a polynucleotide encoding a heterologous protein (e.g., CAR) of the Disclosure may be cultured in a culture medium for 24 to 72 hours (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours). In certain embodiments, T cells contacted with a polynucleotide encoding a heterologous protein of the Disclosure (e.g., CAR) are cultured in a culture medium for approximately 48 hours (e.g., 46, 47, 48, 49, or 50 hours).

[0131] Collection and storage After activation, introduction of heterologous nucleic acids, and culturing, the T cell population can be harvested for storage and subsequent therapeutic use. In certain embodiments, the T cells are harvested at the start of the activation step at 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, In certain embodiments, T cells are harvested for storage after 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, or 36 hours or less. In certain embodiments, T cells are harvested for storage after approximately 64 hours (e.g., 62, 63, 64, 65, or 66) hours or less from the start of the activation step. In certain embodiments, T cells are harvested for storage after 64 hours or less from the start of the activation step. In certain embodiments, T cells are harvested for storage approximately 72 hours (e.g., 70, 71, 72, 73, or 74) hours or less after the start of the activation step.

[0132] In certain embodiments, T cells are harvested for storage 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 hours or less after the completion of the activation step (i.e., the start of the transfection step). In certain embodiments, T cells are harvested for storage approximately 48 (e.g., 46, 47, 48, 49, or 50) hours after the T cells have been in contact with a polynucleotide encoding a heterologous protein (e.g., CAR).

[0133] In certain embodiments, the sampling step is achieved by one or more assays intended to test, among other things, one or more parameters such as cell viability, cell count, purity (e.g., the proportion of T cells in the total cell population), the proportion of cells expressing heterologous proteins, T cell phenotype, T cell activation / exhaustion status, the amount of T cell proliferation compared to the starting population, T cell cytotoxicity, and cytokine release.

[0134] The harvesting step may be followed by a storage step, thereby maintaining the T cells produced according to the disclosed method under conditions suitable for preserving the cells, including their viability, as well as their functional and molecular profiles, until subsequent therapeutic application or quality control testing. In certain embodiments, the storage step includes one or more of the following: (1) reconstitution of the cell population in a storage medium (e.g., refrigerated medium, frozen medium, or cryopreservation medium); (2) transferring the cells to a suitable container for storage under appropriate storage conditions; and (3) maintaining the cells under suitable conditions.

[0135] Properties of manipulated T cells In certain embodiments, engineered T cells that can be generated or obtained according to the methods of this disclosure are disclosed herein. In certain embodiments, T cells are engineered (e.g., genetically engineered) to express a heterologous protein, such as CAR. In certain embodiments, engineered T cells or a population of engineered T cells stably express CAR, for example, by genomic integration of a heterologous nucleic acid sequence encoding CAR in the T cells.

[0136] In certain embodiments, the method disclosed herein generates a population of engineered T cells expressing a heterologous protein (e.g., CAR) from a starting population of T cells, thereby engineering at least 10% (e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more) of the cells in the starting population of T cells to express the heterologous protein. In certain embodiments, the expression of the heterologous protein (e.g., CAR) is measured in T cells at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or later from the harvesting step. In certain embodiments, the expression of the heterologous protein (e.g., CAR) is measured in T cells at 3 days from the harvesting step. In certain embodiments, the expression of heterologous proteins (e.g., CARs) is measured in T cells at least 3, 4, 5, 6, 7, 8, 9, 10 days after the start of the transfection step, or later, and consequently, transient expression from unintegrated vectors is not significantly detected. Methods for quantifying heterologous protein expression at the genomic, transcriptomics, and proteomics levels in cells are well known in the art and include, but are not limited to, flow cytometry (e.g., fluorescence-assisted cell sorting; FACS), quantitative (q)PCR, digital (d)PCR, fluorescence imaging, integration site analysis, RNA sequencing, in situ hybridization, immunoprecipitation, and Topanga assays.

[0137] In certain embodiments, the method disclosed herein involves increasing the amount of IFNγ, granzyme B, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP-10, MCP after contact with target cells expressing a CAR-bound antigen (e.g., CD19), compared to T cells that have not been in contact with the target cells or have been in contact with control target cells that do not express the antigen. 1. Generate a population of engineered T cells that express CAR and secrete one or more proteins selected from the group consisting of MCP4, TNFα, TNFβ, TGFβ, GM-CSF, MIP1α, MIP1β, CCL11, perforin, RANTES, sCD137, and VEGF (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32). In certain embodiments, the secretion of one or more proteins after contact with target cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or higher compared to the secretion of one or more proteins in the absence of target cells or in the presence of control target cells that do not express the antigen. In certain embodiments, cytokine secretion is measured in T cells at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or later from the harvesting step. Methods for quantifying cytokine release from T cells are well known in the art, and include, but are not limited to, ELISA, flow cytometry (e.g., cytometry bead array assay), and proteomics analysis (e.g., multiplicated single-cell chip analysis).

[0138] In certain embodiments, the method disclosed herein generates a population of CAR-expressing engineered T cells that, after contact with target cells expressing a CAR-bound antigen (e.g., CD19), exhibit increased expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137, compared to the expression of one or more T cell activation markers in the absence of target cells or in the presence of control target cells that do not express the antigen. In certain embodiments, the expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 increases by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more, compared to the expression of one or more T cell activation markers in the absence of target cells or in the presence of control target cells that do not express the antigen. In certain embodiments, the expression of one or more T cell activation markers is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days after the collection step, or later. Methods for quantifying the expression of T cell activation markers are well known in the art, and include, but are not limited to, ELISA, flow cytometry, quantitative (q)PCR, digital (d)PCR, fluorescence imaging, in situ hybridization, and proteomics analysis.

[0139] In certain embodiments, the method disclosed herein generates a population of CAR-expressing engineered T cells that exhibit increased cytotoxicity against target cells expressing a CAR-bound antigen (e.g., CD19) compared to cytotoxicity against cells that do not express the antigen. In certain embodiments, the cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more compared to cytotoxicity against cells that do not express the antigen. In certain embodiments, the cytotoxicity of the engineered T cells against target cells expressing the antigen (e.g., CD19) is measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or later from the harvesting step. Methods for evaluating the cytotoxicity of engineered T cells expressing heterologous proteins (e.g., CARs) against target cells are well known in the art and include, but are not limited to, chromium emission assays, bioluminescence assays (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analysis, flow cytometry (e.g., in combination with viability dyes, e.g., CTV), and CFSE / PI assays.

[0140] In certain embodiments, the method disclosed herein generates a population of CAR-expressing engineered T cells that exhibit increased proliferation after contact with target cells expressing a CAR-bound antigen (e.g., CD19) compared to proliferation in the absence of target cells or in the presence of control target cells that do not express the antigen. In certain embodiments, proliferation increases by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more, compared to proliferation in the absence of target cells or in the presence of control target cells that do not express the antigen. In certain embodiments, T cell proliferation after contact with target cells expressing the antigen (e.g., CD19) is measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or later, after the harvesting step. Methods for evaluating the proliferation of engineered T cells expressing heterologous proteins (e.g., CARs) are well known in the art and include, but are not limited to, MTT assays, MTS assays, cell counting (e.g., via flow cytometry), CFSE / flow cytometry analysis, and [3H]thymidine incorporation.

[0141] In certain embodiments, the methods disclosed herein involve manipulating T cells of various phenotypes, such as naive T cells characterized as CD45RO-, CCR7+, and CD95-. N )Central memory T(T) cells characterized as CD45RO+ and CCR7+ CM Effector memory T(T) cells characterized as CD45RO+ and CCR7- EM Stem memory T(T) cells characterized as CD45RO-, CCR7+, and CD95+ SCM ) cells, as well as CD45RA T(T) cells characterized as CD45RO- and CCR7- EMRA This generates a population of effector memory cells that reexpress ) cells. Alternative characterizations of these T cell subsets include, but are not limited to, naive T(T) cells characterized as CD45RA+, CCR7+, and CD95-. N)Central memory T(T) cells characterized as CD45RA- and CCR7+ CM Effector memory T(T) cells characterized as CD45RA- and CCR7- EM Stem memory T(T) cells characterized as CD45RA+, CCR7+, and CD95+ SCM ) cells, as well as CD45RA T(T) cells characterized as CD45RA+ and CCR7- EMRA Effector memory cells, which reexpress cells, are an example of this.

[0142] In certain embodiments, the method disclosed herein increases the amount of naive and stem cell memory T(T) cells compared to the starting population of T cells. NSCM This method generates a population of engineered T cells expressing heterologous proteins (e.g., CAR), including cells identified by markers such as CD45RA+ / CD45RO- / CCR7+ / CD62L+; CD45RA+ / CCR7+; or CD45RO- / CCR7+. In certain embodiments, the T cells generated according to the disclosed method are T cells from the starting population of T cells. NSCM Compared to the number of cells, the amount of T is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 2 times, 2.5 times, 3 times, 3.5 times, 4 times higher, or higher. NSCM Includes cells. In a particular embodiment, T cells generated according to the disclosed method are T cells from a starting population of T cells. SCM Compared to the number of cells, the amount of stem cell memory T(T) is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 2 times, 2.5 times, 3 times, 3.5 times, 4 times higher, or higher. SCMcomprising cells (identified by markers such as CD45RA+ / CD45RO- / CCR7+ / CD62L+ / CD95+; CD45RA+ / CCR7+ / CD95+; or CD45RO- / CCR7+ / CD95+). In certain embodiments, the T cells generated according to the disclosed methods comprise at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the T NSCM cells among all the T cells collected. In certain embodiments, the T cells generated according to the disclosed methods comprise at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the T SCM cells. In certain embodiments, the amount of T <( NSCM cells in the engineered population of T cells is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or later, after the step of collection. Methods for quantifying T NSCM cells are well known in the art and include, among others, without limitation, flow cytometry (e.g., FACS) and fluorescence microscopy.

[0143] In certain embodiments, the methods disclosed herein generate a population of engineered T cells that express a heterologous protein (e.g., a CAR) and comprise a reduced amount of effector memory (T EM ) T cells (identified by markers such as D45RA- / CD45RO+ / CCR7- / CD62L-; D45RA- / CCR7-; or CD45RO+ / CCR7-) compared to the starting population of T cells. In certain embodiments, the T cells generated according to the disclosed methods comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or less of the amount of T [[ID=!5]] EM cells in the starting population of T cells. In certain embodiments, the amount of T EM cells in the engineered population of T cells EMThe cell count is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days after the harvesting step, or later. EM Methods for quantifying cells are well known in the art, and include, but are not limited to, flow cytometry (e.g., FACS) and fluorescence microscopy.

[0144] Chimeric antigen receptor In certain embodiments, T cells that have been engineered (e.g., genetically modified) to express a heterologous protein are disclosed herein. In certain embodiments, the heterologous protein is a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises (1) an extracellular domain containing an antigen-binding site that specifically binds to a target antigen; (2) a transmembrane domain; (3) an intracellular signaling domain; and optionally, (4) a costimulatory domain. In certain embodiments, the CAR disclosed herein further comprises a hinge region.

[0145] In certain embodiments, the CAR is a human CAR, containing a complete human sequence, e.g., a naturally occurring human sequence. In certain embodiments, the extracellular domain is ligated to one or more intracellular signaling domains that can mediate cell activation through the antigen receptor complex. In certain embodiments, the transmembrane domain is ligated to the extracellular domain. In certain embodiments, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In certain embodiments, the transmembrane domain is selected or modified by amino acid substitution to minimize interaction with other members of the receptor complex by avoiding binding of such domain to transmembrane domains of the same or different surface membrane proteins.

[0146] The CARs disclosed herein may contain any number of amino acids, provided that the CAR retains biological activity, such as the ability to specifically bind to an antigen, the ability to mediate cytotoxic activity, the ability to detect affected cells in mammals, or the ability to treat or prevent a disease in mammals. In certain embodiments, the CAR contains 50 or more (e.g., 60 or more, 100 or more, or 500 or more) amino acids, but less than 1,000 (e.g., 900 or less, 800 or less, 700 or less, or 600 or less) amino acids. In certain embodiments, the CAR is in the range of about 50 to about 700 amino acids (e.g., about 300 to about 1,000 amino acids (e.g., about 300 to about 800, about 300 to about 600, or about 400 to about 600 amino acids)), or any two of the aforementioned values.

[0147] In certain embodiments, the CAR contains additional amino acids at the amino or carboxyl terminus of a portion, or at both ends, which are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the CAR, such as recognizing target cells, mediating cytotoxic activity, or treating or preventing disease or injury. More preferably, the additional amino acids enhance the biological activity of the CAR compared to the biological activity of the parent CAR.

[0148] Extracellular domain In certain embodiments, the CAR disclosed herein comprises an extracellular antigen-binding domain containing an antibody or an antigen-binding fragment thereof. Anticarin or other alternative scaffolds are also intended. The antigen-binding domain of the CAR may be a whole antibody or an antibody fragment (e.g., scFv). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have a similar general structure, and each region contains three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody, which is responsible for antigen recognition and binding. The three CDR regions are connected to four framework regions, and their sequences are relatively conserved.

[0149] Antigen-binding fragments of an antibody retain the antibody's ability to specifically bind to its antigen. Antibody fragments preferably include, for example, one or more CDRs or variable regions (or portions thereof). Examples of antibody fragments, but not limited to, include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fab' fragments, which are monovalent fragments consisting of VL, VH, CL, CH1 domains, and disulfide-bridged thiols; (iii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks in a hinge region; (iv) Fv fragments, which consist of the VL and VH domains of a single arm of the antibody; (v) single-chain Fv(scFv), which are monovalent molecules consisting of two domains (i.e., VL and VH) of an Fv fragment joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain; and (vi) single-domain antibodies containing a single monomeric variable antibody domain, e.g., V H H or V NAR; and (vii) a diabody which is a dimer of polypeptide chains, where each polypeptide chain contains a VH connected to the VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to create a dimeric molecule having two functional antigen-binding sites. In certain embodiments, the antigen-binding domain of the CAR contains an scFv which binds to a target antigen.

[0150] In certain embodiments, the antibody or its antigen-binding fragment in the extracellular domain of the CAR may be obtained from or derived from a mammal, including, but not limited to, mouse, rat, or human. In certain embodiments, the antigen-binding domain includes a variable region of a mouse or human monoclonal antibody or its antigen-binding fragment that binds to an antigen. In this regard, the antigen-binding domain includes a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of a mouse or human monoclonal antibody or its antigen-binding fragment that binds to an antigen.

[0151] In certain embodiments, the extracellular domain of the CAR disclosed herein includes a signal sequence. The signal sequence may be located at the amino terminus of the antigen recognition domain (e.g., the variable region of an antibody or its antigen-binding fragment). The signal sequence may include any preferred signal sequence. In one embodiment, the signal sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence or a CD8α signal sequence. For example, a CAR containing mouse scFv may include a GM-CSF signal sequence, while a CAR containing human scFv may include a CD8α signal sequence. The N-terminal signal sequence is typically cleaved from the CAR protein after expression, but it is understood that the nucleic acid encoding the CAR generally includes a sequence encoding the signal sequence.

[0152] In some embodiments, the antigen-binding domain binds to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain specifically binds to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain binds to a CD19 polypeptide (e.g., the CD19 polypeptide is present on the surface of cells, e.g., B cells). In some embodiments, the antigen-binding domain specifically binds to a CD19 polypeptide. In some embodiments, the antigen-binding domain includes an antibody that binds to a CD19 polypeptide, or an antigen-binding fragment thereof. In some embodiments, the antigen-binding domain includes an scFv sequence that binds to a CD19 polypeptide (e.g., anti-CD19 scFv).

[0153] Generally, CD19 expression is understood to be primarily limited to B cell lymphocytes. CD19 has two N-terminal extracellular Ig-like domains separated by a non-Ig-like domain, a hydrophobic transmembrane domain, and a large C-terminal cytoplasmic domain. The CD19 protein forms a complex with several membrane proteins, including complement receptor 2 (CD21) and tetraspanin (CD81), and this complex lowers the threshold for antigen-initiated B cell activation. Activation of this B cell antigen-receptor complex activates the phosphatidylinositol 3-kinase signaling pathway and the subsequent release of intracellular calcium ions. Examples of human CD19 polypeptide sequences, but not limited to, the NCBI reference sequence: NP_001171569.1, as well as its fragments and derivatives.

[0154] In some embodiments, the antigen-binding domain includes the variable region of an anti-CD19 antibody. In some embodiments, the antigen-binding domain includes the variable region of an anti-CD19 monoclonal antibody. In some embodiments, the antigen-binding domain includes the variable region of a mouse or human anti-CD19 monoclonal antibody. The anti-CD19 monoclonal antibody may be obtained from or derived from a subject (e.g., mouse, rat, rabbit, human, etc.) using any preferred method. In some embodiments, the antigen-binding domain includes the light chain variable region and the heavy chain variable region of a mouse, human, or humanized anti-CD19 monoclonal antibody. In some embodiments, the antigen-binding domain includes the light chain variable region of a mouse, human, or humanized anti-CD19 monoclonal antibody. In some embodiments, the antigen-binding domain includes the heavy chain variable region of a mouse, human, or humanized anti-CD19 monoclonal antibody. The 47G4 antibody (described in U.S. Patent Application Publication No. 2010 / 0104509, which is incorporated herein by whole reference) is an example of a human anti-CD19 monoclonal antibody that can be used in accordance with this disclosure. An exemplary method for generating a fully human antibody is described in Lu et al., (2020) J. Biomed. Sci. (2020) 27(1):1.

[0155] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain includes the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable domain includes the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0156] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain includes the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 3, respectively, and the light chain variable domain includes the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0157] In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain, which contains CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0158] In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain, which contains CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with respect to SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 3, respectively.

[0159] In some embodiments, the antigen-binding domain that binds to CD19 includes a light chain variable domain, which contains CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0160] In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 7, and the light chain variable domain includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7, and the light chain variable domain includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8.

[0161] In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain, which contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 7. In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain, which contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain, which contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain, which contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7.

[0162] In some embodiments, the antigen-binding domain that binds to CD19 includes a light chain variable domain, which contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds to CD19 includes a light chain variable domain, which contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds to CD19 includes a light chain variable domain, which contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds to CD19 includes a light chain variable domain, which contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8.

[0163] In some embodiments, the antigen-binding domain that binds to CD19 includes a spacer sequence between two domains or components. In some embodiments, the antigen-binding domain includes a spacer sequence between the heavy chain variable domain and the light chain variable domain. In some embodiments, the spacer includes the sequence shown in Sequence ID No. 9.

[0164] In some embodiments, the antigen-binding domain that binds to CD19 includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 17. In some embodiments, the antigen-binding domain that binds to CD19 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 17. In some embodiments, the antigen-binding domain that binds to CD19 includes the amino acid sequence shown in SEQ ID NO: 17.

[0165] In some embodiments, the antigen-binding domain that binds to CD19 is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 18. In some embodiments, the antigen-binding domain that binds to CD19 is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, the antigen-binding domain is encoded by the nucleic acid sequence shown in SEQ ID NO: 18.

[0166] Other antigen-binding domains that bind to CD19 may also be included in the CARs disclosed herein. Exemplary antigen-binding domains are described in International Patent Publication WO2017062952 and U.S. Patent Publication US20220220200.

[0167] In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domains each containing the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs. 35, 36, and 37, respectively, and the light chain variable domains each containing the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs. 38, 39, and 40, respectively. In some embodiments, the heavy chain variable domains each contain an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NOs. 29, and the light chain variable domains each contain an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NOs. 30.

[0168] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprising the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs. 41, 42, and 43, respectively, and the light-chain variable domain comprising the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs. 44, 45, and 46, respectively. In some embodiments, the heavy-chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NOs. 31, and the light-chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NOs. 32.

[0169] In some embodiments, the antigen-binding domain that binds to CD19 includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domains each containing the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs. 47, 48, and 49, respectively, and the light chain variable domains each containing the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs. 50, 51, and 52, respectively. In some embodiments, the heavy chain variable domains each contain an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NOs. 33, and the light chain variable domains each contain an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NOs. 34.

[0170] Hinged Domain In certain embodiments, the CAR further includes a hinge or spacer between the antigen-binding domain and the transmembrane domain. The hinge or spacer may be, or include, at least a portion of the immunoglobulin constant region or a variant or modified version thereof, e.g., a hinge region, e.g., a CD8a hinge, an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In certain embodiments, the constant region or portion is of human IgG, e.g., IgG4 or IgG1. In certain embodiments, a portion of the constant region acts as a spacer region between the antigen-recognition component, e.g., scFv, and the transmembrane domain. The spacer may be of a length that provides increased cellular responsiveness after antigen binding compared to the absence of the spacer. In certain embodiments, the spacer is 12 amino acids or about 12 amino acids long, or 12 amino acids or less in length. Examples of spacers include those containing any integer between any of the endpoints of the listed ranges, having at least about 10–229 amino acids, about 10–200 amino acids, about 10–175 amino acids, about 10–150 amino acids, about 10–125 amino acids, about 10–100 amino acids, about 10–75 amino acids, about 10–50 amino acids, about 10–40 amino acids, about 10–30 amino acids, about 10–20 amino acids, or about 10–15 amino acids. In certain embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids. Examples of spacers include the CD8α hinge, the IgG4 hinge alone, the IgG4 hinge linked to the CH2 and CH3 domains, or the IgG4 hinge linked to the CH3 domain. In certain embodiments, the CAR hinge includes CD8α, truncated CD8α, or CD28 hinge domains.

[0171] In some embodiments, the hinge region is a short sequence of amino acids that can facilitate structural flexibility between polypeptide domains, for example, between the extracellular domain and the transmembrane domain (see, e.g., Woof et al., Nat. Rev. Immunol. 4(2):89-99 (2004)). In some embodiments, the hinge region may comprise all or part of the extracellular domain of any suitable transmembrane protein (e.g., CD8α).

[0172] In some embodiments, the hinge region is derived from the CD8α protein or the CD28 protein. In some embodiments, the hinge region is derived from the CD8α protein. In some embodiments, the hinge region is derived from the CD28 protein. In some embodiments, the hinge region is a hinge region derived from the CD28 protein or a functional fragment thereof, or includes it. In some embodiments, the hinge region is a hinge region derived from the CD8α protein or a functional fragment thereof, or includes it. In some embodiments, the hinge region is derived from the human CD8α protein or the human CD28 protein. In some embodiments, the hinge region is derived from the human CD8α protein. In some embodiments, the hinge region is derived from the human CD28 protein. In some embodiments, the hinge region is a hinge region derived from the human CD28 protein or a functional fragment thereof, or includes it. In some embodiments, the hinge region is a hinge region derived from the human CD8α protein or a functional fragment thereof, or includes it.

[0173] In some embodiments, the hinge region includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with respect to SEQ ID NO: 28. In some embodiments, the hinge region includes the amino acid sequence shown in SEQ ID NO: 28.

[0174] In some embodiments, the hinge region is derived from the same polypeptide as the transmembrane domain. In some embodiments, the hinge region and transmembrane domain are derived from the CD8 polypeptide. In some embodiments, the hinge region and transmembrane domain are derived from the CD8α polypeptide. In some embodiments, the hinge region and transmembrane domain contain an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 19. In some embodiments, the hinge region and transmembrane domain contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, the hinge region and transmembrane domain contain the amino acid sequence shown in SEQ ID NO: 19.

[0175] In some embodiments, the hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 20. In some embodiments, the hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, the hinge region and transmembrane domain are encoded by the nucleic acid sequence shown in SEQ ID NO: 20.

[0176] transmembrane domain In certain embodiments, modified T cells expressing a CAR comprising a transmembrane domain operably connected to the extracellular domain and intracellular signaling domain of the CAR are disclosed herein. The transmembrane domain may be derived from or obtained from any molecule known in the art (e.g., a type I transmembrane protein).

[0177] In some embodiments, the transmembrane domain of the CAR is derived from a natural source (e.g., a natural or wild-type polypeptide). In some embodiments, the transmembrane domain is derived from any suitable transmembrane protein or polypeptide known in the Art, when used in accordance with the Disclosure. In some embodiments, the transmembrane domain is derived from CD3 epsilon polypeptide, CD4 polypeptide, CD5 polypeptide, CD8 polypeptide, CD9 polypeptide, CD16 polypeptide, CD22 polypeptide, CD28 polypeptide, CD33 polypeptide, CD37 polypeptide, CD45 polypeptide, CD64 polypeptide, CD80 polypeptide, CD86 polypeptide, CD134 polypeptide, CD137 polypeptide, CD154 polypeptide, T cell receptor alpha chain polypeptide, T cell receptor beta chain polypeptide, T cell receptor zeta chain polypeptide, or any derivative thereof and / or combination thereof. In some embodiments, a transmembrane is a transmembrane domain or a functional fragment thereof derived from or including the CD3 epsilon polypeptide, CD4 polypeptide, CD5 polypeptide, CD8 polypeptide, CD9 polypeptide, CD16 polypeptide, CD22 polypeptide, CD28 polypeptide, CD33 polypeptide, CD37 polypeptide, CD45 polypeptide, CD64 polypeptide, CD80 polypeptide, CD86 polypeptide, CD134 polypeptide, CD137 polypeptide, CD154 polypeptide, T cell receptor alpha chain polypeptide, T cell receptor beta chain polypeptide, T cell receptor zeta chain polypeptide, or any combination thereof. In some embodiments, a transmembrane is synthetically induced or manipulated. In some embodiments, the synthetically induced or manipulated transmembrane domain mainly comprises hydrophobic residues (e.g., leucine, valine, etc.). In some embodiments, the manipulated transmembrane domain is any manipulated transmembrane domain known in the art, or includes such a domain.

[0178] Alternatively, in certain embodiments, the transmembrane domain is synthetic. In some embodiments, the synthetic transmembrane domain mainly consists of hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. In certain embodiments, linkage is by linkers, spacers, and / or transmembrane domains.

[0179] This disclosure recognizes that CD8 is a transmembrane glycoprotein that functions as a co-receptor for T cell receptors (TCRs) and is primarily expressed on the surface of T cells, such as cytotoxic T cells. The most common form of CD8 exists as a dimer composed of CD8α and CD8β chains. In some embodiments, the transmembrane domain is derived from the CD8α protein. In some embodiments, the transmembrane protein contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 11. In some embodiments, the transmembrane protein contains the amino acid sequence shown in SEQ ID NO: 11.

[0180] This disclosure further recognizes that CD28 is expressed on T cells and provides the costimulatory signals required for T cell activation. CD28 is a receptor for CD80(B7.1) and CD86(B7.2). In some embodiments, the CAR of this disclosure comprises a CD28 transmembrane domain. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane protein comprises the amino acid sequence shown in SEQ ID NO: 12.

[0181] Intracellular signal transduction domains In certain embodiments, upon binding of a CAR to a target antigen, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of an immune effector cell, e.g., a T cell engineered to express the receptor. For example, in some contexts, the receptor induces T cell function, e.g., cytolytic activity or T-helper activity, e.g., secretion of cytokines or other factors. In certain embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used in place of the complete immunostimulatory chain, e.g., if it transmits an effector function signal. In certain embodiments, one or more intracellular signaling domains include the cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments, also include the cytoplasmic sequence of a co-receptor that, under natural circumstances, acts in coordination with such receptor after antigen receptor binding to initiate signal transduction, and / or any derivatives or variants of such molecules, and / or any synthetic sequence having the same functional capacity.

[0182] In some embodiments, the receptor includes a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex. The primary cytoplasmic signaling sequence acting in a stimulating manner may contain a signaling motif known as an immunoreceptor-activating tyrosine motif (ITAM). Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, FcεRI (e.g., FcεRI gamma chain polypeptide), FcγRI, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b, LIGHT, NKG2C, OX40, PD-1, CD66d, or any derivative or combination thereof.

[0183] In certain embodiments, the receptor includes intracellular components of the TCR complex. It is understood that the most common intracellular signaling domain used in CAR therapy is the intracellular signaling domain of CD3 zeta (CD3ζ). CD3 zeta associates with the T cell receptor to produce a signal and contains ITAM. In certain embodiments, the intracellular signaling molecule in CAR includes an intracellular signaling domain, a portion thereof, or a sequence derived from CD3 zeta. In certain embodiments, the intracellular signaling domain includes the CD3ζ domain. In certain embodiments, the intracellular signaling domain includes the human CD3 zeta-stimulated signaling domain or a functional fragment thereof, for example, the 112 AA cytoplasmic domain of isoform 3 of human CD3 zeta (UniProt accession number: P20963.2).

[0184] In some embodiments, the intracellular signaling domain includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 23. In some embodiments, the intracellular signaling domain includes the amino acid sequence shown in SEQ ID NO: 23.

[0185] In some embodiments, the intracellular signaling domain is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 24. In some embodiments, the intracellular signaling domain is encoded by the nucleic acid sequence shown in SEQ ID NO: 24.

[0186] In some embodiments, the intracellular signaling domain includes at least one intracellular signaling domain or a functional fragment thereof derived from 4-1BB polypeptide, B7-H3 polypeptide, CD2 polypeptide, CD3 gamma polypeptide, CD3 delta polypeptide, CD3 zeta polypeptide, CD7 polypeptide, CD27 polypeptide, CD28 polypeptide, CD30 polypeptide, CD40 polypeptide, FcεRI polypeptide (e.g., FcεRI gamma chain polypeptide), FcγRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, OX40 polypeptide, PD-1 polypeptide, or any derivative thereof or any combination thereof. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain or a functional fragment thereof derived from CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain or a functional fragment thereof derived from CD28 polypeptide. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain or a functional fragment thereof derived from CD28 polypeptide, and an intracellular signaling domain or a functional fragment thereof derived from CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain includes, from the N-terminus to the C-terminus, an intracellular signaling domain or functional fragment derived from a CD28 polypeptide, and an intracellular signaling domain or functional fragment derived from a CD3 zeta polypeptide.

[0187] In some embodiments, the intracellular signaling domain of the Disclosure comprises at least one signaling sequence derived from 4-1BB polypeptide, B7-H3 polypeptide, CD2 polypeptide, CD3 gamma polypeptide, CD3 delta polypeptide, CD3 zeta polypeptide, CD7 polypeptide, CD27 polypeptide, CD28 polypeptide, CD30 polypeptide, CD40 polypeptide, FcεRI polypeptide (e.g., FcεRI gamma chain polypeptide), FcγRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, OX40 polypeptide, PD-1 polypeptide, or any combination thereof. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence derived from CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence derived from CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence derived from CD28 polypeptide and at least one signaling sequence derived from CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence derived from CD28 polypeptide and at least one signaling sequence derived from CD3 zeta polypeptide from the N-terminus to the C-terminus.

[0188] Co-stimulatory domain In certain embodiments, the CAR of the Disclosure includes, for example, an intracellular domain of a T cell costimulatory molecule located between a transmembrane domain and an intracellular signaling domain. In certain embodiments, the intracellular domain includes an intracellular costimulatory signaling domain of CD28 or 4-1BB, or a functional variant or portion thereof, for example, the 41-amino acid cytoplasmic domain of human CD28 (UniProt accession number P10747.1) or the 42-amino acid cytoplasmic domain of human 4-1BB (UniProt accession number Q07011.1), or a functional variant or portion thereof.

[0189] In certain embodiments, the receptor, in its cytoplasmic portion, includes one or more, for example, two or more, costimulatory domains in addition to an activating domain, such as a primary activating domain. Exemplary receptors include intracellular components of CD3-zeta and CD28, or intracellular components of CD3-zeta and 4-1BB.

[0190] In certain embodiments, the receptor includes the signaling domain and / or transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some embodiments, the same receptor includes both the activating and co-stimulatory components.

[0191] In certain embodiments, the intracellular signaling domain comprises a CD8a transmembrane domain and a signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain, and further comprises a CD28 or 4-1BB (CD137, TNFRSF9) costimulatory domain linked to the CD3-zeta intracellular domain.

[0192] In some embodiments, the intracellular signaling domain includes a CD28 intracellular signaling domain. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain or a functional fragment thereof derived from a CD28 polypeptide. In some embodiments, the CD28 polypeptide intracellular signaling domain or a functional fragment thereof includes a co-stimulatory domain.

[0193] In some embodiments, the intracellular signaling domain disclosed herein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 21. In some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 21.

[0194] In some embodiments, the intracellular signaling domain is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 22. In some embodiments, the intracellular signaling domain is encoded by the nucleic acid sequence shown in SEQ ID NO: 22.

[0195] Chimeric antigen receptor - multiple domains In some embodiments, the CAR of the Disclosure comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR of the Disclosure comprises a signal peptide sequence (also referred to as a targeting signal, localization signal, localization sequence, leader sequence, or leader peptide), an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR of the Disclosure comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus. In some embodiments, the CAR of the Disclosure comprises a signal peptide sequence, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus. In some embodiments, the signal peptide sequence is cleaved from the CAR during the synthesis of the CAR protein, during or after its insertion into a membrane (e.g., the ER membrane). In some embodiments, the domains or components of the CAR (e.g., the extracellular domain, hinge region, transmembrane domain, intracellular signaling domain, etc.) are directly ligated or adjacent. In some embodiments, the domains or components of the CAR are not directly connected or are not in close proximity.

[0196] In some embodiments, the CAR described herein includes an intracellular signaling domain, the intracellular signaling domain comprising (a) a CD3 zeta intracellular signaling domain or a functional fragment thereof; and (b) at least one of the 4-1BB, OX40, or CD28 intracellular signaling domain or a functional fragment thereof. In some embodiments, the 4-1BB intracellular signaling domain or a functional fragment thereof, the OX40 intracellular signaling domain, and / or the CD28 intracellular signaling domain or a functional fragment thereof are or include a co-stimulatory domain.

[0197] In some embodiments, the CAR of the Disclosure comprises (a) a CD28 transmembrane domain; and (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an intracellular signaling domain comprising a CD28 intracellular signaling domain or a functional fragment thereof. In some embodiments, the CD28 intracellular signaling domain or a functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0198] In some embodiments, the CAR of the Disclosure comprises (a) a CD8α transmembrane domain; (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an intracellular signaling domain comprising a CD28, FcεRI gamma chain, and / or a 4-1BB intracellular signaling domain or a functional fragment thereof. In some embodiments, the CAR of the Disclosure comprises (a) a CD8α transmembrane domain; (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an intracellular signaling domain comprising a CD28, FcεRI gamma chain, and a 4-1BB intracellular signaling domain or a functional fragment thereof. In some embodiments, the CAR of the Disclosure comprises (a) a CD8α transmembrane domain; (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an intracellular signaling domain comprising an FcεRI gamma chain intracellular signaling domain or a functional fragment thereof. In some embodiments, the CAR of the Disclosure comprises an intracellular signaling domain including (a) a CD8α transmembrane domain; (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) a 4-1BB intracellular signaling domain or a functional fragment thereof. In some embodiments, the CD28 intracellular signaling domain or a functional fragment thereof is or includes a CD28 costimulatory domain. In some embodiments, the FcεRI intracellular signaling domain or a functional fragment thereof is or includes an FcεRI costimulatory domain. In some embodiments, the 4-1BB intracellular signaling domain or a functional fragment thereof is or includes a 4-1BB costimulatory domain.

[0199] In some embodiments, the CAR of the Disclosure comprises (a) a CD8α transmembrane domain, and (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof, and (ii) an intracellular signaling domain comprising a CD27 and / or CD28 intracellular signaling domain or a functional fragment thereof. In some embodiments, the CD27 intracellular signaling domain or a functional fragment thereof is or comprises a CD27 co-stimulatory domain. In some embodiments, the CD28 intracellular signaling domain or a functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0200] In some embodiments, the CAR of the Disclosure comprises (a) a CD28 transmembrane domain, and (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an intracellular signaling domain comprising a CD27, 4-1BB, and / or FcεRI gamma chain intracellular signaling domain or a functional fragment thereof. In some embodiments, the CAR of the Disclosure comprises (a) a CD28 transmembrane domain, and (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an intracellular signaling domain comprising a CD27, 4-1BB, and FcεRI gamma chain intracellular signaling domain or a functional fragment thereof. In some embodiments, the CAR of the Disclosure comprises (a) a CD28 transmembrane domain, and (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an intracellular signaling domain comprising a CD27 intracellular signaling domain or a functional fragment thereof. In some embodiments, the CAR of the Disclosure comprises an intracellular signaling domain including (a) a CD28 transmembrane domain, and (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) a 4-1BB intracellular signaling domain or a functional fragment thereof. In some embodiments, the CAR of the Disclosure comprises an intracellular signaling domain including (a) a CD28 transmembrane domain, and (b)(i) a CD3ζ intracellular signaling domain or a functional fragment thereof; and (ii) an FcεRI gamma chain intracellular signaling domain or a functional fragment thereof. In some embodiments, the CD27 intracellular signaling domain or a functional fragment thereof is or includes a CD27 costimulatory domain. In some embodiments, the FcεRI intracellular signaling domain or a functional fragment thereof is or includes an FcεRI costimulatory domain. In some embodiments, the 4-1BB intracellular signaling domain or a functional fragment thereof is or includes a 4-1BB costimulatory domain.

[0201] This disclosure further provides CARs comprising an extracellular domain for any target molecule of interest (e.g., any known antigen-binding domain, e.g., an antibody, scFv, etc.), and further comprising any transmembrane domains described herein (including any hinge domains described herein), and any intracellular signaling domains described herein (including any signal sequence or motif, any costimulatory domains, etc.), which are present in any combination.

[0202] In some embodiments, the CAR comprises an intracellular signaling domain including (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c)(i) a human CD3ζ intracellular signaling domain or a fragment thereof, and (ii) a human CD28 intracellular signaling domain or a fragment thereof, where the CD28 intracellular signaling domain or a fragment thereof is or comprises a co-stimulatory domain. In some embodiments, the CAR comprises an intracellular signaling domain including (a) a hinge region derived from a human CD8α polypeptide, (b) a transmembrane domain derived from a human CD8α polypeptide, (c)(i) a human CD3ζ intracellular signaling domain, and (ii) a human CD28 intracellular signaling domain. In some embodiments, the CAR comprises the sequence shown in Sequence ID No. 27.

[0203] In some embodiments, the CAR comprises (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c)(i) a human CD3ζ intracellular signaling domain or a fragment thereof, and (ii) an intracellular signaling domain comprising a CD27 and / or CD28 intracellular signaling domain or a fragment thereof, wherein the CD27 and / or CD28 intracellular signaling domain or a fragment thereof is or comprises a co-stimulatory domain.

[0204] In some embodiments, the CAR comprises (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c)(i) a human CD3ζ intracellular signaling domain or a fragment thereof, and (ii) an intracellular signaling domain comprising a human CD28, human CD27, and / or FcεRI gamma chain intracellular signaling domain or a fragment thereof, wherein the human CD28, human CD27, and / or FcεRI gamma chain intracellular signaling domain or a fragment thereof is or comprises a co-stimulatory domain.

[0205] In some embodiments, the CAR may include (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c)(i) a human CD3ζ intracellular signaling domain, and (ii) an intracellular signaling domain including a human CD28 and / or FcεRI gamma chain intracellular signaling domain, where the CD28 and / or FcεRI gamma chain intracellular signaling domain, or a fragment thereof, is or includes a co-stimulatory domain.

[0206] In some embodiments, the CAR described herein further comprises a signal peptide sequence. In some embodiments, the signal peptide is located at the amino terminus of the extracellular domain (e.g., the N terminus of the antigen-binding domain). The signal peptide used in accordance with this disclosure may comprise any preferred signal peptide sequence. In some embodiments, the signal peptide sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal peptide sequence or a CD8α signal peptide sequence. In some embodiments, the CAR provided herein comprises a human or humanized scFv containing a CD8α signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO: 15.

[0207] In some embodiments, the provided CAR comprises (a) a CD8α hinge region containing SEQ ID NO: 28, (b) a CD8α transmembrane domain containing SEQ ID NO: 11, (c) a CD28 intracellular signaling domain containing SEQ ID NO: 21, and (d) a CD3ζ intracellular signaling domain containing SEQ ID NO: 23. In some embodiments, the provided CAR comprises, from N-terminus to C-terminus, (a) a CD8α hinge region containing SEQ ID NO: 28, (b) a CD8α transmembrane domain containing SEQ ID NO: 11, (c) a CD28 intracellular signaling domain containing SEQ ID NO: 21, and (d) a CD3ζ intracellular signaling domain containing SEQ ID NO: 23.

[0208] In some embodiments, the provided CAR comprises (a) an antigen-binding domain including SEQ ID NO: 17, (b) a CD8α hinge region including SEQ ID NO: 28, (c) a CD8α transmembrane domain including SEQ ID NO: 11, (d) a CD28 intracellular signaling domain including SEQ ID NO: 21, and (e) a CD3ζ intracellular signaling domain including SEQ ID NO: 23. In some embodiments, the provided CAR comprises, from N-terminus to C-terminus, (a) an antigen-binding domain including SEQ ID NO: 17, (b) a CD8α hinge region including SEQ ID NO: 28, (c) a CD8α transmembrane domain including SEQ ID NO: 11, (d) a CD28 intracellular signaling domain including SEQ ID NO: 21, and (e) a CD3ζ intracellular signaling domain including SEQ ID NO: 23.

[0209] In some embodiments, the provided CAR comprises (a) a CD8α signal peptide sequence including SEQ ID NO: 15, (b) an antigen-binding domain including SEQ ID NO: 17, (c) a CD8α hinge region shown in SEQ ID NO: 28, (d) a CD8α transmembrane domain shown in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain shown in SEQ ID NO: 21, and (f) a CD3ζ intracellular signaling domain shown in SEQ ID NO: 23. In some embodiments, the provided CAR comprises, from the N-terminus to the C-terminus, (a) a CD8α signal peptide sequence including SEQ ID NO: 15, (b) an antigen-binding domain including SEQ ID NO: 17, (c) a CD8α hinge region shown in SEQ ID NO: 28, (d) a CD8α transmembrane domain shown in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain shown in SEQ ID NO: 21, and (f) a CD3ζ intracellular signaling domain shown in SEQ ID NO: 23.

[0210] In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the CAR of the Disclosure includes the amino acid sequence shown in SEQ ID NO: 10.

[0211] In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR of the Disclosure includes the amino acid sequence shown in SEQ ID NO: 13.

[0212] In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the CAR of the Disclosure is encoded by the nucleic acid sequence shown in SEQ ID NO: 14.

[0213] Modification The CAR may contain one or modified synthetic amino acids in place of one or more naturally occurring amino acids. Exemplary modified amino acids include, but are not limited to, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, (3-phenylserine, (3-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbomane)-carboxylic acid, α,γ-diaminobutyric acid, α,γ-diaminopropionic acid, homophenylalanine, and α-tert butylglycine.

[0214] The CAR (including its functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, disulfide bridging, or converted to an acid addition salt, and / or dimerized or multimerized, or conjugated as necessary.

[0215] Pharmaceutical composition The engineered T cells disclosed herein can be incorporated into pharmaceutical compositions. These compositions can include, in addition to the engineered T cells disclosed herein, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those of skill in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material can depend on the intended route of administration, e.g., intravenous, cutaneous, or subcutaneous, nasal, intramuscular, intraperitoneal routes. The pharmaceutical compositions may include one or more pharmaceutical excipients. Any suitable pharmaceutical excipient can be used and those of skill in the art can select a suitable pharmaceutical excipient. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative and not limiting. Additional pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), which is hereby incorporated by reference in its entirety.

[0216] Kit In certain embodiments, kits are disclosed herein that include a population of one or more doses of engineered T cells (e.g., CAR-T cells) generated or obtained according to the methods disclosed herein in a suitable container means.

[0217] In certain embodiments, the kit includes a container means containing the engineered T cells described herein. In certain embodiments, the container means is any suitable container for containing a liquid or lyophilized composition, including, but not limited to, vials, syringes, bottles, and intravenous (IV) bags or ampoules. The syringe holds any volume of liquid suitable for injection into a subject, including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more. In certain embodiments, the packaging and kit include a label specifying information required by the US FDA or similar regulatory authority, such as a product description, dosage and method of administration, and / or indication for treatment. In certain embodiments, the packaging provided herein includes any of the compositions described herein.

[0218] In certain embodiments, the packaging and kit further include buffers, preservatives, and / or stabilizers in the pharmaceutical formulation. In certain embodiments, each component of the kit is sealed in an individual container, and all of the various containers are contained within a single package. In certain embodiments, the kit of the present disclosure is designed for cold storage or room temperature storage.

[0219] In addition, in certain embodiments, the preparation contains a stabilizer to increase the shelf life of the kit, for example, bovine serum albumin (BSA). If the composition is lyophilized, the kit, in certain embodiments, contains a further preparation of a solution for reconstituting the lyophilized preparation. Acceptable reconstitution solutions are well known in the art and include, for example, pharmaceutically acceptable phosphate-buffered saline (PBS).

[0220] In certain embodiments, the kit may further include instructions for performing any of the methods described herein. The term “packaging material” refers to the physical structure that contains the components of the kit. In certain embodiments, the packaging material is made of a material commonly used for such purposes, such as paper, corrugated fiber, glass, plastic, foil, or ampoule, that keeps the components sterile. In certain embodiments, the label or accompanying documentation includes appropriate written instructions, such as explaining to the user of the kit how to perform one or more of the methods disclosed herein. In certain embodiments, the kit may further include a label or instructions for using the kit components in any of the methods of this disclosure. In certain embodiments, the kit contains the compound in a pack or dispenser, along with instructions for administering the compound in any of the methods described herein. [Examples]

[0221] The following examples are provided to those skilled in the art to describe how the compositions and methods described herein can be used, prepared, and evaluated, and are intended to be purely illustrative of the disclosure and not intended to limit the scope of what the inventors consider to be their invention.

[0222] (Example 1) Preparation of T cells engineered to express chimeric antigen receptors. T cells were extracted from peripheral blood mononuclear cells (PBMCs) isolated from Donor 1 (a 27-year-old Asian male with a BMI of 25.4, a smoker), or from Donor 2 (a 31-year-old Caucasian male with a BMI of 42.0, a non-smoker) or Donor 3 (a 52-year-old mixed-race male with a BMI of 78.1, a non-smoker) using leukocyte apheresis. T cells from the samples were isolated using Miltenyi StraightFrom CD3 microbeads (Miltenyi Biotec; catalog number 130-090-874) and stored at 2–8°C until further processing.

[0223] The isolated T cells were then independently treated in the amounts of 6×10 7 cells (enriched T cell population containing 85% - 95% T cells) for conditions A - C and F, and 1×10 7 cells (enriched T cell population containing 85% - 95% T cells) for conditions D and E, as shown in Figure 9. The cells were seeded at a density of 3×10 6 / cm 2 (conditions B, C, and F) or 1×10 6 / cm 2 (conditions A, D, and E) in a G-Rex bioreactor. The TransAct (T cell TransAct, human; Miltenyi Biotec; catalog number 130 - 111 - 160) activation reagent was added to the cells for conditions A, B, C, D, and E. The cells for condition F were not activated. Cytokines were added together with the activation reagent for condition B (100 ng / mL human IL-2), condition C (10 ng / mL human IL-7 and 10 ng / mL human IL-15), condition D (10 ng / mL human IL-7 and 10 ng / mL human IL-15), and condition E (10 ng / mL human IL-7 and 10 ng / mL human IL-15). The cells were incubated in a CO2 incubator in a culture medium (TexMACS medium supplemented with CTS Immune Cell SR).

[0224] T cells were transduced with KL-h198a28z, a self-inactivated (SIN) bullous stomatitis virus (VSV)-G pseudotyped third-generation lentiviral vector encoding a CD19-binding chimeric antigen receptor. This CAR construct, named Hu19-CD828Z, has the amino acid sequence shown in SEQ ID NO: 13. The lentiviral vector contained the MSCV promoter, as well as other regulatory factors including a central polypurine / central termination sequence upstream of the promoter and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence. The lentiviral vector KL-h198a28z was prepared using a HEK 293 T cell line transiently transfected with a conventional 4-plasmid system. The envelope protein encoding the plasmid (pLTG1292) expresses the heterologous spike protein, VSV-G protein, under the control of a cytomegalovirus (CMV) promoter. The transduction step was initiated simultaneously with activation under condition A; 18 hours after the start of the activation step under conditions B and C; 18 hours after the seeding step under condition F; and 24 hours after the start of the activation step under conditions D and E.

[0225] Cells were incubated with lentiviral vectors for varying durations: 30 hours under condition A; 48 hours under conditions B, C, and E; 5 days under condition D; and 8 days under condition E. At harvesting, approximately 3 × 10⁶ cells were found under conditions A-C and F, respectively. 7 Cells were obtained, and under condition D, approximately 1 × 10⁻⁶ cells were produced. 8 Cells were obtained, and under condition E, approximately 3 × 10 8 Cells were obtained.

[0226] Transduced T cells were frozen using a standard protocol. The cells were later thawed and characterized according to Example 2 below.

[0227] (Example 2) Characterization of T cells transduced by lentiviral vectors survival rate Viability and cell counts were measured using the NucleoCounter NC-200™ (ChemoMetec A / S, Allerod, Denmark), an automated cell counter that utilizes fluorescence detection to distinguish between viable and non-viable cells. Each test item was loaded into a dedicated cassette containing two separate dyes to stain total nucleated and non-viable cells. The software then calculated viability percentages and cell counts. Total cell counts were performed at 0 and 72 hours after thawing. As shown in Figure 1, each batch produced using the activated KYV 3-day process v1 and v2 showed good overall T cell expansion and proliferation over 72 hours after thawing, better than the 9-day process and at least comparable to the T-Charge process. Donor 1 showed the least overall expansion and proliferation, likely due to the lower viability of the starting cell population.

[0228] CAR expression and T cell phenotype T cells were then evaluated for CAR expression and memory T cell phenotype by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer, employing a fluorescent antibody that recognizes CD19 CARs, as well as cell surface markers associated with Tn, Tscm, Tcm, Tem, and Temra memory T cell subsets. Specifically, Tn cells were identified as CD45RO- / CCR7+ / CD95-; Tscm cells as CD45RO- / CCR7+ / CD95+; Tcm cells as CD45RO+ / CCR7+; Tem cells as CD45RO+ / CCR7-; and Temra cells as CD45RO- / CCR7-. Fluorescent signals associated with each marker were acquired using CytExpert software (Beckman Coulter), and final data analysis was performed using FlowJo (BD Biosciences). As shown in Figure 2, CAR expression for KYV 3-day processes v1 and v2 exceeded T-Charge levels and also showed higher CAR expression levels within CD4+ and CD8+ T cell subpopulations (data not shown). KYV-101C 6-day and KYV-101C also showed slightly better transduction efficiency than T-Charge.

[0229] Regarding the memory T cell subset, as shown in Figures 3A and 3B, cells generated by the T-Charge and KYV 3-v day v3 methods maintained the highest percentage of Tnscm. Notably, Tnscm cells (CD45RO- / CCR7+) were observed to be almost exclusively CD95+ (Tscm). The percentage of CAR+ cells in the T-Charge and KYV 3-v3 methods was <5% upon thawing.

[0230] Additional memory phenotypes of CAR+ T cells were evaluated. T-Charge and KYV 3-day v3 cells were excluded from phenotyping due to low CAR expression at thawing, and KYV 3-day v3 cells were excluded overall from phenotyping in run 2 due to the lack of transduction. As shown in Figures 4A–4D, at full CAR expression 72 hours after thawing, T-Charge and KYV 3-day v3 cells showed similar Tnscm percentages to KYV 3-day v1 and KYV 3-day v2 cells. "Post-enriched" samples were not transduced with lentivirus.

[0231] Overall, T cells processed using the 3-day process showed comparable or higher CAR expression than T cells produced using the T-Charge process. In addition, taking into account donor viability and CAR expression, the three 3-day processes showed similar percentages of Tnscm among themselves, as well as higher percentages of Tnscm than the longer KYV101C (9-day) and KYV101C 6-day processes.

[0232] Cytotoxicity The activity of CAR-T cells that kill CD19+ target cells was measured by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer. Briefly, CD19+ Raji or Nalm6 cells were labeled with cell trace violet (CTV) and co-cultured with effector CAR-T cells at several effector-to-target (E:T) cell ratios. The cell lysis percentage was determined by the ratio of live CTV-positive cells co-cultured with effector cells to live CTV-positive cells cultured in the absence of effector cells, at each E:T ratio. Data analysis was performed using FlowJo (BD Biosciences). The cell lysis percentage was measured and plotted against the E:T ratio, as shown in Figure 5. As provided in Table 1 below, the cytotoxic activity at a 1:1 E:T ratio was comparable among cells produced using processes A-E. [Table 1-1]

[0233] Single-cell cytokine secretome analysis The single-cell cytokine secretome was evaluated using a human adaptive immune cytokine panel. CAR-T cells from condition C were compared to condition E by overnight co-incubation (E:T = 3:1) with NALM-6 cells. The human adaptive immune panel included the following cytokines: CCL-11, GM-CSF, Granzyme B, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP-10, MCP-1, MCP-4, MIP-1α, MIP-1β, Perforin, RANTES, sCD137, TNF-α, TNF-β, TGF-β, and VEGF. Figures 6A - 6D show the percentage of cells secreting each of the 32 cytokines, indicating higher levels of IFN-γ and TNF-β observed in cells produced using the KYV 3-day v2 process compared to the KYV 9-day process.

[0234] Next, the multifunctionality of CAR-T cells was evaluated. Multifunctionality was represented by the percentage of a subset of higher multifunctional cells that simultaneously secreted multiple sets of cytokines after co-culture with CD19+ target cells. As shown in Figure 7, CAR-T cells produced using the KYV 3-day v2 process showed higher multifunctionality than CAR-T cells produced using the 9-day process. Additionally, the percentage of multifunctional cells that simultaneously secreted multiple sets of cytokines was evaluated for each cytokine. Figure 8 shows the top 15 cytokines, and cells produced using the KYV 3-day v2 process showed the highest percentage of multifunctional cells that simultaneously secreted multiple sets of cytokines.

[0235] (Example 3) Functional characteristics of CAR T cells engineered using a 3-day process Anti-CD19 CAR T cells were generated using a similar version of the KYV 3-day process, as outlined in Examples 1 and 2, except for the TransACT activation step which is advantageous for simultaneous activation and isolation using CD3 / CD28 Dynabeads.

[0236] In short, T cells were transduced with KL-h198a28z, a self-inactivated (SIN) bullous stomatitis virus (VSV)-G pseudotyped third-generation lentiviral vector encoding a CD19-binding chimeric antigen receptor. This CAR construct, named Hu19-CD828Z, has the amino acid sequence shown in SEQ ID NO: 13. The lentiviral vector contained the MSCV promoter, as well as other regulatory factors including a central polypurine / central termination sequence upstream of the promoter and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence. The lentiviral vector KL-h198a28z was prepared using a HEK 293 T cell line transiently transfected with a conventional 4-plasmid system. The envelope protein encoding the plasmid (pLTG1292) expresses the heterologous spike protein, VSV-G protein, under the control of a cytomegalovirus (CMV) promoter. The isolation and activation steps were initiated simultaneously for the KYV 3-day "v1" and "v2" processes, as shown in Figure 9. Eighteen hours after the isolation / activation steps, the cells were incubated with the lentiviral vector for 48 hours, and transduced T cells were harvested, frozen using a standard protocol, and later thawed for characterization.

[0237] The short-term ability of CAR-T cells to specifically kill CD19+ target cells was measured by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer. Nalm6 cells were labeled with cell trace violet (CTV) and co-cultured with effector CAR-T cells for 24 hours at several effector-to-target (E:T) cell ratios. The cell elimination percentage was determined by the ratio of live CTV-positive cells co-cultured with effector cells to live CTV-positive cells cultured in the absence of effector cells at each E:T ratio. Data analysis was performed using FlowJo (BD Biosciences).

[0238] As shown in Figure 10, after 24 hours of in vitro co-culture with CD19+ NALM6 target cells at an E:T (effector:target) ratio of 0.1:1, the results showed that mock untransduced (UT) cells that did not express CAR exhibited clear target-dependent cytotoxicity, and also showed lower nonspecific killing activity compared to engineered anti-CD19 CAR T cells generated using the KYV 3-day process with isolated and activated T cells.

[0239] Target-dependent cytokine release by CAR T cells was also evaluated. Figure 11 shows the results for target-dependent cytokine release by CAR T cells derived from healthy donors (HD) after 24 hours of in vitro co-culture with CD19+ NALM6 target cells at the indicated E:T (effector:target) ratio. The supernatant was collected and analyzed by the ELLA assay. Mock untransduced (UT) cells that do not express CAR showed low background levels of cytokine release. In contrast, CAR T cells showed clear target-dependent cytokine release and demonstrated their target-dependent cytotoxic behavior.

[0240] Based on positive results from short-term killing assays, the exhaustion tolerance of CAR T cells and their durability against target-dependent cytotoxicity were evaluated using long-term in vitro serial rechallenge assays.

[0241] As shown in Figure 12, CD19-targeted CAR-T cells and CD19+ NALM6-targeted cells were co-cultured at the indicated E:T ratio (CAR-T effector:NALM6 target), and the percentage of target cell toxicity was measured by flow cytometry at the respective indicated time points. At each time point, a fresh series of target cells were added to the co-culture to evaluate the successive recurrent cytotoxicity of CAR-T cells over time. Mock transduction ("UT") cells that did not express CAR did not demonstrate any cytotoxic activity. In contrast, CAR-T cells exhibited a durable immune response, providing a repeating cytotoxic response upon rechallenge at least 30 days after the initial exposure.

[0242] Therefore, cells generated using the 2-3 day method of this disclosure, which includes a method for the combined isolation and activation of T cells, provide a fatigue-tolerant phenotype exhibiting desired target-specific cytotoxic activity.

[0243] Advantageously, the resulting CAR T cells were not only effective, but they also showed a higher rate of expansion and proliferation upon contact with target cells than CAR T cells produced using the “KYV 8-day” or “KYV 6-day” processes outlined in Figure 9. As described, the 8-day process uses (i) T cells obtained from an enriched population (e.g., from leukocyte apheresis samples or isolated PBMCs); (2) isolation and activation steps for the isolated T cells; and (3) a longer period for culture and expansion.

[0244] In short, anti-CD19 CAR-T cells were prepared using either a conventional 8-day manufacturing process ("Conv") or the KYV 3-day process outlined in this example. Both sets of CAR T cells were derived from healthy donor (HD) starting material. The CAR-T cells were stimulated by co-culturing with mitomycin C-treated CD19+ NALM6 target cells, added in a 1:1 ratio every 3–4 days. The expansion of viable T cells was calculated at each indicated time point using Vicell cell count analysis.

[0245] The results are provided in Figure 13. As shown, cells produced using the 3-day process exhibited a higher rate of target-specific expansion and proliferation, resulting in a desirable in vivo immune profile. Thus, the short CAR T production method of this disclosure comprises co-activation and T cell isolation, as well as a short period of culture, which, upon targeted stimulation, produces cells with a desirable phenotype: target-specific cytotoxicity, a durable immune response, and a high rate of expansion and proliferation.

[0246] As shown, CAR T cells produced using the method of this disclosure exhibit high CAR expression, including higher CAR expression within CD4 and CD8 subsets, compared to other existing methods. The cells also showed a high proportion of the desired Tcm and Tscm cell subsets. Single-cell cytokine analysis revealed that cells generated using the method of this disclosure possessed higher levels of IFNg and TNF-β compared to more conventional processes, including other shortened processes such as KYV 3-day alternatives relying on leukocyte apheresis starting materials. Furthermore, cells generated using the method of this disclosure also exhibited higher levels of pluripotency compared to CAR T cells produced using alternative methods, which may be attributable to the shorter time between the step of isolating T cells from the starting material and the step of harvesting the final CAR T product. Additionally, the resulting CAR T product was shown to have increased levels of antigen-induced proliferation compared to other methods as described.

[0247] Therefore, as shown, the method of this disclosure provides the shortest pathway from sample to CAR T product while providing cells of higher quality than existing CAR T manufacturing processes.

[0248] (Example 4) Subgroup of T cells produced on day 3 To evaluate subpopulations of CAR T cells generated using the abbreviated method of this disclosure, three sets of CAR T cells were generated using: “AR037” with T cells isolated from starting material: donor sample (1 week old at 2-8°C) and processed downstream of the classical 9-day process from Examples 1-2; “AR039” without any downstream processing of fresh sample material; and “AR050” applied to the 3-day v2 (CPD-23-007) process for isolating and manipulating T cells from fresh donor sample.

[0249] Figures 14-15 show the TBNK / memory phenotypes of AR037 before and after enrichment, as well as the TBNK / memory phenotype of the final CAR T cell product (e.g., after expansion and proliferation). Figure 16 shows the TBNK / memory phenotypes of AR037 before and after enrichment. Figure 17 shows the TBNK / memory phenotypes of AR050 before and after enrichment.

[0250] As shown, different starting materials produce viable CAR T cells in which a high percentage are Tnscm when using the method of this disclosure. As shown in Figure 15, the number of Tnscm is shown to increase during cell expansion and proliferation. Surprisingly, using a shortened 3-day process (Figure 17), the initial number of Tnscm exceeds that of the 8-day process. Given the increase shown in the final product of the 8-day process, the 3-day process should produce a final product with an exceptionally high percentage of Tnscm cells.

[0251] (Example 5) Ingenui-T platform Conventional CAR T cell production involves time-consuming 7-10 day cultures of patient apheresis material designed to maximize expansion and proliferation, resulting in a final product with a highly differentiated T cell phenotype. However, studies in oncology show that younger T cells with a more stem-like phenotype correlate with improved clinical benefits compared to those with differentiated memory, effector function, or exhausted phenotypes, which are signatures of more differentiated T cell types. Shorter production processes can mitigate these issues and result in improved CAR T cell products. 3 .

[0252] By shortening the time required for CAR T cell production and starting from fresh starting donor samples, this approach addresses key challenges associated with conventional methods, retaining the potential to revolutionize the patient experience value of CAR T cell therapy. This optimization reduces production costs. 4 This could increase the accessibility of the treatment and the overall feasibility of CAR T-cell therapy.

[0253] Ingenui-T is a next-generation CAR T cell manufacturing platform, originally developed for autoimmune diseases, that utilizes the same fully human anti-CD19 CAR construct as KYV-101. KYV-101 is an investigational autoanti-CD19 CAR T cell therapy (manufactured using conventional methods) in patients with B-cell driven autoimmune diseases, including lupus nephritis, systemic sclerosis, myasthenia gravis, multiple sclerosis, and other diseases with strong rationale for B-cell involvement in disease pathogenesis. This example provides an exemplary use of the novel Ingenui-T manufacturing platform of this disclosure, which highlights the platform's ability to produce high-purity and functional CAR T cells.

[0254] Importantly, the Ingenui-T platform yields CAR T cells with a potent functional profile and less differentiated phenotype compared to CAR T cells produced in conventional manufacturing processes using apheresis-derived cell starting materials. By avoiding the challenges associated with apheresis, the Ingenui-T platform offers a promising means to enhance the efficacy and availability of CAR T cell therapy, reduce costs, and ultimately advance its application in the field of autoimmune diseases.

[0255] method Patient whole blood and leukocyte apheresis collection

[0256] Peripheral whole blood (up to 200 mL) was collected from healthy donors (n=9, AllCells or Bloodworks Northwest, USA) and transported fresh for immediate processing. Cryopreservation was intentionally avoided to maximize cell viability. Cell counts were performed to quantify the incoming blood cell population. Donor-matched cryopreserved leukocyte apheresis material (n=4, AllCells, USA) was also obtained, and CAR T cells were created according to conventional manufacturing processes.

[0257] Ingenui-T Manufacturing Platform

[0258] Figure 18 provides an overview of an exemplary use of the Ingenui-T manufacturing platform of this disclosure for generating engineered immune cells.

[0259] As shown in Figure 18, in short, up to 200 mL of collected whole blood was directly added to a Gibco® CTS® DynaCellect® magnetic separation system (Thermo Fisher Scientific, Waltham, MA), and anti-CD3 / CD28 Dynabeads (CTS® Detachable Dynabeads® CD3 / CD28 kit; Thermo Fisher Scientific, Waltham, MA) were used in defined ratios for T cell enrichment and activation. Isolated and activated T cells were counted, and CD3 + T cell purity was analyzed by flow cytometry, and the cells were seeded in containers containing culture medium enriched with cytokines (human interleukin-2 [IL-2], IL-21, IL-15, IL-7, or a combination thereof). Less than 24 hours after seeding, the T cells were transduced with a fixed multiple of infection (MOI) using a lentiviral vector encoding the Hu19-CD828Z anti-CD19 CAR construct. This is the same construct used in the KYV-101 first-in-class, a fully human autoanti-CD19 CAR T cell therapy (Kyverna Therapeutics, Emeryville, CA). After a targeted in vitro culture period of less than 72 hours after seeding, the beads were removed from the culture, the cells were harvested, formulated into final product containers, and cryopreserved. In parallel, untransduced cells were also prepared by the same manufacturing process in the absence of lentiviral transduction for use as control cells.

[0260] Conventional research-grade CAR T cell production

[0261] For the production of anti-CD19 CAR T cells for conventional research use only (RUO), cell products were prepared to correspond to the KYV-101 product. Cryopreserved leukocyte apheresis material was thawed, washed, and subjected to antibody-driven T cell isolation using magnetic beads (Miltenyi Biotec). Isolated T cells were counted and CD3 +T cell purity was analyzed by flow cytometry, and the cells were activated using T cell TransAct (Miltenyi Biotec) in the presence of supporting cytokines (human IL-2, IL-21, IL-15, IL-7, or a combination thereof). Similar to the Ingenui-T platform, T cells were transduced using the same lentiviral vector incorporating the Hu19-CD828Z anti-CD19 CAR construct (Kyverna Therapeutics, Emeryville, CA) at a fixed MOI. The cells were then cultured for 8–10 days before harvesting, formulation, and cryopreservation of the final product.

[0262] in vitro CAR T cell phenotyping

[0263] Using flow cytometry, CD3 + T cell purity, CD4 + and CD8 + T cell populations and anti-CD19 CAR expression were analyzed. CD4 and CD8 T cell memory phenotypes of the Ingenui-T final product were compared to T cell memory populations in whole blood starting materials based on the expression of CD45RO, CCR7, and CD95 surface markers. Similarly, T cell memory phenotypes in conventional CAR T cells were compared to T cell memory populations in apheresis starting materials (whole blood Ingenui-T cells and donor-matched cells). CAR expression was analyzed at 0 and 72 hours after thawing of the final drug product to ensure accurate determination of stably incorporated expression.

[0264] in vitro CAR T cell functional activity

[0265] To evaluate the functional activity of CAR T cells in a short-term single-challenge cytotoxicity assay, donor-matched Ingenui-T cells or conventional CAR T cells were co-cultured for 120 hours with CD19+ NALM6 target cells expressing the mCherry fluorescent reporter protein at the indicated effector-to-target (E:T) ratio. Target-specific cytotoxic activity was assessed by imaging the co-culture using an Incucyte Sx5 (Sartorius) instrument and by calculating the survival or outgrowth of fluorescently targeted cells over time, normalized to the signal intensity at the start of the co-culture.

[0266] To evaluate the long-term functionality of CAR T cells, donor-matched Ingenui-T cells or conventional CAR T cells were rechallenged with CD19+ NALM6 target cells every 2-3 days at the indicated E:T ratio. At each time point, the sample was divided in half, the cytotoxicity percentage was assessed, and the halves were reseeded with fresh target cells. The cytotoxicity percentage was calculated at each time point by measuring target cell survival using flow cytometry and normalized to target cell survival in the absence of CAR T effector cells.

[0267] To evaluate the cytolytic activity of CAR T cells against autologous primary B cells, Ingenui-T cells or control untransduced T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukocyte apheresis material. The effector-to-target (E:T) ratio was used to evaluate CAR T cells against total PBMCs (target). + Ingenui-T cells (effectors) were defined according to their number. After 48 hours, target-specific cytolytic activity against B cells was measured by flow cytometry. B cells were gated CD3 - We defined B cells by either CD19 or CD20 surface expression within the cell to ensure proper detection, even in the presence of interactions with anti-CD19 CAR T cells. The cytolysis percentage of B cells was calculated by normalizing it to the viability of B cells in PBMC-only cultures.

[0268] result Ingenui T-cell manufacturing platform The goal was to demonstrate the technical feasibility of creating anti-CD19 CAR T cells from various types of fresh or cryopreserved starting materials in a shortened manufacturing process. As shown in Figure 18, fresh donor samples from healthy donors were loaded onto a DynaCellect platform using anti-CD3 / CD28 Dynabeads at a defined bead:cell ratio for simultaneous isolation and activation of T cells. The isolated / activated T cells were sampled and their isolation purity (≥95% CD3+) was ensured by flow cytometry, and then seeded into cultures using media containing cytokines IL-2, IL-7, IL-15, IL-21, or combinations thereof. Transduction using lentiviral vectors encoding anti-CD19 CAR constructs was performed with a fixed MOI within the first 24 hours of culture, followed by short-term culture to allow cell retrieval and transgene integration (<72 hours after seeding). Following this short culture period, CAR T cells were collected for bead removal and subsequent formulation in cryopreservation medium. T cell purity analysis of the final Ingenui-T cell product showed a T cell percentage of 93.9 ± 1.6%, derived from a starting T cell frequency of 42.3 ± 6.8% in whole blood. These results were comparable to T cell enrichment obtained via conventional CAR T cell production processes using donor-matched cells (final T cell purity of 94.0 ± 3.3%, derived from a starting T cell frequency of 46.3 ± 7.2% in conventional apheresis; Table 1).

[0269] Given the short culture time, Ingenui-T cells demonstrated minimal expansion during the manufacturing process, resulting in a 0.68 ± 0.09-fold change in total T cell number from culture seeding to final formulation (including any losses due to washing and bead removal procedures). Nevertheless, the final yield of Ingenui-T cell product was 38.5 ± 6.6 × 10¹⁶ cells per 100 mL of fresh starting sample. 6The cells were T cells. Product attributes were tested at the time of collection and 72 hours after thawing and culture to simulate the product's performance in patients. 72 hours after thawing, CAR+ expression in Ingenui-T cells ranged from 45.1% to 54.5%, which was statistically similar to the CAR+ expression of 37.4% to 56.3% obtained from conventional CAR T cell production processes derived from apheresis (Table 1). This demonstrates that anti-CD19 CAR T cells can be successfully produced directly from fresh starting donor samples using the Ingenui-T platform, in a shortened production process, at a scale sufficient for therapeutic administration to patients with B cell-driven autoimmune diseases.

[0270] Comparison of phenotype and function between Ingenui-T cells and conventional CAR T cells

[0271] To demonstrate the pharmacological activity of anti-CD19 CAR T cells produced on the Ingenui-T platform, the inventors performed a series of phenotypic and functional in vitro characterization experiments and compared Ingenui-T cells with CAR T cells expressing the same anti-CD19 CAR construct produced using a conventional manufacturing process. Whole blood (e.g., fresh starting material) for the Ingenui-T process and apheresis for the conventional process were sourced from the same donor as part of the same collection. As expected, due to the shortened culture period on the Ingenui-T platform, Ingenui-T cells contained a less differentiated T cell memory phenotype than CAR T cells produced using a conventional manufacturing process. Ingenui-T cells from fresh starting material exhibited a more overall effector / memory compartment (combined T cells). CM , T EM , and T E With a slight increase in population, the T-cell memory phenotype was preserved, closely resembling the phenotype observed in the starting material.

[0272] From the starting materials to the final Ingenui-T product, the effector / memory section is TN +T SCM While maintaining the substantial proportion of cells within each compartment, the CD4+ T cell fraction shifted from an average of 48.8±5.6% to 69.4±4.8%, and the CD8+ T cell fraction shifted from an average of 42.9±3.9% to 46.6±5.5% (Figure 19A).

[0273] In contrast, CAR T cells obtained from conventional manufacturing processes (cultured for approximately 9 days) were almost entirely converted to effector / memory compartments, shifting from 58.4±3.5% to 94.0±2.8% and from 40.1±5.2% to 86.2±4.9% respectively within the CD4+ and CD8+ T cell fractions.

[0274] Importantly, minimizing the differentiation of Ingenui-T cells in vitro is expected to preserve their potential for expansion and proliferation in vivo and activation in patients. This, in turn, allows for significantly lower doses for equivalent therapeutic efficacy in clinical applications, which is a significant attribute of Ingenui-T compared to apheresis, given the inherently lower starting number of T cells that can be obtained from whole blood collections.

[0275] The functional activity of Ingenui-T cells was evaluated in both short-term and long-term in vitro preclinical assays to demonstrate target-specific cytotoxicity against CD19-expressing cells. The cytotoxic activity of Ingenui-T cells and conventional CAR T cells derived from donor-matched apheresis (derived from conventional 9-day cultures expressing the same CAR construct) was compared against CD19+ NALM6 tumor cells, a representative target cell line, in a short-term cytotoxicity assay.

[0276] As shown in Figure 19B, in an Incucyte imaging-based assay, Ingenui-T cells controlled NALM6 target cell outgrowth over a 120-hour period with a lower E:T ratio than conventional CAR T cells. This reflects the expected increase in CAR T cell efficacy and target-mediated CAR T cell proliferation, due to the less differentiated memory phenotype of Ingenui-T cells. Minimal cytotoxicity was observed against the control CD19-negative target cell line (CEM / C1), and no cytotoxic activity was observed in untransduced Ingenui-T cells (i.e., without CAR expression; data not shown for CD19-positive targets). These results confirm the anti-CD19 target-specific activity and increased functional efficacy of Ingenui-T cells compared to conventional CAR T cells produced using conventional manufacturing processes.

[0277] To further evaluate the functional activity of Ingenui-T cells, the inventors performed a long-term continuous rechallenge assay in vitro. In this assay, CAR T cells and CD19+ NALM6 target cells were co-cultured at the indicated E:T ratio starting on day 0, and the same number of NALM6 target cells were subsequently added every 2 or 3 days to evaluate the efficacy and durability of long-term target-specific continuous cell injury.

[0278] As shown in Figure 19C, Ingenui-T cells continued to kill target cells for a significantly longer period at a given E:T ratio and required a >4-fold lower E:T ratio than donor-matched CAR T cells created from a 9-day culture process to maintain the same duration of killing. This finding again aligns with our expectation that the younger differentiation phenotype of Ingenui-T cells results in higher functional potency, higher proliferation (data not shown), and more prolonged cytolytic activity compared to conventional CAR T cells, which tend to become more exhausted and lose functionality over time.

[0279] Finally, the in vitro cytolytic activity of Ingenui-T cells was evaluated against autologous primary B cells, which are targeted for depletion in the treatment of patients with B-cell-driven autoimmune diseases. When Ingenui-T cells were co-cultured with autologous total peripheral blood mononuclear cells (PBMCs) for 48 hours, B cells were eliminated in a specific and dose-dependent manner (Figure 19D). As expected, Ingenui-T cells demonstrated higher efficacy in B cell killing than conventional CAR T cells when tested with dose-limited E:T ratios (e.g., 0.011:1; Figure 19D and data not shown).

[0280] These preclinical assays provide proof-of-concept data demonstrating that highly functional anti-CD19 CAR T cells can be generated from newly collected donor material using our Ingenui-T platform. These results pave the way for the clinical development of Ingenui-T cells as a treatment for autoimmune diseases, which would improve patient experience value, increase treatment availability, and reduce costs by eliminating the need for patients to undergo apheresis. [Table 1-2]

[0281] (Example 10) Ingenui-T Platform - Fresh Departure Materials SM This embodiment provides further data on cells produced from freshly collected starting material, e.g., leukocyte apheresis or WB SM, using the method disclosed herein and the Ingenui-T platform. The goal was to characterize anti-CD19 CAR T cells starting from fresh SM (e.g., whole blood or leukocyte apheresis SM) in a shortened production process compared to those produced using a longer 9-day process.

[0282] In short, the manufactured CAR T cells were prepared using the method outlined in Figure 18, where the starting material obtained from the subjects in Figure 18 was derived from leukocyte apheresis samples.

[0283] Peripheral whole blood and leukocyte apheresis SM were obtained from healthy donors, collected, and transported in a fresh state for immediate processing.

[0284] As shown in Figure 18, in short, 100 mL of whole blood or leukocyte apheresis sample was collected and aliquots were directly added to a Gibco® CTS® DynaCellect® magnetic separation system (Thermo Fisher Scientific, Waltham, MA), and anti-CD3 / CD28 Dynabeads (CTS® Detachable Dynabeads® CD3 / CD28 kit; Thermo Fisher Scientific, Waltham, MA) were used in defined ratios for T cell enrichment and activation. Isolated and activated T cells were counted, and CD3 + T cell purity was analyzed by flow cytometry, and the cells were seeded in containers containing culture medium enriched with cytokines (human interleukin-2 [IL-2], IL-21, IL-15, IL-7, or a combination thereof). Less than 24 hours after seeding, the T cells were transduced with a fixed multiple of infection (MOI) using a lentiviral vector encoding the Hu19-CD828Z anti-CD19 CAR construct. This is the same construct used in KYV-101 described above. After targeted in vitro cell culture, the beads were removed from the culture, the cells were harvested, formulated into final product containers, and cryopreserved. In parallel, untransduced cells were also prepared using the same manufacturing process in the absence of lentiviral transduction for use as control cells.

[0285] As shown in Figure 20A, flow cytometry was used to analyze the overall T cell expansion, T cell viability, and T cell purity of CAR-T cells produced using a 3-day process on the Ingenui-T platform with fresh starting leukocyte apheresis samples. Across various different cytokine cultures, the 3-day process consistently produced T cells with more than 1x expansion, more than 90% viability, and more than 95% purity. Importantly, these results remained consistent regardless of whether the T cells were transduced with exogenous immune receptors or not.

[0286] Similarly, as shown in Figure 20B, when starting with a fresh whole blood sample, expanded T cells with high final concentrations were produced with very high T cell purity using the 3-day process of this disclosure.

[0287] These results were comparable to T cell enrichment obtained via conventional CAR T cell production processes using donor-matched cells. In particular, for cells produced from fresh SM, Ingenui-T cells demonstrated minimal expansion during the production process despite short culture times, and also produced a high final yield. Thus, not only was a sufficient quantity of final product produced, but it was also produced with limited expansion, which should produce T cells with the desirable phenotype described above.

[0288] Figure 21A shows the CAR+ expression percentage, analyzed by flow cytometry, in CAR-T cells produced in the KYV 3-day process starting from fresh leukocyte apheresis material. CAR expression was analyzed in total CD3+ T cells or CD4+ or CD8+ T cells at harvesting. KYV 3-day conditions "A", "B", "C", and "D" indicate the different cultured cytokine(s) used. N=4 healthy donors per condition. As described in Example 3, CAR+ expression in Ingenui-T cells (leukocyte apheresis SM) 72 hours after thawing of the drug product was statistically similar to CAR+ expression obtained from conventional CAR T cell production processes.

[0289] Similarly, as shown in Figure 21B, CAR+ expression % was similarly high when starting with fresh whole blood starting samples.

[0290] The CD4 and CD8 T cell memory phenotypes of Ingenui-T end products, generated using either fresh leukocyte apheresis starting material (SM) or whole blood (WB) SM, were compared to the T cell memory population in whole blood / leukocyte apheresis starting material based on the expression of CD45RO, CCR7, and CD95 surface markers. Similarly, the T cell memory phenotype in conventional CAR T cells was compared to the T cell memory population in leukocyte apheresis starting material (whole blood Ingenui-T cells and donor-matched cells). CAR expression was analyzed at 0 and 72 hours after thawing of the final drug product to ensure accurate determination of stably incorporated expression.

[0291] Figure 22A shows the CD4+ to CD8+ ratio for CAR-T cells using fresh leukocyte apheresis SM and the 3-day process, compared to similar cells produced using the conventional 9-day process. As shown, the 3-day process produces CAR-T cells with a CD4:CD8 ratio similar to that of the much longer 9-day process.

[0292] Figure 22B shows similar results for cells produced using a 3-day process, starting from fresh whole blood (WB).

[0293] To demonstrate the pharmacological activity of anti-CD19 CAR T cells produced on the Ingenui-T platform, a series of phenotypic and functional in vitro characterization assays were performed to compare Ingenui-T cells (using fresh WB or leukocyte apheresis SM) with CAR T cells expressing the same anti-CD19 CAR construct produced using a conventional (9-day) manufacturing process.

[0294] As shown in Figures 23A–23B, compared to the 9-day process, the 3-day process of this disclosure using leukocyte apheresis SM produced fewer effector T cells and a higher proportion of TNSCM cells, particularly T cells with Tscm cells. As shown in Figure 23C, when using WB SM, the 3-day method of this disclosure also produced T cells with a high proportion of TNSCM cells.

[0295] Fresh whole blood for the Ingenui-T process, as well as leukocyte apheresis SM for the 3-day and 9-day processes, were sourced from the same donor as part of the same collection. As expected, due to the shortened culture period on the Ingenui-T platform, Ingenui-T cells contained less differentiated T cell memory phenotypes than CAR T cells produced using conventional manufacturing processes. Ingenui-T cells derived from fresh donor samples exhibited an overall effector / memory compartment (combined T cells). CM , T EM , and T E With a slight increase in population, the cells retained a T-cell memory phenotype very similar to that observed in the starting material. There was a dramatic improvement over the existing 9-day process. Therefore, although the cells were transduced with the same exogenous CAR, they are nevertheless fundamentally different end products.

[0296] The functional activity of Ingenui-T cells was evaluated using both fresh WB SM and leukocyte apheresis SM to demonstrate target-specific cytotoxicity against CD19-expressing cells. The cytotoxic activity of Ingenui-T cells and conventional CAR T cells derived from donor-matched apheresis (derived from conventional 9-day cultures expressing the same CAR construct) was compared against CD19+ NALM6 tumor cells, a representative target cell line, in a short-term cytotoxicity assay.

[0297] Figure 24 shows the cytolysis percentage of CD19+ NALM6 target cells or CD19- CEM / C1 control cells after co-culture with anti-CD19 CAR-T cells produced from the KYV 3-day process at the indicated E:T (effector:target) ratio. N=2 donors are shown. Cytolytic activity was measured by a luminescence assay and normalized against target cells alone (0:1). As shown, cells from the 3-day process exhibited a clear target-dependent cytotoxic response against CD19+ expressing cells.

[0298] Figure 25A shows the results of killing or outgrowth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells produced in the KYV 3-day process from leukocyte apheresis starting material, at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time=0. KYV 3-day conditions "C1", "C2", "C3", and "C4" indicate different cultured cytokines used. "NT" = untransduced control T cells. One representative donor is shown from n=4. Similarly, Figure 25B provides similar results for the killing or outgrowth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells produced in the KYV 3-day process from freshly collected whole blood, at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time=0. "NT" = untransduced control T cells. "Conv 9 days" refers to donor-matched CAR-T cells produced in the conventional 9-day culture process from leukocyte apheresis starting material. One representative donor is shown from n=4.

[0299] As shown in Figure 25A, in an Incucyte imaging-based assay, Ingenui-T cells (leukocyte apheresis SM) controlled NALM6 target cell outgrowth over a 120-hour period with a lower E:T ratio than conventional CAR T cells. Similar results were found for cells generated using WB SM (Figure 25B). T cells generated using the 3-day process exhibited a more robust, target-specific cytotoxic response than comparable cells generated using longer (e.g., 9-day) processes, regardless of whether they originated from WB SM or leukocyte apheresis samples. Cells generated using WB SM exhibited a more effective and robust cytotoxic response even when compared to cells generated using leukocyte apheresis samples.

[0300] This reflects the expected efficacy of CAR T cells and target-mediated CAR T cell proliferation. Furthermore, minimal cytotoxicity was observed against the control CD19-negative target cell line (CEM / C1), and no cytotoxic activity was observed in untransduced Ingenui-T cells. These results confirm the anti-CD19 target-specific activity of Ingenui-T cells generated from fresh starting donor material.

[0301] The in vitro cytolytic activity of Ingenui-T cells was evaluated against autologous primary B cells, which are targeted for depletion in the treatment of patients with B-cell-driven autoimmune diseases. To evaluate the cytolytic activity of CAR T cells against autologous primary B cells, Ingenui-T cells or control untransduced T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukocyte apheresis material. The effector-to-target (E:T) ratio was measured against CAR T cells against total PBMCs (target). + Ingenui-T cells (effectors) were defined according to their number. After 48 hours, target-specific cytolytic activity against B cells was measured by flow cytometry. B cells were gated CD3 - We defined B cells by either CD19 or CD20 surface expression within the cell to ensure proper detection, even in the presence of interactions with anti-CD19 CAR T cells. The cytolysis percentage of B cells was calculated by normalizing it to the viability of B cells in PBMC-only cultures.

[0302] When Ingenui-T cells generated from fresh WB SM were co-cultured with autologous total peripheral blood mononuclear cells (PBMCs) for 48 hours, B cells were eliminated in a specific and dose-dependent manner (Figure 26). Ingenui-T cells demonstrated higher B cell killing efficacy than conventional CAR T cells when tested with a dose-limited E:T ratio.

[0303] Figure 27 shows IFN-gamma production by anti-CD19 CAR-T cells produced from freshly collected whole blood via the KYV 3-day process in co-culture with CD19+ NALM6 target cells or CD19- CEMC1 control cells at the indicated E:T (effector:target) ratio. Culture supernatant was collected and analyzed by ELLA. N=2 donors are shown. These results demonstrate target-dependent cytokine release by anti-CD19 CAR-T cells produced from the KYV 3-day process in response to CD19+ expressing target cells.

[0304] Figure 28A shows cytokine release by anti-CD19 CAR-T cells produced from leukocyte apheresis starting material via the KYV 3-day process in co-culture with CD19+ NALM6 target cells at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. Figures 28B-28C show cytokine release by anti-CD19 CAR-T cells produced from freshly collected whole blood via the KYV 3-day process in co-culture with CD19+ NALM6 target cells at indicated E:T (effector:target) ratios of 0.3:1 or 1:1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD. The KYV 3-day conditions "C1", "C2", "C3", and "C4" indicate different cultured cytokines used in the production process. "Conv 9 days" refers to donor-matched CAR-T cells produced from leukocyte apheresis starting material via the conventional 9-day culture process. "NT" = untransduced control T cells. N=4 healthy donors per condition. As shown, cells generated using the 3-day method of this disclosure provide effector dose-dependent CAR-mediated cytokine release in response to CD19+ expressing target cells.

[0305] The long-term continuous cytotoxic activity of anti-CD19 CAR-T cells produced from the KYV 3-day process was compared to that of a conventional 9-day process in response to CD19+ expressing target cells. Figure 29 shows the duration of in vitro cytotoxicity by KYV 3-day or Conv 9-day anti-CD19 CAR T cells from healthy donors in a continuous rechallenge assay against CD19+ NALM6 tumor cells. KYV 3-day CAR T cells were derived from leukocyte apheresis starting material ("APH") or freshly collected whole blood ("WB"). CAR T cells were co-cultured with NALM6 target cells in three replicates at the indicated effector:target (E:T) ratio, and NALM6 cell viability was analyzed every 2-3 days by flow cytometry. Time to loss of CAR-mediated cytotoxic activity (days), defined as the assay time point at which >95% survival of target cells was detected, was measured for each individual replicate. Data are representative of n=4 donors. As demonstrated, the cells developed a durable, long-lasting cytotoxic response.

[0306] Figure 30 shows data comparing in vitro expansion of anti-CD19 CAR-T cells produced from a KYV 3-day process with that produced from a conventional 9-day process, in response to CD19+ expressing target cells. The data provide responses of KYV 3-day or conventional ("Conv") 9-day anti-CD19 CAR T cells to repeated stimulation in co-culture with CD19+ expressing REH target cells. KYV 3-day CAR T cells were derived from leukocyte apheresis starting material ("APH", n=4) or other freshly collected starting material ("WB", n=3) and compared with donor-matched Conv 9-day CAR T cells derived from leukocyte apheresis material. CAR T cells were co-cultured with mitomycin C-treated REH target cells in a 1:1 ratio, and the cells were re-seed every 3-4 days with new target cells. The total expansion proliferation of CAR+ T cells (gated by flow cytometry analysis) was measured on day 16. As demonstrated, cells exhibit the potential for expansion and proliferation upon contact with a suitable target.

[0307] Based on these promising results, we evaluated the in vivo activity of cells using newly collected starting material after 3 days in mice.

[0308] Figures 31A-31B show data on the in vivo activity of anti-CD19 CAR-T cells produced using the KYV 3-day process compared to the conventional 9-day process in NSG mice with CD19+ NALM6 tumors.

[0309] Figure 31A shows mean NALM6 tumor growth in NSG mice treated with indicated doses of donor-matched anti-CD19 CAR T cells, both produced from leukocyte apheresis ("APH") starting material using either the KYV 3-day process or the conventional ("Conv") 9-day process. NALM6-luciferase tumor cells were intravenously injected into mice on day 7 prior to T cell transfer. On day 0, mice were given a single intravenous injection of indicated doses of CAR T cells. Tumor volume in each animal was measured twice weekly using IVIS bioluminescence imaging and is shown as total flux (photons / second). Data are shown as mean ± SEM for all animals per group. Data are representative of two studies using n=2 independent donors.

[0310] Figure 31B shows individual NALM6 tumor growth curves in NSG mice treated with donor-matched anti-CD19 CAR T cells at a dose of 1e6 CAR+ T cells. CAR T cells were prepared from freshly collected whole blood ("WB") using the KYV 3-day process, or from leukocyte apheresis starting material using the conventional ("Conv") 9-day process. Tumor cells were inoculated, mice treated, and analyzed as described in Figure 31A. N=5 animals per group.

[0311] These preclinical assays provide proof-of-concept data, including in vivo data, demonstrating that highly functional anti-CD19 CAR T cells can be generated using the Ingenui-T platform, starting from fresh donor starting material. These results pave the way for the clinical development of Ingenui-T cells as a treatment for autoimmune diseases, which would improve patient experience value, increase treatment availability, and reduce costs by eliminating the need for patients to undergo apheresis.

[0312] Consideration The Ingenui-T platform focuses on enhancing patient experience value and reducing the cost of manufacturing CAR T cell therapies. The next-generation manufacturing process starts from fresh starting materials, utilizes a rapid (<3 day) manufacturing process, and yields potent CAR T cell products with demonstrated target-specific killing activity. This manufacturing process represents a significant departure from conventional methods, which inevitably involve apheresis, a difficult and resource-intensive process, and prolonged cell culture. 2

[0313] The Ingenui-T platform delivers both a shorter process and a more potent product, potentially providing equivalent therapeutic benefits at lower doses while minimizing cell differentiation in vitro by reducing culture time and enabling feasibility using limited volumes of fresh donor material. By simplifying the process and minimizing culture time, it not only reduces and optimizes resource utilization but also reduces the time spent in specialized facilities, reduces the involvement of highly skilled personnel, and enhances the cost-effectiveness of CAR T cell therapy. This reduction in overall costs for manufacturing the product and the decrease in the burden on patients maintains the basis for wider availability and affordability. This optimization also aligns with the goal of scalability in CAR T cell therapy, addressing a critical need in this field.

[0314] The results of Ingenui-T cell production demonstrate the ability to enrich T cells from fresh starting material and to successfully create potent anti-CD19 CAR T cells. Phenotypic characterization shows that Ingenui-T cells exhibit a less differentiated phenotype compared to conventionally produced CAR T cells. This characteristic may have clinical significance as less differentiated T cells are associated with enhanced in vivo expansion, proliferation, and efficacy.

[0315] The Ingenui-T anti-CD19 CAR T cell product, currently under development for the treatment of B cell-driven autoimmune diseases, introduces a novel treatment paradigm that addresses key challenges associated with conventional manufacturing methods. Reduced patient burden, cost-effectiveness, and the platform's unique approach highlight its potential to significantly impact the feasibility and availability of CAR T cell therapy for the treatment of autoimmune diseases.

[0316] Other Embodiments

[0317] Various modifications and variations of the disclosed information will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. Although this disclosure is described in relation to specific embodiments, it should be understood that the disclosure in the claims should not be unduly limited to such specific embodiments. In fact, various modifications of the described methods for making this disclosure, which are obvious to those skilled in the art, are intended to be within the scope of this disclosure. Other embodiments are included in the claims. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] References (1) Mikhael, J., Fowler, J. & Shah, N. Chimeric Antigen Receptor T-Cell Therapies: Barriers and Solutions to Access. JCO Oncology Practice 18, 800-807 (2022). (2) Qayed, M. et al. Leukapheresis guidance and best practices for optimal chimeric antigen receptor T-cell manufacturing. Cytotherapy 24, 869-878 (2022). (3) Bulliard, Y., Andersson, B. S., Baysal, M. A., Damiano, J. & Tsimberidou, A. M. Reprogramming T cell differentiation and exhaustion in CAR-T cell therapy. J Hematol Oncol 16, 108 (2023). (4) Ghassemi, S. et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118-128 (2022).

Claims

1. A method for producing a population of manipulated T cells that express a heterologous protein, wherein the method is performed in the following order: (a) A step of contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) Contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding the heterologous protein, and culturing the T cells in a serum-free culture medium containing interleukin-2 (IL-2) protein for 29 to 71 hours; and (c) Step of collecting the T cells Includes, A method comprising collecting T cells, wherein the collected T cells include one or more T cells that have been manipulated to express the heterologous protein.

2. A method for producing a population of manipulated T cells that express a heterologous protein, wherein the method is performed in the following order: (a) A step of contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) Contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding the heterologous protein, and culturing the T cells for 31 to 71 hours in a culture medium containing interleukin-7 (IL-7) and interleukin-15 (IL-15) proteins; and (c) Step of collecting the T cells Includes, A method comprising collecting T cells, wherein the collected T cells include one or more T cells that have been manipulated to express the heterologous protein.

3. A method for producing a population of manipulated T cells that express a heterologous protein, wherein the method is performed in the following order: (a) A step of contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) Contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding the heterologous protein; (c) Culturing the T cells for 31 to 71 hours in a serum-free culture medium containing interleukin-7 (IL-7) and / or interleukin-15 (IL-15) protein; and (c) Step of collecting the T cells Includes, A method comprising collecting T cells, wherein the collected T cells include one or more T cells that have been manipulated to express the heterologous protein.

4. A method for producing a population of manipulated T cells that express a heterologous protein, wherein the method is performed in the following order: (a) A starting population of T cells is brought into contact with one or more agents that activate CD3 and CD28 in the absence of cytokines; (b) Contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding the heterologous protein, and culturing the T cells in a culture medium containing at least one cytokine for 24 to 72 hours; and (c) Step of collecting the T cells Including; Step (b) is performed approximately 18 hours after step (a). A method comprising collecting T cells, wherein the collected T cells include one or more T cells that have been manipulated to express the heterologous protein.

5. The method according to any one of claims 4, wherein the at least one cytokine in step (c) comprises one or more of IL-2, IL-7, IL-15, and / or IL-21.

6. The method according to claim 4, wherein the at least one cytokine in step (c) includes IL-2.

7. The method according to claim 4, wherein the at least one cytokine in step (c) includes IL-21.

8. The method according to claim 4, wherein the at least one cytokine in step (c) comprises IL-7 and IL-15.

9. The method according to claim 4, wherein the at least one cytokine in step (c) comprises IL-2, IL-7, and IL-15.

10. The method according to claim 4, wherein the at least one cytokine in step (c) comprises IL-21, IL-7, and IL-15.

11. A method for producing a population of manipulated T cells that express a heterologous protein, wherein the method is performed in the following order: (a) A step of contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) Contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding the heterologous protein, and culturing the T cells in a culture medium containing interleukin-21 (IL-21) protein for 31 to 71 hours; and (c) Step of collecting the T cells Includes, A method comprising collecting T cells, wherein the collected T cells include one or more T cells that have been manipulated to express the heterologous protein.

12. The method according to claim 11, wherein the culture medium further comprises IL-7 and / or IL-15.

13. A method for producing a population of manipulated T cells that express a heterologous protein, wherein the method is performed in the following order: (a) A step of contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) The step of contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding the heterologous protein, and culturing the T cells in a culture medium for 24 to 72 hours; and (c) Step of collecting the T cells Includes, The culture medium does not contain IL-2, IL-7, IL-15, or IL-21. A method comprising collecting T cells, wherein the collected T cells include one or more T cells that have been manipulated to express the heterologous protein.

14. The method according to claim 13, wherein the culture medium does not contain cytokines.

15. The method according to claim 13 or 14, wherein step (a) does not involve the use of IL-2, IL-7, IL-15, or IL-21.

16. The method according to any one of claims 13 to 15, wherein step (a) is carried out in the absence of cytokines.

17. The method according to any one of claims 13 to 16, wherein step (c) does not involve the use of IL-2, IL-7, IL-15, or IL-21.

18. The method according to any one of claims 13 to 17, wherein step (c) is carried out in the absence of cytokines.

19. The method according to any one of claims 13 to 18, carried out in the absence of cytokines.

20. The method according to any one of claims 13 to 19, wherein the culture medium is a basal culture medium.

21. The method according to any one of claims 2 or 4 to 19, wherein the culture medium comprises serum.

22. The method according to claim 21, wherein the culture medium further contains added cytokines or growth factors.

23. The method according to any one of claims 2 or 4 to 20, wherein the culture medium is serum-free.

24. The method according to any one of claims 1 to 23, wherein step (b) is performed at least one hour after the start of step (a).

25. The aforementioned starting population of T cells is approximately 1 × 10⁶ per 1 mL. ６ The method according to any one of claims 1 to 24, wherein the cells are seeded into a culture at a concentration.

26. The aforementioned starting population of T cells is approximately 2 × 10⁶ per 1 mL. ６ The method according to any one of claims 1 to 24, wherein the cells are seeded into a culture at a concentration.

27. The aforementioned starting population of T cells is approximately 5 × 10⁶ per 1 mL. ６ The method according to any one of claims 1 to 24, wherein the cells are seeded into a culture at a concentration.

28. (a) The starting population of T cells is approximately 1 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium contains IL-2, The method according to any one of claims 1, 4 to 10, or 14 to 25.

29. (a) The starting population of T cells is approximately 1 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium contains IL-7 and IL-15, The method according to any one of claims 2 to 10 or 14 to 25.

30. (a) The starting population of T cells is approximately 1 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium comprises IL-7, IL-15, and IL-21, The method according to any one of claims 2 to 12 or 14 to 25.

31. (a) The starting population of T cells is approximately 1 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium contains IL-21, The method according to any one of claims 4 to 12 or 14 to 25.

32. (a) The starting population of T cells is approximately 1 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium does not contain IL-2, IL-7, IL-15, or IL-21. The method according to any one of claims 13 to 25.

33. (a) said starting population of T cells is seeded into a culture at a concentration of about 2×10 ６ cells per mL; (b) The culture medium contains IL-2, The method according to any one of claims 1, 4 to 10, 14, 21 to 24, or 26.

34. (a) The starting population of T cells is approximately 2 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium contains IL-7 and IL-15, The method according to any one of claims 2 to 10, 14, 21 to 24, or 26.

35. (a) The starting population of T cells is approximately 2 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium comprises IL-7, IL-15, and IL-21, The method according to any one of claims 2 to 12, 14, 21 to 24, or 26.

36. (a) The starting population of T cells is approximately 2 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium contains IL-21, The method according to any one of claims 4 to 12, 14, 21 to 24, or 26.

37. (a) The starting population of T cells is approximately 2 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium does not contain IL-2, IL-7, IL-15, or IL-21. The method according to any one of claims 13 to 24 or 26.

38. (a) The starting population of T cells is approximately 5 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The method according to any one of claims 1, 4 to 10, 14, 21 to 24, or 27, wherein the culture medium comprises IL-2.

39. (a) The starting population of T cells is approximately 5 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium contains IL-7 and IL-15, The method according to any one of claims 2 to 10, 14, 21 to 24, or 27.

40. (a) The starting population of T cells is approximately 5 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium comprises IL-7, IL-15, and IL-21, The method according to any one of claims 2 to 12, 14, 21 to 24, or 27.

41. (a) The starting population of T cells is approximately 5 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium contains IL-21, The method according to any one of claims 4 to 12, 14, 21 to 24, or 27.

42. (a) The starting population of T cells is approximately 5 × 10 per 1 mL. ６ The cells are seeded into the culture at a specific concentration; (b) The culture medium does not contain IL-2, IL-7, IL-15, or IL-21. The method according to any one of claims 13 to 24 or 27.

43. (d) The method according to any one of claims 1 to 42, further comprising the step of maintaining the T cells collected in step (c) at a temperature of 38°C or lower, between 2 and 8°C, or -80°C or lower.

44. The method according to any one of claims 1 to 43, wherein the one or more agents that activate CD3 and / or CD28 comprises an anti-CD3 antibody, an anti-CD28 antibody, or both.

45. The method according to claim 44, wherein the one or more agents that activate CD3 and / or CD28 include a first agent that binds to CD3 and a second agent that binds to CD28.

46. The method according to any one of claims 1 to 45, comprising the step of obtaining a sample containing the T cell starting population from a subject prior to step (a).

47. The method according to any one of claims 1 to 45, comprising the step of obtaining a sample containing the T cell starting population from a subject prior to step (a).

48. The method according to claim 46 or 47, wherein the sample is a whole blood sample obtained from the subject.

49. The method according to claim 46 or 47, wherein the sample is a leukocyte apheresis sample obtained from the subject.

50. The aforementioned starting population of T cells includes helper T(Th) cells and cytotoxic T(T) cells. Ｃ ) cells, memory T(T) Ｍ ) cells, regulatory T (T ｒｅｇ The method according to any one of claims 1 to 49, comprising ) cells, innate immune-like T cells.

51. The method according to claim 50, wherein the Th cells include Th1 cells, Th2 cells, Th17 cells, Th9 cells, Tfh cells, and / or Th22 cells.

52. The memory T cells mentioned above are central memory T cells (T ＣＭ ) Cells, effector memory T (T ＥＭ ) Cells, tissue-resident memory T(T) ＲＭ ) cells, and virtual memory T(T) ＶＭ The method according to claim 50 or 51, comprising ) cells.

53. The method according to any one of claims 50 to 52, wherein the innate immune-like T cells are natural killer T (NKT) cells, mucosa-associated invariant T (MAIT) cells, and γδ T cells.

54. The method according to any one of claims 1 to 53, wherein the polynucleotide is contained in the delivery vehicle.

55. The method according to claim 54, wherein the delivery vehicle is lipid nanoparticles.

56. The method according to claim 54, wherein the delivery vehicle is a nucleic acid vector.

57. The method according to claim 55, wherein the nucleic acid vector is a viral vector.

58. The method according to claim 57, wherein the viral vector is a lentiviral vector.

59. The method according to any one of claims 1 to 58, wherein, after step (c), the following expansion and proliferation of the starting population of T cells is 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times or less.

60. The method according to any one of claims 1 to 59, wherein after step (c), at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 75% of the cells of the starting population of T cells are manipulated to express the heterologous protein.

61. The method according to any one of claims 1 to 60, wherein the heterogeneous protein comprises a chimeric antigen receptor (CAR).

62. The aforementioned CAR, (a) Antigen-binding fragment of an anti-CD19 antibody; (b) Transmembrane domain; (c) Intracellular T cell signaling domain derived from human CD3ζ; and (d) Intracellular T cell signaling domain derived from human CD28 The method according to claim 61, including the method described in claim 61.

63. The method according to claim 62, wherein one or more of the T cells collected in step (c) secrete an increased amount of one or more proteins selected from the group consisting of IFNγ, granzyme B, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP-10, MCP1, MCP4, TNFα, TNFβ, TGFβ, GM-CSF, MIP1α, MIP1β, CCL11, perforin, RANTES, sCD137, and VEGF after contact with the target cells expressing CD19, compared to T cells that have not been in contact with the target cells.

64. The method according to claim 63, wherein the secretion of the one or more proteins after contact with the target cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or higher compared to the secretion of the one or more proteins in the absence of the target cells.

65. The method according to any one of claims 62 to 64, wherein one or more of the T cells collected in step (c) exhibit increased expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 after contact with the target cells expressing CD19, compared to the expression of one or more T cell activation markers in the absence of the target cells.

66. The method according to claim 65, wherein the expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more, compared to the expression of the one or more T cell activation markers in the absence of the target cells.

67. The method according to any one of claims 62 to 66, wherein one or more of the T cells collected in step (c) exhibit increased cytotoxicity against target cells expressing CD19 compared to cytotoxicity against cells that do not express CD19.

68. The method according to claim 67, wherein the cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more, compared to the cytotoxicity to cells that do not express CD19.

69. The method according to any one of claims 62 to 68, wherein one or more of the T cells collected in step (c) exhibit increased proliferation after contact with the target cells expressing CD19, compared to proliferation in the absence of the target cells.

70. The method according to claim 69, wherein proliferation increases by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, or more, compared to proliferation in the absence of the target cells.

71. The T cells collected in step (c) showed an increased amount of naive and stem cell memory T cells compared to the starting population of T cells. ＮＳＣＭ The method according to any one of claims 62 to 70, wherein the cells are shown.

72. The T cells collected in step (c) are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% more than the starting population of T cells. ＮＳＣＭ The method according to claim 71, comprising cells.

73. The T cells collected in step (c) showed a reduced amount of T compared to the starting population of T cells. ＥＭ The method according to any one of claims 62 to 72, comprising cells.

74. The T cells collected in step (c) are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less than the starting population of T cells. ＥＭ The method according to claim 73, comprising cells.

75. The method according to any one of claims 1 to 74, performed using ex vivo.

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