Method of expanding t cell population

Abstract

Provided herein are methods for making, expanding and / or producing genetically modified T cells comprising chimeric antigen receptors (CARs) or T cell receptors (TCRs).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 368,550, filed on July 15, 2022, which is incorporated herein by reference in its entirety.

[0003] References to sequence listings submitted electronically

[0004] The contents of the electronically submitted sequence listing submitted in this application (CARTSTEAP2-300-WO-PCT.xml; size: 81,152 bytes; and creation date: July 10, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] The generation of tumor-specific T lymphocytes genetically modified to express chimeric antigen receptors (CARs) is gaining attention as a form of synthetic biology that elicits powerful antitumor effects (Jena et al., 2010, Blood. 116: 1035-1044; Bonini et al., 2011, Biol Blood Marrow Transplant 17(1 Suppl): S15-20; Restifo et al., 2012, Nat Rev Immunol 12: 269-281; ​​Kohn et al., 2011, Mol Ther 19: 432-438; Savoldo et al., 2011, J Clin Invest 121: 1822-1825; Ertl et al., 2011, Cancer Res 71: 3175-3181). Because specificity is conferred by antibody fragments, CAR-T cells are not MHC-restricted and are therefore more practical than T cell receptor-based approaches that require MHC matching.

[0006] Therefore, CAR-T cell therapy represents a major advance in personalized cancer treatment. In this strategy, the patient's own T cells are genetically engineered to express synthetic receptors that bind to tumor antigens. The CAR-T cells are then expanded for clinical use and infused back into the patient to attack and destroy chemotherapy-resistant cancers. Significant clinical responses and high complete remission rates have been observed in the context of CAR-T cell therapy for B-cell malignancies. This led to the FDA's recent approval of two CAR-T cells for the CD19 protein for the treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. Therefore, CAR-T cells are arguably one of the first successful examples of synthetic biology and personalized cell cancer therapy becoming commercially available.

[0007] Despite the recent success of CAR-T cell therapy, there remains a need in the art for better methods of T cell expansion. Summary of the invention

[0008] The present disclosure relates to a method for expanding a T cell population, the method comprising: (a) isolating CD3+T cells from a sample; (b) culturing CD3+T cells in a culture medium comprising human interleukin 21 (IL-21); (c) activating CD3+T cells; (d) transducing CD3+T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) to produce CAR-T cells or T cell receptor (TCR) cells; (e) culturing CAR-T cells in a culture medium; and (f) harvesting CAR-T cells or T cell receptor (TCR) cells. The present disclosure also relates to a method for manufacturing a T cell therapeutic agent, which comprises: (a) obtaining a sample comprising a CD3+T cell population; (b) culturing CD3+T cells in a culture medium comprising human interleukin 21 (IL-21); (c) activating CD3+T cells; (d) transducing CD3+T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) to produce CAR-T cells or T cell receptor (TCR) cells; (e) culturing CAR-T cells or T cell receptor (TCR) cells in a culture medium; and (f) harvesting CAR-T cells or T cell receptor (TCR) cells. The present disclosure also relates to a method for expanding a T cell group, the method comprising: (a) separating CD4+ and CD8+T cells from a sample to form a CD3+T cell group; (b) culturing CD3+T cells in a culture medium containing human interleukin 21 (IL-21); (c) activating CD3+T cells; (d) transducing CD3+T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) to produce CAR-T cells or T cell receptor (TCR) cells; (e) culturing CAR-T cells or T cell receptor (TCR) cells in a culture medium; and (f) harvesting CAR-T cells or T cell receptor (TCR) cells. In some aspects, the culture medium also includes human interleukin 2 (IL-2).

[0009] In some aspects, part (d) comprises transducing CD3+T cells with a vector comprising a nucleic acid encoding a CAR to produce a CAR-T cell. In some aspects, part (d) comprises transducing CD3+T cells with a vector comprising a nucleic acid encoding a TCR to produce a TCR cell. In some aspects, about 1×10 6 to about 1×10 9CD3+T cells. In some aspects, the sample is an enriched single collection product collected via leukocyte apheresis. In some aspects, the CD3+T cells in step (c) are cultured for about one day or about two days. In some aspects, the CD3+T cells in step (c) are activated with an agonist of CD2, CD3, CD28 or any combination thereof. In some aspects, the CD3+T cells in step (c) are activated with magnetic microbeads. In some aspects, the CD3+T cells in step (c) are activated with anti-CD3 antibodies or their CD3 binding fragments and anti-CD28 antibodies or their CD28 binding fragments. In some aspects, the anti-CD3 antibodies or their CD3 binding fragments and the anti-CD28 antibodies or their CD28 binding fragments are coupled with magnetic microbeads. In some aspects, CAR-T cells or TCR cells are cultured in step (e) for about two days to about ten days. In some aspects, CAR-T cells or TCR cells are cultured in step (e) for about four days to about six days. In some aspects, CAR-TT cells are cultured in step (e) for about four days. In some aspects, CAR-T cells or TCR cells are cultured in step (e) for about six days. In some aspects, the concentration of human IL-21 is about 0.01U / mL to about 0.3U / mL, and the concentration of human IL-2 is about 5IU / mL to about 100IU / mL. In some aspects, the concentration of human IL-21 is about 0.19U / mL. In some aspects, the concentration of human IL-2 is about 40IU / mL. In some aspects, CD3+T cells are stirred during step (b). The method disclosed herein relates to a method for manufacturing a T cell therapeutic agent, which comprises: (a) separating CD4+ and CD8+ T cells from a sample to form a CD3+ T cell population; (b) culturing CD3+ T cells in a culture medium comprising human interleukin 2 at a concentration of 40 IU / mL and human interleukin 21 at a concentration of 0.19 U / mL; (c) activating CD3+ T cells with magnetic beads comprising anti-CD3 antibodies or CD3 binding fragments thereof and anti-CD28 antibodies or CD28 binding fragments thereof; (d) virally transducing CD3+ T cells with a lentiviral vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) to produce CAR-T cells; (e) culturing the CAR-T cells in a culture medium for about four days; and (f) harvesting the CAR-T cells.

[0010] In some aspects, CD4+ and CD8+T cells are separated by positive selection. In some aspects, the vector is a virus, a slow virus, an adenovirus, a retrovirus, an adeno-associated virus (AAV), a transposon, a DNA vector, an mRNA, a lipid nanoparticle (LNP) or a CRISPR-Cas system. In some aspects, the vector is a slow virus. In some aspects, a slow virus is added with a multiplicity of infection (MOI) of about 0.25 to about 20. In some aspects, a slow virus is added with an MOI of about 1 to about 4. In some aspects, a slow virus is added with an MOI of about 2 or about 4. In some aspects, the volume of the cell culture medium increases after step (d). In some aspects, the volume of the cell culture medium increases by at least 6 times.

[0011] In some aspects, the culture medium in step (e) is replaced at least once a day. In some aspects, the culture medium in step (e) is replaced every 12 hours. In some aspects, the CAR-T cells or TCR cells expand at least about 1 times to about 5 times during step (e). In some aspects, the CAR-T cells or TCR cells expand at least about 1 times to about 3 times during step (e). In some aspects, the CAR-T cells or TCR cells expand about 2 times during step (e). In some aspects, the CAR-T cells or TCR cells expand about 3 times during step (e). In some aspects, CAR binds to STEAP2 or Glypican-3 (GPC3). In some aspects, CAR encodes an antigen binding domain that binds to STEAP2, and wherein the antigen binding domain comprises:

[0012] (a) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5

[0013] CDR2, VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:6;

[0014] (b) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13

[0015] , a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a VH-

[0016] CDR3;

[0017] (c) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 21, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 23

[0018] , a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 24, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 26

[0019] CDR3;

[0020] (d) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 32, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 33

[0021] , a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 34, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36

[0022] CDR3; or

[0023] (e) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 41, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 42, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 43

[0024] , a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 44, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 45, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 46, a VH-

[0025] CDR3. In some aspects, the CAR comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:9. In some aspects, the CAR encodes an antigen binding domain that binds to GPC3, and wherein the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising an amino acid sequence of SEQ ID NO: 112, a CDR2 comprising an amino acid sequence of SEQ ID NO: 113, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 114, and wherein the VL comprises: a CDR1 comprising an amino acid sequence of SEQ ID NO: 115 or SEQID NO: 118, a CDR2 comprising an amino acid sequence of SEQ ID NO: 116 or SEQID NO: 119, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 117 or SEQID NO: 120. In some aspects, the VH comprises an amino acid sequence of SEQID NO: 108 or SEQ ID NO: 110, and the VL comprises an amino acid sequence of SEQID NO: 109 or SEQ ID NO: 111. In some aspects, the nucleic acid also encodes an armoring molecule.

[0026] In some aspects, the armor molecule comprises a dominant negative TGFβ receptor type 2 (TGFβRIIDN). In some aspects, the armor molecule comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 105. In some aspects, the armor molecule comprises the amino acid sequence shown in SEQ ID NO: 105. In some aspects, the CAR-T cells or TCR cells are formulated in an isotonic solution. In some aspects, the isotonic solution comprises a plasmalyte containing human serum albumin. In some aspects, the isotonic solution contains about 1×10 6 to about 1×10 9 CAR-T cells or TCR cells. In some aspects, the isotonic solution contains about 3.4×10 6 In some aspects, the CAR-T cell or TCR cell is a T CM Cells and T SCMIn some aspects, about 20% to about 50% of the CAR-T cells or TCR cells express CD45RA, CCR7, and CD27 but do not express CD45RO. In some aspects, about 20% to about 30% of the CAR-T cells or TCR cells are T cells. SCM Cells, and express CD45RA, CCR7 and CD27 but do not express CD45RO. In some aspects, more than 50% of CAR-T cells or TCR cells express chimeric antigen receptors or T cell receptors. In some aspects, about 40% to about 60% of CAR-T cells or TCR cells express chimeric antigen receptors or T cell receptors. In some aspects, more than 50% of CAR-T cells or TCR cells express CD8. In some aspects, about 40% to about 60% of CAR-T cells or TCR cells express CD8.

[0027] In some aspects, CAR-T cells or TCR cells have an oxygen consumption rate (OCR) higher than 100pmol / min. In some aspects, CAR-T cells or TCR cells have an OCR of about 50pmol / min to about 200pmol / min. In some aspects, CAR-T cells or TCR cells have an extracellular acidification rate (ECAR) higher than 30mpH / min. In some aspects, CAR-T cells or TCR cells have an ECAR of about 30mpH / min to about 60mpH / min. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A-Figure 1D It was shown that GPC3 CAR-T cells expanded under IL-10 or IL-21 alone were less activated than IL-2. Figure 1C-Figure 1D Cells are shown to be enriched for CAR in the presence of IL-21 alone + and CD8 + cell.

[0029] Figure 2A-2F . Figure 2A-2B It was shown that the cell growth (population doubling time) and cell viability of GPC3 CAR-T cells expanded for 8 days in cell culture medium supplemented with IL-2 alone, IL-21 alone, or a combination of IL-2 and IL-21 were similar. Figure 2C and Figure 2D It was shown that CD4GPC3CAR-T cells and CD8GPC3CAR-T cells expanded in cell culture medium supplemented with IL-21 alone or a combination of IL-2 and IL-21 were less differentiated than those supplemented with IL-2 alone. Figure 2E and Figure 2FWe showed that GPC3 CAR-T cells were able to further robustly expand beyond day 8 to day 13 when cultured in medium supplemented with IL-2 alone or a combination of IL-2 and IL-21.

[0030] Figure 3A-Figure 3F . Figure 3A-3B It was shown that the cell growth (population doubling time) and cell viability of GPC3 CAR-T cells expanded for 8 days in cell culture medium supplemented with IL-2 alone, IL-21 alone, or a combination of IL-21 in a concentration range of 2 ng / mL-10 ng / mL and IL-2 in a concentration range of 25 IU / mL to 100 IU / mL were similar. Figure 3C The percentages of GPC3 CAR+ cells in CD4 T cells and CD8 T cells were shown to be similar between different IL-2 and IL-21 concentrations. Figure 3D and Figure 3F It was shown that high IL-2 concentrations (>=50 IU / mL) would mask the effects of IL-21 on GPC3 CAR-T cells. Figure 3F It was shown that low IL-2 concentration (25 IU / mL) together with IL-21 could enrich CD8 T cells during GPC3 CAR-T cell expansion.

[0031] Figure 4A-4B For CAR-T cells expressing STEAP2 and GPC3, 1×10 9 Seeds were used for T cell expansion.

[0032] Figure 5A-Figure 5B For CAR-T cells expressing STEAP2 and GPC3, 1×10 9 T cell activity of each seed.

[0033] Figure 6A-6B The 4-day SMART bioproduction process was shown to consistently produce the minimum target of 30% CAR+ cells.

[0034] Figure 7A-7B We show that a 4-day SMART bioproduction process can consistently produce 400 × 10 6 The dose of CAR+T cells.

[0035] Figure 8A-B It was shown that the 4-day SMART process produced highly pure CD3+ T cells with a purity greater than 98% in both STEP2-expressing CAR-T cells and GPC3-expressing CAR-T cells.

[0036] Figure 9A-9C The percentage of STEAP2 CAR expression and the percentage of TGFβRII armor expression are shown. Figure 9A-9BShowing high levels of CAR expression and TGFβRII in cells treated on day 4. Day 4 cells showed a linear correlation between CAR and TGFβRII expression. Fig. 9C The percentage of GPC3 CAR expression in cells treated on day 4 is shown.

[0037] Fig.10 The differentiation curves of live CAR+T cells are shown, and central memory (T CM )(CCR7+CD45RO+) was the dominant phenotype of CAR-positive T cells harvested on day 6, while CAR-T cells harvested on day 4 showed stem cell memory (T SCM ) and T CM Phenotypes of both.

[0038] Figure 11A-11B Activation and exhaustion curves of live CAR+ T cells are shown. CAR+ T cells showed increased late activation (CD25+), which was reduced in cells harvested on day 6 compared to cells harvested on day 4 ( Fig.11A The percentage of cells expressing exhaustion markers was less than 4% for PD1 / LAG3 / Tim3 double positive and less than 1% for PD1 / LAG3 / Tim3 triple positive ( Fig. 11B ).

[0039] Figure 12A-12B Display STEAP2( Fig. 12A )CAR + T cells and GPC3 ( Fig. 12B )CAR + T cells exhibited killing activity against target-positive cell lines over a range of E:T ratios.

[0040] Figure 13A-13B The results show that STEAP2 ( Fig.13A )CAR-T cells or GPC3( Fig. 13B ) IFN-γ, TNF-α, and IL-2 cytokine release during target activation of CAR-T cells with target-expressing cells. Cytokine release between cells harvested on day 4 or day 6, such as Fig.13A shown.

[0041] Figure 14A-14E The results showed that SMART GPC3-CAR+T cells showed higher expression of stemness markers and effector function. In addition, SMART cells showed fewer T cell exhaustion markers.

[0042] Figures 15A-15DA schematic diagram of OCR over time is shown. Basal respiration is the energy demand of the cell under baseline conditions; Maximum respiration: respiratory chain working at maximum capacity → maximum respiratory rate that the cell can reach; and Spare respiratory capacity (SRC): the ability of the cell to respond to energy demands or under stress → cell fitness indicator ΔOCR = OCR max -OCR 基础 . Figures 15A-15D SMART CAR-T cells were shown to have higher SRC than 12-day TNT CAR-T cells, indicating increased mitochondrial energy reserves, adaptability, and compliance.

[0043] Figures 16A-16D A schematic diagram of ECAR is shown. Glycolysis is the rate of glucose consumption at rest; Glycolytic capacity is the maximum extracellular acidification (ECAR) rate after oxidative phosphorylation stops → the cell uses glycolysis to its maximum capacity; and Glycolytic reserve is the cell's ability to glycolyze in response to energy demands or under stress. Figures 16A-16D The 4-day SMART CAR-T cells were shown to have a higher glycolytic reserve than the 12-day TNT CAR-T cells, indicating an increased ability to perform glycolysis in response to energy demands.

[0044] Figure 17A-17B It was shown that SMART STEAP2-CAR+T cells exhibited a higher level of CAR expression on day 4.

[0045] Figure 18A-18E The results showed that SMART GPC3-CAR+T cells showed significant effects on effector cytokines ( Fig.18A )IFN-γ;( Fig.18B )IL-2; and ( Fig. 18C )Antigen-specific secretion of IL-21 is increased. Figure 18D-18E It was shown that in two different donors, the SMART GPC3 CAR demonstrated enhanced tumor control and increased expansion levels in a serial killing assay compared to the traditional (TNT) approach.

[0046] Figure 19A-19E Display GPC-3SMART CAR + T cells demonstrated dose-dependent tumor control in vivo, including TNT and SMART untransduced (UT) controls ( Fig.19B ). Figure 19C-Figure 19D Display 3×10 6 and 6×10 6 Dose curve of IFN-γ. Fig.19E SHOWS STEAP2SMART CAR +T cells demonstrated dose-dependent tumor control (300,000 cells to 6 million cells).

[0047] Figure 20A-20B Shown is that the CD4 / CD8 ratio is maintained during cell expansion in prostate cancer (B) compared to healthy donors (A).

[0048] Figure 21A-21B Prostate cancer STEAP2 CAR-T cells were shown to be less differentiated, as indicated by CD62L / CD45RO expression.

[0049] Fig. 22 Shown is the in vivo efficacy of SMART CAR-T administration on tumor volume in the NSG mouse model.

[0050] Fig.23 Shown in vivo efficacy of SMART CAR-T administered versus traditional 12-day approach CAR-T on tumor volume in NSG MHC class 1 / 2 knockout mouse model minimizing GvHD.

[0051] Figure 24A-Figure 24D It was shown that 4-day SMART CAR-T had a higher SRC than 11-day TNT CAR-T.

[0052] Figure 25A-Figure 25D showed that higher concentrations of carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone (FCCP) in 4-day-old SMART CAR-T cells resulted in a greater increase in the amount of OCR compared to 11-day-old TNT CAR-T cells.

[0053] Figure 26A-26D The results showed that SMART CAR-T cells at day 4 had increased glycolysis, glycolytic amount, and glycolytic reserve compared with TNT CAR-T cells at day 11. DETAILED DESCRIPTION

[0054] The present disclosure relates to methods for culturing T cells transduced with chimeric antigen receptors (CARs) that produce persistent T cell populations that exhibit increased antigen-independent activation.

[0055] In order to more easily understand the present disclosure, some terms are first defined. As used in this specification, unless otherwise explicitly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0056] It should be noted that the term "a" or "an" refers to one or more of the entity; for example, "a feed medium" should be understood to mean one or more feed media. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0057] As used herein, the term "and / or" should be understood to specifically disclose each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] It should be understood that wherever herein an aspect is described with the language "comprising," other similar aspects described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure for the skilled person.

[0060] Units, prefixes and symbols are expressed in their SI recognized form. Numerical ranges include numerical values ​​that limit the range. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire content of the specification.

[0061] The use of alternatives (eg, "or") should be understood to mean any one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated components.

[0062] The term "about" or "substantially comprising" means that a value or composition is within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to ±10%. In addition, particularly with respect to biological systems or processes, these terms may mean values ​​of up to an order of magnitude or up to 5 times. When a specific value or composition is provided in the present application and claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value or composition.

[0063] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range, and where appropriate, include fractions thereof (such as tenths and hundredths of integers).

[0064] The term "T cell" or "T lymphocyte" is recognized in the art and is intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, dormant T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subpopulation of T cells. Other illustrative T cell populations suitable for use in certain aspects include naive T cells and memory T cells.

[0065] As used herein, the term "proliferation" refers to an increase in cell division (symmetrical or asymmetrical cell division). In particular aspects, "proliferation" refers to the symmetrical or asymmetrical division of T cells. "Increased proliferation" occurs when the number of cells in a treated sample increases compared to cells in an untreated sample.

[0066] The term "expansion" in the methods of the present invention refers to the process of increasing the number of cells in a cell culture. In the expansion step, in one aspect, the cells are regularly fed and the culture medium is replaced according to a feeding regimen. The specific time and amount of adding culture medium in a particular feeding regimen will depend on the number of cells and metabolite levels in the culture.

[0067] As used herein, the term "differentiation" refers to a process of reducing the potential or proliferation of a cell or causing a cell to move to a more developmentally restricted state. In certain aspects, the differentiated T cells acquire immune effector cell function.

[0068] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Illustrative immune effector cells contemplated herein are T lymphocytes, particularly cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTL; CD4 + T cells).

[0069] "Modified T cells" refer to T cells that have been modified by introducing polynucleotides encoding the engineered CARs contemplated herein. Modified T cells include both genetic modifications and non-genetic modifications (e.g., episomal or extrachromosomal modifications).

[0070] As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell.

[0071] The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0072] As used herein, the term "gene therapy" refers to the introduction of additional genetic material in the form of DNA or RNA into the total genetic material in a cell, which restores, corrects or alters the expression of a gene, or is used to express a therapeutic polypeptide, such as a CAR and / or one or more cytokines. In a particular aspect, a T cell is modified to express an engineered TCR or CAR without modifying the genome of the cell, such as by introducing an additional vector expressing a CAR into the cell.

[0073] As used herein, "chimeric antigen receptor (CAR)" means a fusion protein comprising an extracellular domain capable of binding to a predetermined antigen, an intracellular segment comprising a cytoplasmic domain of a signaling protein derived from one or more polypeptides different from the extracellular domain, and a transmembrane domain. "Chimeric Antigen Receptor (CAR)" is sometimes referred to as a "chimeric receptor", "T-body" or "chimeric immune receptor (CIR)". The phrase "extracellular domain capable of binding to a predetermined antigen" means any protein molecule or portion thereof that can specifically bind to a predetermined antigen. "Intracellular signaling domain" means any oligopeptide or polypeptide domain known to function to transmit signals, causing activation or inhibition of biological processes in cells, such as activation of immune cells such as T cells. Examples include ILR chains, CD28 and / or CD3ζ.

[0074] As used herein, "lentivirus" refers to the genus of the Retroviridae family. Slow viruses are unique among retroviruses that can infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most effective methods of gene delivery vectors. HIV, SIV, and FIV are all examples of slow viruses. Vectors derived from slow viruses provide a means of achieving significant levels of gene transfer in vivo.

[0075] The term "ex vivo" generally refers to an activity occurring outside an organism, such as an experiment or measurement performed in or on a living tissue in an artificial environment outside an organism, preferably with minimal natural condition changes. In a particular aspect, an "ex vivo" procedure involves obtaining from an organism and cultivating or regulating living cells or tissues in a laboratory device, usually under aseptic conditions, and usually lasting for a few hours or about 24 hours at most, but including 48 or 72 hours at most, depending on the situation. In some aspects, such tissues or cells can be collected and frozen, and then thawed for ex vivo processing. Using tissue culture experiments or procedures that last more than a few days using living cells or tissues is generally considered to be "in vitro", but in some aspects, the term can be used interchangeably with ex vivo.

[0076] The term "in vivo" generally refers to activities that occur inside an organism, such as cell self-renewal and cell expansion. In one aspect, the term "in vivo expansion" refers to the ability of a cell population to increase in number in vivo.

[0077] The acronym "SMART" (Short-term manipulated autonomously replicating T cells) refers to a T cell expansion process in which cells are cultured in the presence of IL-2 and IL-21.

[0078] The acronym "TNT" (traditional cultured T cells) refers to traditional T cell expansion methods that do not employ IL-21 and typically involve culturing cells for more than 7 days and / or typically involve the use of IL-2.

[0079] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (eg, TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, including but not limited to signal transduction via the TCR / CD3 complex.

[0080] "Stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand.

[0081] As used herein, "stimulatory ligand" means a ligand that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response of the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include but are not limited to CD3 ligands (e.g., anti-CD3 antibodies) and CD2 ligands (e.g., anti-CD2 antibodies) and peptides (e.g., CMV, HPV, EBV peptides).

[0082] The term "activation" refers to a T cell state that has been fully stimulated to induce detectable cell proliferation. In particular aspects, activation can also be associated with induced cytokine production and detectable effector functions. The term "activated T cell" refers in particular to a T cell that is proliferating. The signal produced by a single TCR is not enough to fully activate the T cell, and one or more secondary or costimulatory signals are also required. Therefore, T cell activation includes a primary stimulation signal and one or more secondary costimulatory signals through a TCR / CD3 complex. Costimulation can be demonstrated by the proliferation and / or cytokine production of T cells, and these T cells have received primary activation signals, such as stimulation by CD3 / TCR complexes or by CD2.

[0083] A "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR / CD3 ligation), results in T cell proliferation, cytokine production, and / or up- or down-regulation of specific molecules (eg, CD28).

[0084] A "costimulatory ligand" refers to a molecule that binds to a costimulatory molecule. A costimulatory ligand can be soluble or provided on a surface. A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand (e.g., an anti-CD28 antibody).

[0085] As used herein, "autologous" refers to cells derived from the same subject.

[0086] As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared.

[0087] As used herein, "isogenic" refers to cells from a different subject that are genetically identical to the cells being compared.

[0088] As used herein, "allogeneic" refers to cells of a different species than the cell being compared. In a preferred aspect, the cells of the invention are allogeneic.

[0089] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits cancer symptoms that can be treated with the gene therapy vectors, cell-based therapeutic agents and methods disclosed elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits or guinea pigs), farm animals, and livestock or pets (such as cats or dogs). Non-human primates are included, and preferably human patients are included. Typical subjects include human patients who have cancer, have been diagnosed with cancer, or are at risk of cancer or are suffering from cancer.

[0090] "Enhancement" or "promote" or "increase" or "enlargement" generally refers to the ability of the compositions considered herein to produce, induce or cause a greater physiological response (i.e., downstream effect) compared to the response caused by the vehicle or control molecule / composition. Measurable physiological responses may include an increase in T cell expansion, activation, persistence, and / or an increase in cancer cell death and killing ability, as well as other factors that are apparent from the understanding of the art and the description herein. An "increased" or "enhanced" amount is generally a "statistically significant" amount, and may include an increase in the response produced by a vehicle or control composition by 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500 times, 1000 times) (including all integers and decimal points between and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0091] "Reduce" or "lower" or "mitigate" or "lower" or "attenuate" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a lower physiological response (i.e., downstream effect) compared to the response elicited by a vehicle or control molecule / composition. A "reduced" or "reduced" amount is generally a "statistically significant" amount and can include a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500-fold, 1000-fold) reduction in the response produced by a vehicle, a control composition (reference response), or in a specific cell lineage (including all integers and decimal points between and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0092] "Maintain" or "maintain" or "no change" or "no substantial change" or "no substantial reduction" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a lower physiological response (i.e., downstream effect) in a cell compared to the response elicited by vehicle, a control molecule / composition, or a response in a particular cell lineage. An equivalent response is one that is not significantly different or measurably different from a reference response.

[0093] Source of T cells

[0094] Prior to T cell amplification and gene modification of the present invention, a T cell source is obtained from a subject. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present invention, any number of T cell lines available in the art can be used. In certain aspects of the present invention, T cells can be obtained using many techniques known to those skilled in the art, such as Ficoll. TM Separation, obtained from a unit of blood collected from a subject. In one aspect, cells from individual circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected by apheresis can be washed to remove plasma fractions and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In one aspect of the invention, cells are washed with phosphate buffered saline (PBS). In an alternative aspect, the washing solution lacks calcium and may lack magnesium or may lack many (if not all) divalent cations. Similarly, the initial activation step in the absence of calcium leads to amplified activation. As will be readily understood by those of ordinary skill in the art, the washing step can be completed by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free 2+ , Mg-free 2+ Alternatively, the apheresis may be stripped of unwanted components and the cells resuspended directly in culture medium.

[0095] In another aspect, the erythrocytes are lysed and the monocytes are depleted, for example by PERCOLL TM Isolate T cells from peripheral blood lymphocytes by gradient centrifugation or by counterflow centrifugal elutriation. Specific subsets of T cells, such as CD3 +、CD28 + 、CD4 + 、CD8 + 、CD45RA + and CD45RO + T cells can be further separated by positive or negative selection techniques. In some aspects, T cells are separated by positive selection for CD4 and CD8 expression. For example, in one aspect, T cells are separated by hatching the time period of the T cells expected by positive selection with the beads conjugated with anti-CD4 / anti-CD8. In one aspect, the time period is about 30 minutes. In another aspect, the time period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In another aspect, the time period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. In yet another aspect, the time period is 10 hours to 24 hours. In any case where there are only a few T cells compared with other cell types, for example, when separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals, longer incubation times can be used to separate T cells. In addition, longer incubation times can be used to increase the capture efficiency of CD8+T cells. Thus, by simply shortening or extending the time that T cells are allowed to bind to the CD4 / CD8 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), T cell subsets can be preferentially selected at the beginning of the culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD4 and / or anti-CD8 antibodies on beads or other surfaces, T cell subsets can be preferentially selected at the beginning of the culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In some aspects, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and amplification process. "Unselected" cells can also be subjected to other rounds of selection.

[0096] Enrichment of T cell populations by negative selection can be accomplished with a combination of antibodies against surface markers unique to the negatively selected cells. One approach is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4 + Cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, and HLA-DR. In some aspects, it may be desirable to enrich or positively select for cells that normally express CD4 + 、CD25 + 、CD62L hi GITR + and FoxP3 +Alternatively, in certain aspects, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0097] In order to separate the desired cell population by positive selection or negative selection, the concentration of cells and surfaces (for example, particles such as beads) can be changed. In some aspects, it may be desirable to significantly reduce the volume (that is, increase the concentration of cells) that beads and cells are mixed together to ensure the maximum contact of cells and beads. For example, in one aspect, a concentration of 2 billion cells / ml is used. In one aspect, a concentration of 1 billion cells / ml is used. In another aspect, greater than 100 million cells / ml is used. In another aspect, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml is used. In yet another aspect, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml is used. In other aspects, a concentration of 125 million or 150 million cells / ml can be used. Use of high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0098] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects cells that express large amounts of the desired antigen to be bound to the particles. For example, with a dilute concentration of CD8 + Compared with T cells, CD4 + T cells express higher levels of CD28 and are captured more efficiently. In one aspect, the cell concentration used is 5×10 6 / ml. In other aspects, the concentration used can be about 1×10 5 / ml to 1×10 6 / ml, and any integer values ​​in between.

[0099] In other aspects, cells can be incubated at 2°C-10°C or at room temperature on a rotator at different speeds for different lengths of time.

[0100] The T cells used for stimulation can also be frozen after the washing steps. In some aspects, the freezing and subsequent thawing steps can provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing steps to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1° / minute and storing in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0101] In certain aspects, cryopreserved cells are thawed and washed and allowed to stand at room temperature for 1 hour prior to activation using the methods of the invention.

[0102] In the context of the present invention, it is also considered that a blood sample or a single sampling product may be collected from a subject for a period of time before the cells of amplification as described herein may be needed. Therefore, the source of the cells to be amplified can be collected at any necessary time point, and the desired cells (such as T cells) are separated and frozen for later use in the T cell therapy of many diseases or conditions (such as those described herein) that will benefit from T cell therapy. In one aspect, a blood sample or a single sampling of blood components is taken from a subject with overall health. In some aspects, a blood sample or a single sampling of blood components is taken from a subject with overall health who is in the risk of developing a disease but has not yet developed a disease, and the cells of interest are separated and frozen for later use. In some aspects, T cells can be amplified, frozen and used at a later time. In some aspects, samples are collected from patients shortly after diagnosing a specific disease as described herein but before any treatment. In another aspect, cells are isolated from a blood sample or apheresis of a subject prior to a number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents (such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228), and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993). In another aspect, the cells are isolated for use in patients and frozen for later use in combination with (e.g., before, at the same time, or after) bone marrow or stem cell transplantation, T cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another aspect, the cells are isolated prior to B cell ablative therapy (such as an agent reactive with CD20, e.g., Rituxan), and the cells can be frozen for later use in treatment.

[0103] In another aspect of the present invention, T cells are obtained directly from patients after treatment. In this regard, it has been observed that after certain cancer treatments, especially after treatment with drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved for their ability to expand in vitro during normal recovery from treatment in the patient soon after treatment. Similarly, after using the method described herein to manipulate in vitro, these cells can be in a preferred state for enhancing implantation and in vivo expansion. Therefore, in the context of the present invention, it is considered to collect blood cells during the recovery period, including other cells of T cells, dendritic cells or hematopoietic lineages. In addition, in some aspects, mobilization (for example, mobilization with GM-CSF) and conditioning schemes can be used to produce in a subject conditions that are conducive to the re-growth, recirculation, regeneration and / or expansion of specific cell types, especially during the limited time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0104] T cell activation and expansion

[0105] Whether before or after genetically modifying T cells to express the desired CAR, T cells can generally be activated and expanded using methods such as those described in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0106] Typically, the T cells of the present invention are expanded by contacting with a surface of a ligand attached with an agent that stimulates a CD3 / TCR complex-related signal and a co-stimulatory molecule on the surface of the T cell. Specifically, a T cell population can be stimulated as described herein, such as by contacting an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody fixed on the surface, or by contacting a protein kinase C activator (e.g., bryostatin) together with a calcium ion carrier. In order to costimulate the auxiliary molecules on the surface of the T cells, a ligand that binds the auxiliary molecule is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. In order to stimulate CD4 + T cells or CD8 +Proliferation of T cells is carried out using anti-CD3 antibodies and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besangon, France). Other methods known in the art may also be used (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0107] In some aspects, the primary stimulation signal and the costimulatory signal of T cell can be provided by different schemes.For example, the medicament providing each signal can be in solution or coupled to the surface.When coupled to the surface, the medicament can be coupled to the same surface (that is, in "cis" form) or to a separate surface (that is, in "trans" form). Alternatively, a kind of medicament can be coupled to the surface, and another medicament is in solution.In one aspect, the medicament providing the costimulatory signal is combined with the cell surface, and the medicament providing the primary activation signal is in solution or coupled to the surface.In some aspects, both medicaments can be in solution.In another aspect, the medicament can be in soluble form, then cross-linked to the surface, such as cells or antibodies expressing Fc receptors or other binding agents that will be combined with the medicament.In this regard, referring to, for example, artificial antigen presenting cells (aAPC) in U.S. Patent Application Publication Nos. 20040101519 and 20060034810, it is considered that these cells are used for activation and amplification of T cells in the present invention.

[0108] In one aspect, two agents are immobilized on the beads, either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead at equal molecular weight. In one aspect, for CD4 + T cell expansion and T cell growth using a 1:1 ratio of each antibody bound to the beads. In certain aspects of the invention, a ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio.

[0109] In other aspects of the present invention, cells (such as T cells) are combined with agent-coated beads, beads and cells are subsequently separated, and cells are then cultured. In an alternative aspect, before culture, agent-coated beads and cells are not separated, but cultured together. In another aspect, beads and cells are first concentrated by applying a force (such as a magnetic force), causing an increase in the connection of cell surface markers, thereby inducing cell stimulation.

[0110] Conditions suitable for T cell culture include an appropriate culture medium (e.g., minimum essential medium or RPMI medium 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), IL-21, insulin, IFN-7, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other additives known to the technician for cell growth. Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder (plasmanate) and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, which are supplemented with amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of defined hormones, and / or cytokines sufficient for the growth and expansion of T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures, but not in the cultures of cells to be infused into the subject. Target cells are maintained under conditions necessary to support growth, e.g., appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2). In one aspect, the culture medium is an X-VIVO 15 serum-free culture medium containing 1% (v / v) recombinant serum replacement (ITSE-A).

[0111] In one aspect, T cells are cultured in a culture medium containing 10IU / mL to 100IU / mL recombinant human IL-2. In one aspect, T cells are cultured in a culture medium containing 10IU / mL, 15IU / mL, 20IU / mL, 25IU / mL, 30IU / mL, 35IU / mL, 40IU / mL, 45IU / mL, 50IU / mL, 55IU / mL, 60IU / mL, 65IU / mL, 70IU / mL, 75IU / mL, 80IU / mL, 85IU / mL, 90IU / mL, 95IU / mL or 100IU / mL recombinant human IL-2. In another aspect, T cells are cultured in a culture medium also containing 0.1U / mL to 0.3U / mL recombinant IL-21. In another aspect, T cells are cultured in medium containing IL-2 and 1 U / mL, 2 U / mL, 5 U / mL, 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 75 U / mL or 100 U / mL of recombinant human IL-21. In another aspect, T cells are cultured in the culture medium containing IL-2 and 0.10U / mL, 0.11U / mL, 0.12U / mL, 0.13U / mL, 0.14U / mL, 0.15U / mL, 0.16U / mL, 0.17U / mL, 0.18U / mL, 0.19U / mL, 0.20U / mL, 0.21U / mL, 0.22U / mL, 0.23U / mL, 0.24U / mL, 0.25U / mL, 0.26U / mL, 0.27U / mL, 0.28U / mL, 0.29U / mL or 0.30U / mL recombinant human IL-21. In one aspect, T cells are cultured in the culture medium containing 40IU / mL recombinant human IL-2 and 0.19U / mL recombinant human IL-21.

[0112] In one aspect of the invention, the cells are cultured for up to 14 days. In another aspect, the mixture can be cultured for 4 days. The T cells can be stirred at any stage of the culture. In one aspect, the cells are stirred in a medium containing IL-2 and IL-21 during cell culture. In some aspects, the T cells harvested on day 4 exhibit higher target-independent killing activity compared to the CAR-T cells harvested on day 6.

[0113] Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)

[0114] TCR engineered T cells express tumor antigen-specific receptors with alpha and beta chains that are produced by high-quality and high-affinity antigen-specific T cell clones. Individually, CARs are recombinant receptors for antigens that redirect the specificity and function of T lymphocytes and other immune cells in a single molecule. The general premise for their use in cancer immunotherapy is the rapid generation of tumor-targeted T cells, bypassing barriers and the incremental dynamics of active immunity. Once expressed in T cells, CAR-modified T cells acquire supraphysiological properties that can exert both immediate and long-term effects. Engineering CARs into T cells requires culturing T cells to allow transduction and expansion. Transduction can utilize a variety of methods, but stable gene transfer is required to achieve persistent CAR expression in clonal expansion and persistent T cells. In principle, any cell surface molecule can be targeted by CAR, thereby transcending tolerance to self-antigens and antigen recognition gaps in the physiological T cell repertoire that limit the range of T cell reactivity.

[0115] However, redirecting immune reactivity to selected antigens is not the only purpose of smarter CARs, which are designed to achieve much more than targeting and priming T cell activation. CARs with different strengths and qualities of signaling have the potential to modulate T cell expansion and persistence, as well as the intensity of T cell activation in the tumor microenvironment, significantly changing the characteristics of efficacy and safety of tumor-targeted T cells.

[0116] Depending on the desired antigen to be targeted, the CAR of the present disclosure can be engineered to include an appropriate antigen binding portion specific for the desired antigen target. In one aspect, the CAR specifically recognizes STEAP2 or Glypican-3 (GPC3).

[0117] Table 1: Sequences

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] armored element

[0127] Disclosed herein are polynucleotides comprising (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen binding domain, and (b) a nucleotide sequence encoding an armor molecule. A method for preparing CAR-T cells that are more resistant to tumor-related immunosuppression is called "armor". Armor is a molecular manipulation of CAR-T cells to express one or more "armor molecules" that can counteract immunosuppression. For example, researchers reported modifying CAR-T cells to secrete a single-chain variable fragment (scFv) that blocks PD-1, which improves the anti-tumor activity of CAR-T cells in mouse models of PD-L1+ hematological tumors and solid tumors (Rafiq, S., Yeku, O., Jackson, H. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 36, 847–856 (2018)). Other studies have demonstrated the effectiveness of armoring T cells with dominant negative TGFβ receptor type 2 (TGFβRIIDN) armor molecules to neutralize the inhibitory effects of TGFβ on T cells (Bollard et al., Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma, J Clin Oncol 36(11):1128-1139(2018)). Currently, at least one clinical study is investigating the effectiveness of armoring anti-PSMA-CAR-T cells with TGFβRIIDN armor molecules for the treatment of castration-resistant prostate cancer (NCT03089203).

[0128] In some aspects, the armor molecule comprises a dominant negative TGFβ receptor type 2 (TGFβRIIDN). In some aspects, the armor molecule comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 105. In some aspects, the armor molecule comprises the amino acid sequence shown in SEQ ID NO: 105.

[0129] Metabolic testing of CAR-T cells or TCR cells

[0130] In some aspects, using Assays to measure the metabolic activity of CAR-T cells or TCR cells. The assay measures the extracellular flux of OCR and ECAR. OCR reflects the rate at which cells consume oxygen during oxidative phosphorylation, a process that occurs in the mitochondria. ECAR measures the production of protons caused by glycolysis, a metabolic pathway that produces energy from glucose. In some aspects, CAR-T cells or TCR cells are added to a specialized microplate with wells containing sensors for detecting changes in OCR and ECAR. The cells are exposed to experimental conditions, such as varying concentrations of a drug or metabolic substrate, and OCR and ECAR are measured at intervals. In some aspects, 0.5 μM FCCP is used for In some aspects, 2 μM FCCP is used for Assay. In some aspects, CAR-T cells or TCR cells have an OCR higher than 100pmol / min. In some aspects, CAR-T cells or TCR cells have an OCR higher than 40pmol / min. In some aspects, CAR-T cells or TCR cells have an OCR higher than 150pmol / min. In some aspects, CAR-T cells or TCR cells have an OCR of about 50pmol / min to about 200pmol / min. In some aspects, CAR-T cells or TCR cells have an ECAR higher than 30mpH / min. In some aspects, CAR-T cells or TCR cells have an ECAR higher than 50mpH / min. In some aspects, CAR-T cells or TCR cells have an ECAR higher than 30mpH / min. In some aspects, CAR-T cells or TCR cells have an ECAR of about 30mpH / min to about 60mpH / min.

[0131] Example

[0132] The foregoing description of specific aspects will so fully reveal the general nature of the invention that others can easily modify and / or adapt such specific aspects for various applications by applying the knowledge in the art without undue experimentation and without departing from the general concepts of the invention. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects. It should be understood that the wording or terminology herein is for the purpose of description rather than limitation, so that the terms or wording of this specification will be interpreted by the skilled person in accordance with the teachings and guidance.

[0133] Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0134] Example 1: IL-2 and IL-21

[0135] Amplification in the presence of IL-21 produces less differentiated cells and a higher proportion of CAR+CD8+ cells. Purified human T cells were inoculated in AIM-V culture medium containing 5% human serum, 1% penicillin-streptomycin (Invitrogen) and 1% antibiotic-antimycotic (Invitrogen) at a concentration of 0.2E6 cells / mL+ interleukin (IL)-2 (300IU / mL) (Peprotech). According to the manufacturer's protocol, T cells were activated with anti-CD3 / CD28 Dynabeads (Invitrogen). After 24 hours, lentivirus was added to the wells, and the plates were centrifuged at 2000g for 2 hours at 37°C. After centrifugation, the cells were washed and resuspended in fresh culture medium containing IL-2 (300IU / mL), IL-21 (10ng / mL, R&D Systems), IL-10 (10ng / mL, R&D Systems) and IL-15 (10ng / mL, R&D Systems) as shown. The plates were placed in a 37°C, 5% CO2 incubator and the cells were split as necessary to maintain a cell density of approximately 1E6 cells / mL. Cells were harvested after 10 days and analyzed by flow cytometry ( Figure 1A-Figure 1D ).

[0136] The combination of IL-2 and IL-21 produced better phenotype and long-term cell expansion in TNT cells harvested on day 8. Total T cells (CD4 and CD8) selected on day 0 were seeded at 1.5E6 viable cells / mL in X-VIVO 15 medium (Lonza) supplemented with 5% CTS serum replacement (Thermo Fisher) in a 125 mL shake flask with a working volume of 10% and agitation at 51 rpm. ImmunoCult CD3 / CD28 / CD2 T cell activator (Stemcell Technologies) was added to the cell culture at 25 μL / mL to activate T cells immediately after inoculation. After two days of culture in an incubator at 37°C and 5% CO2, GPC3 LVV was added to the cell culture at an MOI of 10, and the agitation rate was increased to 169 rpm to enhance LVV transduction. On day 3, 0.9E6 viable cells were transferred and cultured in 100 mL X-VIVO 15 medium + 5% (v / v) CTS serum replacement supplemented with only 100 IU / mL of IL-2 (Akron), or 10 ng / mL of IL-21 (Akron), or 25 IU / mL IL-2 and 10 ng / mL IL-21. On day 6, a second dose of IL-2 was added to each well at the same concentration as on day 3 without mixing. On day 8, 5E6 viable cells were passaged and cultured in 100 mL of the same medium with fresh cytokines at the same concentration. On day 10, a second dose of IL-2 was added to the cell culture. On days 8 and 13, cells were harvested for cell counting and analyzed by flow cytometry for the expression of CD3, CD4, CD8, GPC3 CAR, CD45RO, CD45RA, CD62L, and CCR7 ( Figure 2A-2F ).

[0137] High IL-2 concentrations can mask the effects of IL-21. On day 0, selected total T cells (CD4 and CD8) were inoculated at 1.5E6 viable cells / mL in X-VIVO 15 medium (Lonza) supplemented with 5% CTS serum replacement (Thermo Fisher) in a 125 mL shake flask with a working volume of 10% and agitation at 51 rpm. ImmunoCult CD3 / CD28 / CD2 T cell activator (Stemcell Technologies) was added to the cell culture at 25 μL / mL to activate T cells immediately after inoculation. After two days of culture in an incubator at 37°C and 5% CO2, GPC3 LVV was added to the cell culture at an MOI of 10, and the agitation rate was increased to 169 rpm to enhance LVV transduction. On day 3, 0.9E6 viable cells were transferred and cultured in 100 mL X-VIVO 15 medium + 5% (v / v) CTS serum replacement supplemented with 100 IU / mL IL-2 (Akron) alone, or 25 IU / mL IL-2 and 10 ng / mL IL-21, or 50 IU / mL IL-2 and 10 ng / mL IL-21, or 100 IU / mL IL-2 and 10 ng / mL IL-21, or 25 IU / mL IL-2 and 5 ng / mL IL-21, or 25 IU / mL IL-2 and 2 ng / mL IL-21. On day 6, a second dose of IL-2 at the same concentration as on day 3 was added to each well without mixing. On day 8, cells were harvested for cell counting and analyzed by flow cytometry (LSR Fortessa from BD Biosciences) for the expression of CD3, CD4, CD8, GPC3 CAR, CD45RO, CD45RA, CD62L, and CCR7 ( Figure 3A-Figure 3F ).

[0138] Example 2: SMART 4-day CAR-T cell culture process

[0139] Apheresis biological raw materials (BSM) from patients were received from clinical sites within a pre-determined collection window and shipped at 2°C-8°C. BSM were washed on a Cytiva Sefia S2000 using the Flexcell program to remove most RBCs and platelets and then formulated in 1:1 PlasmaLyte A (Baxter) with 5% (w / v) human serum albumin (HSA): CS10 was divided into two 70 mL / CS250 bags (OriGen) and cryopreserved using a controlled rate freezer before being stored in the gas phase of LN2.

[0140] Day 0: When manufacturing begins, half of the frozen leukopak is thawed under controlled conditions using PlasmaTherm (Plasma Therm) and CD4 plus CD8 T lymphocytes were isolated using GMP anti-CD4 and anti-CD8 CliniMACS beads (Miltenyi). After isolation, 1.0E+09 purified CD3 T cells were added to Miltenyi The tube group was placed in the culture chamber and on the same day, the cells were transfected with Miltenyi T cells at a ratio of v / v = 1:17.5 via CD3 / 28. TM The cells were activated and cultured overnight in 70 mL of complete X-VIVO 15 serum-free medium (Lonza) containing 1% (v / v) recombinant serum replacement (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21.

[0141] Day 1: The next day, transduce cells with lentiviral vectors at a predetermined multiplicity of infection. Two hours after adding lentivirus, add fresh cell culture medium to bring the cell culture volume to 250 mL.

[0142] Day 2 to Day 4: Cells were cultured and expanded continuously on days 2, 3, and 4. Every 12 hours, 180 mL of cell culture medium was replaced with 180 mL of fresh complete culture medium containing 1% (v / v) recombinant serum replacement (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21.

[0143] Day 4: The cells were washed with harvest buffer (PlasmaLyte A (Baxter) with 5% (w / v) human serum albumin (HSA)) and concentrated by volume reduction to produce the drug substance (DS). Samples were taken for analysis.

[0144] Example 3: SMART CAR-T cell culture process (shake flask scale-down model)

[0145] Day 0: For scale-down model studies, CD4 plus CD8 T lymphocytes were enriched from frozen biomass using GMP anti-CD4 and anti-CD8 CliniMACS microbeads (Miltenyi) on a Prodigy or manually. 8 Purified CD3 T cells were added to 125 mL shake flasks and transfected with Miltenyi T cell TransAct via CD3 / 28 at v / v=1:17.5 on the same day. TMActivate and culture overnight in 7 mL complete X-VIVO 15 serum-free medium (Lonza) containing 1% (v / v) recombinant serum replacement (ITSE-A), 40 IU / mL recombinant human IL-2 and 0.19 U / mL recombinant human IL-21. The shake flask was placed on an orbital shaker at 50 rpm.

[0146] Day 1: The next day, transduce cells with lentiviral vectors at a predetermined multiplicity of infection. Two hours after adding lentivirus, add fresh cell culture medium to bring the cell culture volume to 25 mL. After the volume increase, increase the agitation rate of the orbital shaker to 65 rpm.

[0147] Day 2: The cell culture was divided into two equal parts (about 12 mL each) and 5 mL of spent medium was removed from the cell culture. 18 mL (25 mL in total) of complete X-VIVO 15 serum-free medium (Lonza) containing 1% (v / v) recombinant serum replacement (ITSE-A), 40 IU / mL recombinant human IL-2 and 0.19 U / mL recombinant human IL-21 was added to each cell culture fraction in a 125 mL shake flask.

[0148] Day 3: Every 24 hours, the cell culture medium was replaced with 18 mL of fresh complete medium containing 1% (v / v) recombinant serum replacement (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21.

[0149] On day 4, cells were washed with harvest buffer (PlasmaLyte A (Baxter) with 5% (w / v) human serum albumin (HSA)) and concentrated by volume reduction to produce drug substance (DS). Samples were taken for analysis.

[0150] Using the shake flask method, 1 × 10 cells were seeded for both STEAP2 and GPC3 CAR-T cells. 9 Excellent T cell expansion was observed in 100 cells. The total viable cell count was determined, and the expanded T cells also maintained high viability ( Figure 4A-4B and Figure 5A-Figure 5B ). It was confirmed that CAR-T cells expanded under IL-10 or IL-21 were less activated. In addition, in the presence of IL-21 alone, CAR-T cells were enriched for CAR + and CD8 + cell.

[0151] Example 4: Analytical testing of the SMART 4-day method versus the traditional culture method (TNT)

[0152] Evaluate the relative purity of SMART-treated T cells. Figure 8A-BAs shown, for both STEAP2 and GPC3 CAR-T cells, the T cell population was a highly pure cell population with a total CD3 positivity of at least 98%. The level of CAR expression ( Figure 6A-6B ) showed that STEAP2( Figure 9A-9B ) and GPC3( Fig. 9B ) The correlation between CAR and TGFβRII expression in CAR-T cells. Similarly, when PBMCs from prostate cancer patients ( Fig. 9A , run 4C) and healthy donor PBMC ( Fig. 9A At the beginning of the 4-hour run (day 6), high expression levels of STEAP2 CAR were seen. Interestingly, for STEAP2 CAR expression, the percentage of CAR+ T cells increased further when cells were harvested on day 6 compared to day 4 ( Fig. 9A ).

[0153] The differentiation profile of live CAR+ T cells showed a dominant early memory phenotype ( Fig.10 ).like Fig.10 As shown, central memory (T CM )(CCR7+CD45RO+) was the dominant phenotype of CAR-positive T cells harvested on day 6, while CAR-T cells harvested on day 4 showed stem cell memory (T SCM ) and T CM Both. CM Differentiation of the cells increased as the duration of expansion increased to 6 days. Importantly, the phenotype of CAR-positive T cells from cancer patients and healthy donors was comparable.

[0154] Live CAR+ T cells also showed a more advanced activation curve, with less than 1% of cells being PD1 / LAG3 / TIM3 triple positive ( Figure 11A-11B ). In the activation curve, CAR+T cells showed more advanced activation (CD25+). Activation was slightly reduced in CAR-T cells when harvested on day 6 compared to cells harvested on day 4. In the exhaustion curve, overall, the percentage of cells expressing exhaustion markers was extremely low, less than 4% for PD1 / LAG3 / Tim3 double positive, and less than 1% for PD1 / LAG3 / Tim3 triple positive. Slight differences were observed in the expression of exhaustion markers for CAR-T cells generated from cells treated for 4 days versus 6 days.

[0155] The functionality of STEAP2 and GPC3 CAR-T cells is shown in Figure 12A-12B and Figure 11A-11B In. Figure 12A-12B As shown in Figure 2, STEAP2 and GPC3 CAR-T cells exhibited target-dependent killing activity within a certain range of E:T ratios. Figure 13A-13B As shown in Figure 2 for STEAP2 and GPC3 CAR-T cells, cytokine release was observed when the cells were co-cultured with target-expressing cell lines at an E:T ratio of 1:2.

[0156] Example 5: Biological Characterization of SMART CAR-T Cells

[0157] Both GPC3 and STEAP2 SMART CAR-T cells were analyzed to determine the mechanism of their increased activity relative to CAR-T cells generated by traditional methods. It was hypothesized that the shorter SMART expansion process produces cells with higher stemness and adaptability. For TCF7, CD27, CCR7, FOXO1, CD28, and BCL6, the expression of stemness genes from the 4-day and 12-day processes was analyzed. Figure 14A-14E , Figures 15A-15D and Figures 16A-16D As shown, day 4 T cells showed higher expression of stemness genes and high metabolic fitness compared to TNT CAR-T cells. This translated into better expression of STEAP2 CAR by day 4 compared to the traditional TNT approach in an in vitro serial killing assay ( Figure 17A-17B ) and higher fold expansion of SMART 4-day GPC3 CAR-T cells ( Figure 18D-18E ) Both. SMART CAR-T cells also increased antigen-specific secretion of effector cytokines. Figures 18A-18C As shown, SMART CAR-T cells produced higher levels of IFNγ, IL-2, and IL-21 in serial killing assays. Metabolic adaptation showed that 4-day SMART CAR-T cells had higher OCR than 11-day TNT CAR-T ( Figure 24A-Figure 24D , Figure 25A-Figure 25D ) and ECAR( Figure 26A-26D ).

[0158] Example 6: In vivo efficacy of SMART CAR-T cells

[0159] To determine the impact of the in vitro phenotype of TNT and SMART cells on their activity in an in vivo setting, NSG mice were implanted with GPC3-positive HUH7 tumors overexpressing human TGFβ. 3 The mice were randomly divided into groups with an average size of Figure 19A-19B TNT or SMART GPC3 CAR-T cells were administered intravenously (IV) at the indicated doses. Tumor volume and body weight were monitored twice weekly throughout the study to reveal superior tumor control of SMART CAR+T cells compared to TNT CAR+T cells at all doses tested ( Figure 19A-19B Mice were bled on the indicated days and serum was analyzed for IFNγ by MSD ( Figure 19C-Figure 19D ). A significant reduction in tumor volume was seen after administration of SMART CAR-T cells and was associated with higher concentrations of IFNγ in serum.

[0160] Separately, the same experiment was performed using STEAP2 CAR-T cells using implanted STEAP2-positive C4-2 tumors that exogenously expressed human TGFβ. 3 At an average size of , mice were randomized and dosed IV with a titrated range of TNT or SMART STEAP2 CAR-T cells, as Fig.19E Tumor volume and body weight were monitored twice weekly throughout the study to reveal superior tumor control with SMART CAR+T cells compared to TNT CAR+T cells at all doses tested.

[0161] The CD4 / CD8 ratio of prostate cancer T cells undergoing expansion was also analyzed. Figure 20A-20B As shown in , T cells were double stained and analyzed by FACS. These prostate cancer T cells were also less differentiated than cells from healthy donors, as shown by CD62L / CD45RO expression. ( Figure 21A-21B ).

[0162] STEAP2-positive C4-2 cells overexpressing exogenous human TGFb were implanted into male NSG mice. When the tumors reached an average size of 175 mm 3 When the mice were randomly divided into treatment groups, they were administered with different amounts of SMARTCAR-T cells from two different donors, such as Fig. 22 A significant reduction in tumor volume was seen after administration of SMART CAR-T cells from two donors.

[0163] A similar experiment was run in which C4-2 TGFb cells were implanted into NSG MHC class 1 / class 2 knockout mice to mitigate the potential contribution of GvHD. The study compared TNT and SMART 40A3 CAR-T cells at a dose of 6e6 from the same donor. The comparison revealed superior tumor growth inhibition and a more comprehensive set of complete responders in the SMART CAR-T treatment group. 1e6 and 3e6 of the second donor SMART material were also effective in this context, producing 2 / 5 and 4 / 5 complete responders, respectively. The same degree of tumor volume reduction was not seen with the 12-day (TNT) CAR-T cells ( Fig.23 ).

Claims

1. A method for expanding a T cell population, the method comprising: (a) Isolation of CD3 from samples + T cells; (b) culturing the CD3 + T cells; (c) activating the CD3 + (d) transducing the CD3 T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) + T cells to produce CAR-T cells or T cell receptor (TCR) cells; (e) culturing the CAR-T cells in a culture medium; and (f) harvesting the CAR-T cells or T cell receptor (TCR) cells.

2. A method for producing a T cell therapeutic agent, the method comprising: (a) Obtaining CD3 + (b) culturing the CD3 T cell population in a culture medium containing human interleukin 21 (IL-21); + T cells; (c) activating the CD3 + (d) transducing the CD3 T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) + T cells to produce CAR-T cells or T cell receptor (TCR) cells; (e) culturing the CAR-T cells or T cell receptor (TCR) cells in a culture medium; and (f) harvesting the CAR-T cells or T cell receptor (TCR) cells.

3. A method for expanding a T cell population, the method comprising: (a) Isolation of CD4 from samples + and CD8 + CD3 T cells + (b) culturing the CD3 T cell population in a medium containing human interleukin 21 (IL-21) + T cells; (c) activating the CD3 + (d) transducing the CD3 T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) + T cells to produce CAR-T cells or T cell receptor (TCR) cells; (e) culturing the CAR-T cells or T cell receptor (TCR) cells in a culture medium; and (f) harvesting the CAR-T cells or T cell receptor (TCR) cells.

4. The method according to any one of claims 1 to 3, wherein the culture medium further comprises human interleukin 2 (IL-2).

5. The method according to any one of claims 1 to 4, wherein part (d) comprises transducing the CD3 + T cells to produce CAR-T cells.

6. The method according to any one of claims 1 to 4, wherein part (d) comprises transducing the CD3 + T cells to produce TCR cells.

7. The method according to any one of claims 1 to 6, wherein in step (b), about 1×10 6 to about 1×10 9 CD3 + T cells.

8. The method of any one of claims 1 to 7, wherein the sample is an enriched apheresis product collected via leukapheresis.

9. The method according to any one of claims 1 to 8, wherein the CD3 + The T cells are cultured for about one day or about two days.

10. The method according to any one of claims 1 to 9, wherein the CD3+ T cells in step (c) are activated with an agonist of CD2, CD3, CD28 or any combination thereof.

11. The method according to any one of claims 1 to 10, wherein the CD3+ T cells in step (c) are activated using magnetic microbeads.

12. The method according to any one of claims 1 to 11, wherein the CD3+ T cells in step (c) are activated with an anti-CD3 antibody or a CD3-binding fragment thereof and an anti-CD28 antibody or a CD28-binding fragment thereof.

13. The method of claim 12, wherein the anti-CD3 antibody or CD3 binding fragment thereof and the anti-CD28 antibody or CD28 binding fragment thereof are coupled to magnetic microbeads.

14. The method according to any one of claims 1 to 13, wherein the CAR-T cells or TCR cells are cultured in step (e) for about two days to about ten days.

15. The method according to any one of claims 1 to 13, wherein the CAR-T cells or TCR cells are cultured in step (e) for about four days to about six days.

16. The method of claim 15, wherein the CAR-T T cells are cultured in step (e) for about four days.

17. The method of claim 15, wherein the CAR-T cells or TCR cells are cultured in step (e) for about six days.

18. The method of any one of claims 4 to 17, wherein the concentration of human IL-21 is about 0.01 U / mL to about 0.3 U / mL, and the concentration of human IL-2 is about 5 IU / mL to about 100 IU / mL.

19. The method of any one of claims 1 to 18, wherein the concentration of human IL-21 is about 0.19 U / mL.

20. The method of claim 19, wherein the concentration of human IL-2 is about 40 IU / mL.

21. The method according to any one of claims 1 to 20, wherein the CD3 + T cells.

22. A method for producing a T cell therapeutic agent, the method comprising: (a) Isolation of CD4 from samples + and CD8 + CD3 T cells + (b) culturing the CD3 T cell population in a medium containing human interleukin 2 at a concentration of 40 IU / mL and human interleukin 21 at a concentration of 0.19 U / mL + (c) activating the CD3 T cells with magnetic beads comprising an anti-CD3 antibody or a CD3 binding fragment thereof and an anti-CD28 antibody or a CD28 binding fragment thereof + (d) transducing the CD3 T cells with a lentiviral vector containing a nucleic acid encoding a chimeric antigen receptor (CAR) + (e) culturing the CAR-T cells in a culture medium for about four days; and (f) harvesting the CAR-T cells.

23. The method according to any one of claims 3 to 22, wherein the CD4 + and CD8 + T cells were isolated by positive selection.

24. The method of any one of claims 1 to 23, wherein the vector is a virus, a lentivirus, an adenovirus, a retrovirus, an adeno-associated virus (AAV), a transposon, a DNA vector, mRNA, a lipid nanoparticle (LNP), or a CRISPR-Cas system.

25. The method of any one of claims 1 to 24, wherein the vector is a lentivirus.

26. The method of claim 25, wherein the lentivirus is added at a multiplicity of infection (MOI) of about 0.25 to about 20.

27. The method of claim 26, wherein the lentivirus is added at an MOI of about 1 to about 4.

28. The method of claim 27, wherein the lentivirus is added at an MOI of about 2 or about 4.

29. The method of any one of claims 1 to 28, wherein the volume of the cell culture medium is increased after step (d).

30. The method of claim 29, wherein the volume of the cell culture medium is increased at least 6-fold.

31. The method according to any one of claims 1 to 30, wherein the culture medium in step (e) is replaced at least once a day.

32. The method according to any one of claims 1 to 31, wherein the culture medium in step (e) is replaced every 12 hours.

33. The method of any one of claims 1 to 32, wherein the CAR-T cells or TCR cells expand at least about 1-fold to about 5-fold during step (e).

34. The method of any one of claims 1 to 32, wherein the CAR-T cells or TCR cells expand at least about 1-fold to about 3-fold during step (e).

35. The method of claim 34, wherein the CAR-T cells or TCR cells expand approximately 2-fold during step (e).

36. The method of claim 34, wherein the CAR-T cells or TCR cells expand approximately 3-fold during step (e).

37. The method of any one of claims 1 to 36, wherein the CAR binds to STEAP2 or Glypican-3 (GPC3).

38. The method of any one of claims 1 to 37, wherein the CAR encodes an antigen binding domain that binds to STEAP2, and wherein the antigen binding domain comprises: (a) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (b) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; (c) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:21, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:22, a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23, a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:24, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:25, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:26; (d) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:31, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:32, a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:33, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:34, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:35, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:36; or (e) a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:

46.

39. The method of claim 38, wherein the CAR comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:

9.

40. The method of any one of claims 1 to 37, wherein the CAR encodes an antigen binding domain that binds to GPC3, and wherein the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising an amino acid sequence of SEQ ID NO: 112, a CDR2 comprising an amino acid sequence of SEQ ID NO: 113, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 114, and wherein the VL comprises: a CDR1 comprising an amino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 118, a CDR2 comprising an amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 119, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 117 or SEQ ID NO:

120.

41. The method of claim 40, wherein the VH comprises the amino acid sequence of SEQ ID NO: 108 or SEQ ID NO: 110, and the VL comprises the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO:

111.

42. The method of any one of claims 1 to 41, wherein the nucleic acid further encodes an armoring molecule.

43. The method of claim 42, wherein the armor molecule comprises a dominant negative TGFβ receptor type 2 (TGFβRIIDN).

44. The method of claim 42 or claim 43, wherein the armor molecule comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:

105.

45. The method of claim 44, wherein the armor molecule comprises the amino acid sequence shown in SEQ ID NO:

105.

46. ​​The method of any one of claims 1 to 45, wherein the CAR-T cells or TCR cells are formulated in an isotonic solution.

47. The method of claim 46, wherein the isotonic solution comprises plasmalyte containing human serum albumin.

48. The method of claim 46 or claim 47, wherein the isotonic solution contains about 1×10 6 to about 1×10 9 CAR-T cells or TCR cells.

49. The method of claim 48, wherein the isotonic solution contains about 3.4×10 6 CAR-T cells or TCR cells.

50. The method according to any one of claims 1 to 49, wherein the CAR-T cell or TCR cell is a T CM Cells and T SCM A mixture of cells.

51. The method of claim 50, wherein about 15% to about 50% of the CAR-T cells or TCR cells are T SCM cells, and expressed CD45RA, CCR7, and CD27 but not CD45RO.

52. The method of claim 52, wherein about 20% to about 30% of the CAR-T cells or TCR cells are T SCM cells, and expressed CD45RA, CCR7, and CD27 but not CD45RO.

53. The method of any one of claims 1 to 52, wherein greater than 50% of the CAR-T cells or TCR cells express a chimeric antigen receptor or T cell receptor.

54. The method of claim 53, wherein about 40% to about 60% of the CAR-T cells or TCR cells express a chimeric antigen receptor or a T cell receptor.

55. The method of any one of claims 1 to 54, wherein greater than 50% of the CAR-T cells or TCR cells express CD8.

56. The method of claim 55, wherein about 40% to about 60% of the CAR-T cells or TCR cells express CD8.

57. The method of any one of claims 1 to 56, wherein the CAR-T cell or TCR cell has an oxygen consumption rate (OCR) higher than 100 pmol / min.

58. The method of any one of claims 1 to 56, wherein the CAR-T cell or TCR cell has an OCR of about 50 pmol / min to about 200 pmol / min.

59. The method of any one of claims 1 to 58, wherein the CAR-T cell or TCR cell has an extracellular acidification rate (ECAR) greater than 30 mPH / min.

60. The method of claim 59, wherein the ECAR is about 30 mpH / min to about 60 mpH / min.

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