Method for proliferating a T cell population

Abstract

Provided herein are methods for manufacturing, expanding, and / or generating genetically modified T cells comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 368,550, filed on July 15, 2022, which is hereby incorporated by reference in its entirety.

[0002] Reference to Electronically Submitted Sequence Listing The electronically submitted sequence listing (CARTSTEAP2 - 300 - WO - PCT.xml; size: 81,152 bytes, created on July 10, 2023) is hereby incorporated by reference in its entirety.

Background Art

[0003] The generation of tumor - specific T lymphocytes by genetic modification to express chimeric antigen receptors (CARs) has attracted attention as a form of synthetic biology that induces a potent anti - tumor effect (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). Specificity is conferred by antibody fragments, so CAR - T cells are not MHC - restricted and are thus more practical than T - cell receptor - based approaches that require MHC matching.

[0004] Therefore, CAR-T cell therapy represents a major advance in personalized cancer treatment. In this strategy, a patient's own T cells are genetically engineered to express a synthetic receptor that binds 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. Dramatic clinical responses and high rates of complete remission have been observed in the context of CAR-T cell therapy for B cell malignancies. This has led to two recent FDA approvals of CAR-T cells against the CD19 protein for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma. Thus, CAR-T cells are almost certainly one of the first successful examples of commercially viable synthetic biology and personalized cell cancer therapies.

[0005] Despite the recent success of CAR-T cell therapy, there remains a need in the art to better improve methods of T cell expansion. SUMMARY OF THE INVENTION

[0006] The present disclosure relates to a method for expanding a population of T cells, comprising: (a) isolating CD3+ T cells from a sample; (b) culturing the CD3+ T cells in a culture medium containing human interleukin 21 (IL-21); (c) activating the CD3+ T cells; (d) transducing the CD3+ T cells with a vector containing 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 the CAR-T cells in the medium; and (f) harvesting the CAR-T cells or T cell receptor (TCR) cells. The present disclosure also relates to a method for manufacturing a T cell therapeutic agent, comprising: (a) obtaining a sample containing a population of CD3+ T cells; (b) culturing the CD3+ T cells in a culture medium containing human interleukin 21 (IL-21); (c) activating the CD3+ T cells; (d) transducing the CD3+ T cells with a vector containing 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 the CAR-T cells or T cell receptor (TCR) cells in the medium; and (f) harvesting the CAR-T cells or T cell receptor (TCR) cells. The present disclosure also relates to a method for expanding a population of T cells, comprising: (a) isolating CD4+ and CD8+ T cells from a sample to form a population of CD3+ T cells; (b) culturing the CD3+ T cells in a culture medium containing human interleukin 21 (IL-21); (c) activating the CD3+ T cells; (d) transducing the CD3+ T cells with a vector containing 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 the CAR-T cells or T cell receptor (TCR) cells in the medium; and (f) harvesting the CAR-T cells or T cell receptor (TCR) cells. In some embodiments, the culture medium further contains human interleukin 2 (IL-2).

[0007] In some embodiments, part (d) comprises transducing CD3+ T cells with a vector comprising a nucleic acid encoding a CAR to produce CAR-T cells. In some embodiments, part (d) comprises transducing CD3+ T cells with a vector comprising a nucleic acid encoding a TCR to produce TCR cells. In some embodiments, about 1×10 6 ~ about 1×10 9Individual CD3+ T cells are cultured in a culture medium in step (b). In some embodiments, the sample is a concentrated apheresis product collected via leukapheresis. In some embodiments, the CD3+ T cells in step (c) are cultured for about 1 day or about 2 days. In some embodiments, the CD3+ T cells in step (c) are activated with an agonist of CD2, CD3, CD28, or any combination thereof. In some embodiments, the CD3+ T cells in step (c) are activated with magnetic microbeads. In some embodiments, 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. In some embodiments, the anti-CD3 antibody or a CD3-binding fragment thereof, and the anti-CD28 antibody or a CD28-binding fragment thereof are bound to magnetic microbeads. In some embodiments, CAR-T cells or TCR cells are cultured in step (e) for about 2 to about 10 days. In some embodiments, CAR-T cells or TCR cells are cultured in step (e) for about 4 to about 6 days. In some embodiments, CAR-T T cells are cultured in step (e) for about 4 days. In some embodiments, CAR-T cells or TCR cells are cultured in step (e) for about 6 days. In some embodiments, 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. In some embodiments, the concentration of human IL-21 is about 0.19 U / mL. In some embodiments, the concentration of human IL-2 is about 40 IU / mL. In some embodiments, the CD3+ T cells are agitated during step (b).The method of the present disclosure is a method for producing a T cell therapeutic agent, comprising: (a) isolating CD4+ and CD8+ T cells from a sample to form a population of CD3+ T cells; (b) culturing the CD3+ T cells in a culture 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 containing 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 virus containing a nucleic acid encoding a chimeric antigen receptor (CAR) to produce CAR-T cells; (e) culturing the CAR-T cells in the medium for about 4 days; and (f) harvesting the CAR-T cells.

[0008] In some embodiments, the CD4+ and CD8+ T cells are isolated by positive selection. In some embodiments, the vector is a virus, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), transposon, DNA vector, mRNA, lipid nanoparticle (LNP), or CRISPR-Cas system. In some embodiments, the vector is a lentivirus. In some embodiments, the lentivirus is added at a multiplicity of infection (MOI) of about 0.25 to about 20 of the present invention. In some embodiments, the lentivirus is added at an MOI of about 1 to about 4. In some embodiments, the lentivirus is added at an MOI of about 2 or about 4. In some embodiments, the cell culture medium increases in volume after step (d). In some embodiments, the volume of the cell culture medium increases at least 6-fold.

[0009] In some embodiments, the medium in step (e) is replaced at least once a day. In some embodiments, the medium in step (e) is replaced every 12 hours. In some embodiments, the CAR-T cells or TCR cells are expanded at least about 1-fold to about 5-fold during step (e). In some embodiments, the CAR-T cells or TCR cells are expanded at least about 1-fold to about 3-fold during step (e). In some embodiments, the CAR-T cells or TCR cells are expanded about 2-fold during step (e). In some embodiments, the CAR-T cells or TCR cells are expanded about 3-fold during step (e). In some embodiments, the CAR binds to STEAP2 or glypican-3 (GPC3). In some embodiments, the CAR encodes an antigen-binding domain that binds to STEAP2, and the antigen-binding domain is (a) VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (b) VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; (c) VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26; (d) VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; or (e) comprising VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, 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 set forth in SEQ ID NO: 9. In some embodiments, the CAR encodes an antigen-binding domain that binds to GPC3, the antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), VH comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 112, CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and CDR3 comprising the amino acid sequence of SEQ ID NO: 114, VL comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 118, CDR2 comprising the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 119, and CDR3 comprising the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 120. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 108 or SEQ ID NO: 110, and VL comprises the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 111. In some embodiments, the nucleic acid also encodes a protective molecule.

[0010] In some embodiments, the protective molecule comprises a dominant negative TGFβ receptor type 2 (TGFβRIIDN). In some embodiments, the protective 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 set forth in SEQ ID NO: 105. In some embodiments, the protective molecule comprises the amino acid sequence set forth in SEQ ID NO: 105. In some embodiments, the CAR-T cells or TCR cells are formulated in an isotonic solution. In some embodiments, the isotonic solution comprises PlasmaLyte containing human serum albumin. In some embodiments, the isotonic solution contains about 1×10 6 ~ about 1×10 9 CAR-T cells or TCR cells. In some embodiments, the isotonic solution contains about 3.4×10 6 CAR-T cells or TCR cells. In some embodiments, the CAR-T cells or TCR cells are a mixture of T CM and T SCM cells. In some embodiments, about 20% to about 50% of the CAR-T cells or TCR cells express CD45RA, CCR7, and CD27 and do not express CD45RO. In some embodiments, about 20% to about 30% of the CAR-T cells or TCR cells are T SCM cells that express CD45RA, CCR7, and CD27 and do not express CD45RO. In some embodiments, more than 50% of the CAR-T cells or TCR cells express a chimeric antigen receptor or a T cell receptor. In some embodiments, about 40% to about 60% of the CAR-T cells or TCR cells express a chimeric antigen receptor or a T cell receptor. In some embodiments, more than 50% of the CAR-T cells or TCR cells express CD8. In some embodiments, about 40% to about 60% of the CAR-T cells or TCR cells express CD8.

[0011] In some embodiments, the CAR-T cells or TCR cells have an oxygen consumption rate (OCR) exceeding 100 pmol / min. In some embodiments, the CAR-T cells or TCR cells have an OCR of about 50 pmol / min to about 200 pmol / min. In some embodiments, the CAR-T cells or TCR cells have an extracellular acidification rate (ECAR) exceeding 30 mpH / min. In some embodiments, the CAR-T cells or TCR cells have an ECAR of about 30 mpH / min to about 60 mpH / min.

Brief Description of the Drawings

[0012]

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[0013] The present disclosure relates to a method of culturing T cells transduced with a chimeric antigen receptor (CAR) that generates a persistent population of T cells exhibiting increased antigen-independent activation.

[0014] To make the present disclosure more readily understandable, certain terms are first defined. As used herein, each of the following terms shall have the meaning set forth below, unless explicitly provided otherwise herein. Throughout this specification, further definitions are provided.

[0015] Note that terms such as "a" or "an" refer to one or more of the entity. For example, "feed medium" is understood to represent one or more feed media. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0016] As used herein, the term "and / or" should be construed as a specific disclosure where each of the two specified features or components is either accompanied by or not accompanied by the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein 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 encompass 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).

[0017] Whenever an aspect is described in this specification using the word "comprising", it is understood that other similar aspects regarding "consisting of" and / or "consisting essentially of" are also provided.

[0018] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of terms used in this disclosure to those of ordinary skill in the art.

[0019] Units, prefixes, and symbols are shown in the form recognized by the International System of Units (SI). Numerical ranges include the numbers defining the range. The headings provided in this specification do not limit the various aspects of the disclosure, but can be obtained by referring to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0020] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination of their alternatives. As used in this specification, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any listed or enumerated component.

[0021] Terms such as "about" or "essentially comprising" refer to values or compositions within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measuring system. For example, "about" or "essentially comprising" can mean within one or more standard deviations in each practice in the art. Alternatively, "about" or "essentially comprising" can mean a range up to ±10%. Further, especially with respect to biological systems or processes, these terms can mean up to one order of magnitude or up to five times the value, whichever is greater. When a particular value or composition is provided in the present application and the claims, unless otherwise stated, the meaning of "about" or "essentially comprising" should be assumed to be within the acceptable error range for that particular value or composition.

[0022] As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include any integer value within the recited range and, where appropriate, fractions thereof (such as tenths and hundredths of an integer), unless otherwise indicated.

[0023] The terms "T cell" or "T lymphocyte" are recognized in the art and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (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 -It can be a T cell or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells.

[0024] As used herein, the term "proliferation" refers to an increase in cell division that can be either symmetric or asymmetric cell division. In certain embodiments, "proliferation" refers to symmetric or asymmetric division of T cells. An "increase in proliferation" occurs when there is an increase in the number of cells in a treated sample compared to the cells in an untreated sample.

[0025] The term "proliferating" in the methods of the present invention refers to the process of increasing the number of cells in a cell culture. In the proliferation step, in one embodiment, cells are fed according to a feeding regimen and the culture medium is exchanged at regular intervals. The specific timing and amount of medium added in a particular feeding regimen depends on the number of cells and the levels of metabolites in the culture.

[0026] As used herein, the term "differentiation" refers to a method of reducing the potency or proliferation of a cell or transitioning the cell to a more developmentally restricted state. In certain embodiments, differentiated T cells acquire immune effector cell functions.

[0027] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). Exemplary immune effector cells contemplated herein are T lymphocytes, particularly cytotoxic T cells (CTL; CD8 + T cells), TIL, and helper T cells (HTL; CD4 + T cells).

[0028] A "modified T cell" refers to a T cell modified by the introduction of a polynucleotide encoding an engineered CAR as contemplated herein. Modified T cells include both genetically modified and non-genetically modified (e.g., episomal or extrachromosomal) modifications.

[0029] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell.

[0030] The terms "genetically modified cell", "modified cell" and "redirected cell" are used interchangeably.

[0031] As used herein, the term "gene therapy" refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell for the purpose of restoring, correcting, or modifying gene expression, or expressing a therapeutic polypeptide, such as a CAR and / or one or more cytokines. In certain embodiments, T cells are modified to express an engineered TCR or CAR without modifying the cell's genome, for example, by introducing an episomal vector expressing the CAR into the cell.

[0032] As used herein, "chimeric antigen receptor (CAR)" means a fusion protein comprising an extracellular domain capable of binding to a given antigen, an intracellular segment comprising one or more cytoplasmic domains derived from a signal transduction protein different from the polypeptide from which the extracellular domain is derived, and a transmembrane domain. "Chimeric antigen receptor (CAR)" may also be referred to as "chimeric receptor", "T body", or "chimeric immunoreceptor (CIR)". The phrase "extracellular domain capable of binding to a given antigen" means any proteinaceous molecule or portion thereof capable of specifically binding to a given antigen. "Intracellular signal transduction domain" means any oligopeptide or polypeptide domain known to function to transmit a signal that activates or inhibits a biological process in a cell, such as the activation of immune cells such as T cells. Examples include the ILR chain, CD28 and / or CD3ζ.

[0033] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient methods of gene delivery vectors as they can deliver a significant amount of genetic information into the DNA of host cells. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0034] The term "ex vivo" generally refers to activities performed outside the body, such as experiments or measurements performed in living tissue or on living tissue in an artificial environment outside the body, preferably with minimal changes to natural conditions. In certain embodiments, "ex vivo" procedures involve living cells or tissues that are removed from a living body and cultured or conditioned in an experimental apparatus, typically under aseptic conditions, for a period of typically several hours or up to about 24 hours (but including up to 48 hours or 72 hours depending on the circumstances). In certain embodiments, such tissues or cells can be collected and frozen and later thawed for ex vivo processing. Tissue culture experiments or procedures that use living cells or tissues and last longer than a few days are typically considered "in vitro", although in certain embodiments, this term can be used interchangeably with ex vivo.

[0035] The term "in vivo" generally refers to activities that occur within a living organism, such as cell self-renewal and cell proliferation. In one embodiment, the term "in vivo proliferation" refers to the ability of a cell population to increase in number in vivo.

[0036] The acronym "SMART" (Shorty-Manipulated Auto-Replicating T-Cells) refers to a T cell proliferation process in which cells are cultured in the presence of IL-2 and IL-21.

[0037] The acronym "TNT" (Traditional expanded T cells) refers to a traditional T cell proliferation process that does not use IL-21, typically involves cell cultures that exceed 7 days, and / or typically involves the use of IL-2.

[0038] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating signal transduction events including, but not limited to, signal transduction via the TCR / CD3 complex.

[0039] "Stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand.

[0040] 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 on a T cell (referred to herein as a "stimulatory molecule"), thereby mediating a primary response by 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), as well as peptides (e.g., CMV, HPV, EBV peptides).

[0041] The term "activation" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. In certain embodiments, activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers, inter alia, to a proliferating T cell. Signals generated through the TCR alone are insufficient for complete activation of T cells, and one or more secondary or co-stimulatory signals are also required. Thus, T cell activation includes a primary stimulatory signal via the TCR / CD3 complex and one or more secondary co-stimulatory signals. Co-stimulation can be demonstrated by proliferation and / or cytokine production by T cells that have received a primary activation signal such as stimulation via the CD3 / TCR complex or CD2.

[0042] "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 upregulation or downregulation of specific molecules (e.g., CD28).

[0043] "Co-stimulatory ligand" refers to a molecule that binds to a co-stimulatory molecule. The co-stimulatory ligand may be soluble or provided on the surface. "Co-stimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand (e.g., anti-CD28 antibody).

[0044] "Autologous", as used herein, refers to cells derived from the same subject.

[0045] "Allogeneic", as used herein, refers to cells of the same species that are genetically different from the cells in the comparison.

[0046] "Syngeneic", as used herein, refers to cells of different subjects that are genetically identical to the cells in the comparison.

[0047] "Xenogeneic", as used herein, refers to cells of a different species from the cells in the comparison. In a preferred embodiment, the cells of the present invention are allogeneic.

[0048] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that can be treated with the gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere in this specification and that exhibits symptoms of cancer. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and domestic animals or pets (such as cats or dogs). Non-human primates, preferably human patients, are included. Typical subjects include human patients having cancer, diagnosed with cancer, or at risk of cancer or having cancer.

[0049] "Enhance", "promote", "increase", or "augment" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a greater physiological response (i.e., downstream effect) as compared to a response caused by either a vehicle or a control molecule / composition. Measurable physiological responses can include, among others that will be apparent from an understanding in the art and the description herein, an increase in T cell proliferation, activation, persistence, and / or an increase in the ability to kill cancer cells. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (any integer and decimal between and exceeding 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response brought about by a vehicle or control composition.

[0050] "Decrease", "reduce", "lower", "diminish", or "attenuate" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a lesser physiological response (i.e., downstream effect) as compared to a response caused by either a vehicle or a control molecule / composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and can include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (any integer and decimal between and exceeding 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response (reference response) brought about by a vehicle, control composition, or the response in a particular cell lineage.

[0051] "To maintain" or "to preserve" or "maintenance" or "no change" or "substantially no change" or "substantially no decrease" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a lesser physiological response (i.e., downstream effect) in a cell as compared to a response caused by either a vehicle, a reference molecule / composition, or a response in a particular cell lineage. An equivalent response is a response that is not significantly different from, or measurably different from, the reference response.

[0052] Source of T cells Prior to the expansion and genetic modification of the T cells of the present invention, the source of the T cells 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 infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the invention, any number of T cell lines available in the art can be used. In certain embodiments of the invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art such as Ficoll™ separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In another embodiment, the wash solution is calcium-free and may be magnesium-free or may be lacking in many, but not all, divalent cations. Here too, the initial activation step in the absence of calcium results in an expansion of activation. As will be readily understood by those of skill in the art, the washing step can be accomplished by methods known to those of skill in the art such as by using a semi-automatic "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 are, for example, Ca2+ Non-containing Mg 2+ It can be resuspended in various biocompatible buffers such as PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed and the cells can be directly resuspended in the culture medium.

[0053] In another aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elution. CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +Certain subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. In some embodiments, the T cells are isolated by positive selection for CD4 and CD8 expression. For example, in one embodiment, the T cells are isolated by incubation with anti-CD4 / anti-CD8 conjugate beads for a time sufficient for positive selection of the desired T cells. In one embodiment, the time is about 30 minutes. In a further embodiment, the time is in the range of 30 minutes to 36 hours or more and all integer values therebetween. In a further embodiment, the time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time is 10 to 24 hours. In any situation where there are fewer T cells compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or an immunocompromised individual, longer incubation times can be used to isolate the T cells. Further, the use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time for which the T cells are bound to the CD4 / CD8 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), subpopulations of T cells can be preferentially selected at the start of culture or at other points during the process. Further, by increasing or decreasing the ratio of anti-CD4 and / or anti-CD8 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected at the start of culture or at other desired points. One of ordinary skill in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In certain embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. The "unselected" cells can also be subjected to further rounds of selection.

[0054] Enrichment of T cell populations by negative selection can be achieved using combinations of antibodies against surface markers specific to the negatively selected cells. One method is negative magnetic immunoadhesion or cell sorting and / or selection by flow cytometry using 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 certain embodiments, it may be desirable to enrich or positively select regulatory T cells that typically express CD4 + , CD25 + , CD62L hi , GITR + , and FoxP3 + . Alternatively, in certain embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0055] For the isolation of desired cell populations by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the beads and the cells. For example, in one embodiment, a concentration of 2 billion cells / mL is used. In one embodiment, a concentration of 1 billion cells / mL is used. In a further embodiment, a concentration of more than 100 million cells / mL is used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / mL are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 1 billion cells / mL are used. In a further embodiment, concentrations of 125 million or 150 million cells / mL can be used. The use of high concentrations can result in increased cell yields, cell activation, and cell proliferation.

[0056] In related aspects, it may be desirable to use lower concentrations of cells. By significantly diluting a mixture of T cells and surfaces (e.g., particles such as beads), the interaction between the particles and the cells is minimized. Thereby, cells that express a large amount of the desired antigen that binds to the particles are selected. For example, CD4 + T cells express a higher level of CD28 than CD8 + T cells at the dilution concentration and are captured more efficiently. In one aspect, the concentration of cells used is 5×10 6 / mL. In other aspects, the concentration used can be from about 1×10 5 / mL to 1×10 6 / mL, and any integer value therebetween.

[0057] In other aspects, the cells can be incubated on a rotating device at various speeds for various lengths of time at either 2 - 10°C or room temperature.

[0058] T cells for stimulation can also be frozen after the washing step. In some aspects, the freezing and subsequent thawing steps can provide a more homogeneous product by removing granulocytes and some monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and are useful in this context, but one method involves using PBS containing 20% DMSO and 8% human serum albumin, or 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or a culture medium containing 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 media containing, for example, Hespan and PlasmaLyte A. Then, the cells are frozen to -80°C at a rate of 1 o per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as non - controlled freezing immediately at -20°C or in liquid nitrogen, can be used.

[0059] In certain embodiments, cryopreserved cells are thawed, washed, allowed to stand at room temperature for 1 hour, and then activated using the method of the present invention.

[0060] In the context of the present invention, collection of a blood sample or apheresis product from a subject during a period prior to when the proliferating cells described herein may be needed is also contemplated. Thus, the source of the cells to be expanded can be collected at any point when needed, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions (e.g., the diseases or conditions described herein) for which a subject would benefit from T cell therapy. In one aspect, the blood sample or apheresis is taken from a generally healthy subject. In certain aspects, the blood sample or apheresis is taken from a generally healthy subject at risk of developing a disease but who has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain aspects, T cells can be expanded, frozen, and used later. In certain aspects, the sample is collected from a patient immediately following diagnosis of a particular disease described herein but prior to any treatment. In a further aspect, the cells are isolated from a blood sample or apheresis from a subject prior to any number of related treatment modalities including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunosuppressive agents such as CAMPATH, anti-CD3 antibody, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs either inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase, which is important for growth factor-induced signal transduction (rapamycin) (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 a further aspect, the cells are isolated for the patient and frozen for later use in conjunction with (e.g., before, simultaneously with, or after) bone marrow or stem cell transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or T cell depletion therapy using an antibody such as OKT3 or CAMPATH. In another aspect, the cells can be isolated prior to B cell depletion therapy with an agent that reacts with CD20, such as rituxan, and frozen for later use in treatment.

[0061] In a further aspect of the invention, T cells are obtained from the patient immediately following treatment. In this regard, it has been observed that following certain cancer treatments, particularly those with drugs that damage the immune system, the quality of the T cells obtained immediately following treatment, during the period when the patient is normally recovering from the treatment, is optimal or can be improved with respect to their ability to proliferate ex vivo. Similarly, following ex vivo manipulation using the methods described herein, these cells can be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic system, during this recovery period. Further, in certain aspects, mobilization (e.g., with GM-CSF) and pre-treatment regimens can be used to create a preferred state for the re-growth, recirculation, regeneration, and / or proliferation of specific cell types, particularly during a defined time frame following treatment, in a subject. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0062] Activation and Proliferation of T Cells Before or after genetic modification of T cells to express the desired CAR, the T cells can be activated and expanded generally 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.

[0063] Generally, the T cells of the present invention are expanded by contacting them with a surface to which an agent that stimulates CD3 / TCR complex - related signals and a ligand that stimulates co - stimulatory molecules on the surface of the T cells are attached. In particular, a T cell population can be stimulated, for example, by contact with an anti - CD3 antibody or an antigen - binding fragment thereof immobilized on a surface or an anti - CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. For co - stimulation of accessory molecules on the surface of T cells, ligands that bind to the accessory molecules are used. For example, under conditions suitable for stimulating the proliferation of T cells, a population of T cells can be contacted with an anti - CD3 antibody and an anti - CD28 antibody. CD4 + T cells or CD8 + To stimulate the proliferation of either CD4 T cells or CD8 T cells, anti - CD3 antibody and anti - CD28 antibody are used. Examples of anti - CD28 antibodies include 9.3, B - T3, XR - CD28 (Diaclone, Besangon, France), and can be used in the same manner as other methods generally known in the art (Berg et al., Transplant Proc. 30(8):3975 - 3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319 - 1328, 1999; Garland et al., J. Immunol Meth. 227(1 - 2):53 - 63, 1999).

[0064] In certain embodiments, the primary stimulation signal and the co-stimulation signal for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or can be bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent can be bound to a surface and the other agent can be present in solution. In one embodiment, the agent providing the co-stimulation signal is bound to the cell surface and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in a soluble form and then cross-linked to a surface such as a cell expressing an Fc receptor or an antibody or other binding agent that binds to the agent. In this regard, for example, for artificial antigen presenting cells (aAPCs) contemplated for use in the activation and proliferation of T cells in the present invention, see U.S. Patent Application Publication Nos. 20040101519 and 20060034810.

[0065] In one embodiment, the two agents are immobilized on beads either on the same bead (i.e., “cis”) or on separate beads (i.e., “trans”). By way of 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-stimulation signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead in equimolar amounts. In one embodiment, CD4 + T cell proliferation and each antibody bound to the beads in a 1:1 ratio for T cell proliferation are used. In certain embodiments of the present invention, the ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increase in T cell proliferation is observed as compared to the proliferation observed using a 1:1 ratio.

[0066] In a further aspect of the invention, cells such as T cells are combined with beads coated with a drug, the beads and cells are then separated, and the cells are then cultured. In another aspect, the beads coated with the drug and the cells are cultured together without separation prior to culturing. In a further aspect, the beads and cells are first concentrated by the application of a force such as magnetism, resulting in an increase in the ligation of cell surface markers, thereby inducing cell stimulation.

[0067] Conditions suitable for T cell culture include a suitable medium (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) containing factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), IL-21, insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives known to those skilled in the art for the growth of cells. Other additives for cell growth include, but are not limited to, surfactants, Plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with amino acids, sodium pyruvate, and vitamins added, and is either serum-free or has an appropriate amount of serum (or plasma) or a defined set of hormones, and / or a sufficient amount of cytokines added for the growth and proliferation of T cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells to be injected into a subject. The target cells are maintained under conditions necessary to support growth, e.g., at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). In one aspect, the medium is X-VIVO 15 serum-free medium containing 1% (v / v) recombinant serum substitute (ITSE-A).

[0068] In one aspect, T cells are cultured in a medium containing 10 - 100 IU / mL of recombinant human IL-2. In one aspect, T cells are cultured in a medium containing 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / mL of recombinant human IL-2. In another aspect, T cells are cultured in a medium that also contains 0.1 - 0.3 U / mL of recombinant IL-21. In another aspect, T cells are cultured in a medium containing IL-2 and 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 75, or 100 U / mL of recombinant human IL-21. In another aspect, T cells are cultured in a medium containing IL-2 and 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 U / mL of recombinant human IL-21. In one aspect, T cells are cultured in a medium containing 40 IU / mL of recombinant human IL-2 and 0.19 U / mL of recombinant human IL-21.

[0069] In one aspect of the invention, the cells are cultured for up to 14 days. In another aspect, the mixture may be cultured for 4 days. T cells can be agitated during any stage of the culture. In one aspect, the cells are agitated during cell culture in a medium containing IL-2 and IL-21. In a particular aspect, T cells collected on day 4 exhibit higher target-independent killing activity compared to CAR-T cells collected on day 6.

[0070] Chimeric antigen receptor (CAR) and T cell receptor (TCR) T cells engineered with a TCR express a tumor antigen-specific receptor having an α-chain and a β-chain that are produced from high-quality and high-avidity antigen-specific T cell clones. Separately, a CAR is a recombinant receptor for an antigen that redirects the specificity and function of T lymphocytes and other immune cells in a single molecule. A general premise for their use in cancer immunotherapy is to rapidly generate tumor-targeted T cells and avoid the barriers and escalating dynamics of active immunity. When expressed in T cells, CAR-modified T cells acquire supra-physiological properties that can exert both immediate and long-term effects. Engineering of CAR into T cells requires that the T cells be cultured to enable transduction and expansion. Transduction can utilize various methods, but stable gene transfer is required to enable persistent CAR expression in clonally expanding and persistent T cells. In principle, any cell surface molecule can be targeted via a CAR, thus nullifying the antigen recognition gap in the physiological T cell repertoire that restricts tolerance to self-antigens and the range of T cell reactivity.

[0071] However, redirection of immunoreactivity to a selected antigen is not the only purpose of a smarter CAR, and a CAR is designed to achieve much more than just targeting and initiating T cell activation. CARs with different strengths and qualities of signaling have the potential to regulate T cell proliferation and persistence, and the intensity of T cell activation within the tumor microenvironment, which are characteristics that dramatically alter the efficacy and safety of tumor-targeted T cells.

[0072] Depending on the desired antigen to be targeted, the CARs 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).

[0073] [Table 1-1]

[0074]

Table 1-2

[0075]

Table 1-3

[0076]

Table 1-4

[0077]

Table 1-5

[0078]

Table 1-6

[0079]

Table 1-7

[0080] Protective molecule Disclosed herein is a polynucleotide comprising (a) a nucleotide sequence encoding a CAR comprising an antigen-binding domain, and (b) a nucleotide sequence encoding a protective molecule. One approach for generating CAR-T cells that are more resistant to tumor-associated immunosuppression is referred to as "protection." Protection is the molecular engineering of CAR-T cells to express one or more "protective molecules" that can counter immunosuppression. For example, researchers reported improved CAR-T cell antitumor activity in murine models of PD-L1+ blood and solid tumors by modifying CAR-T cells to secrete a PD-1-blocking single-chain variable fragment (scFv) (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 protecting T cells with a dominant negative TGFβ receptor type II (TGFβRIIΔN) protective molecule to neutralize the suppressive effect 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 protecting anti-PSMA-CAR-T cells with a TGFβRIIΔN protective molecule for treating castration-resistant prostate cancer (NCT03089203).

[0081] In some embodiments, the protective molecule comprises a dominant negative TGFβ receptor type 2 (TGFβRIIDN). In some embodiments, the protective 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 set forth in SEQ ID NO: 105. In some embodiments, the protective molecule comprises the amino acid sequence set forth in SEQ ID NO: 105.

[0082] Metabolic tests of CAR-T cells or TCR cells In some embodiments, the metabolic activity of CAR-T cells or TCR cells was measured using a Seahorse® assay. The Seahorse® 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 mitochondria. ECAR measures the production of protons resulting from glycolysis, a metabolic pathway that generates energy from glucose. In some embodiments, CAR-T cells or TCR cells are added to a specialized microplate having wells containing sensors for detecting changes in OCR and ECAR. The cells are exposed to experimental conditions such as different concentrations of drugs or metabolic substrates, and OCR and ECAR are measured at intervals. In some embodiments, the Seahorse® assay is performed using 0.5 μM FCCP. In some embodiments, the Seahorse® assay is performed using 2 μM FCCP. In some embodiments, CAR-T cells or TCR cells have an OCR greater than 100 pmol / min. In some embodiments, CAR-T cells or TCR cells have an OCR greater than 40 pmol / min. In some embodiments, CAR-T cells or TCR cells have an OCR greater than 150 pmol / min. In some embodiments, CAR-T cells or TCR cells have an OCR of about 50 pmol / min to about 200 pmol / min. In some embodiments, CAR-T cells or TCR cells have an ECAR greater than 30 mpH / min. In some embodiments, CAR-T cells or TCR cells have an ECAR greater than 50 mpH / min. In some embodiments, CAR-T cells or TCR cells have an ECAR greater than 30 mpH / min. In some embodiments, CAR-T cells or TCR cells have an ECAR of about 30 mpH / min to about 60 mpH / min.

Example

[0083] The foregoing description of specific embodiments fully discloses the general nature of the present invention, so that others, by applying knowledge within the skill of those in the art and without undue experimentation, can readily modify and / or adapt such specific embodiments to various uses without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the meaning and scope of the equivalents of the disclosed embodiments based on the teachings and guidance presented herein. The phrases or terms herein are for the purpose of description and not of limitation, and thus it should be understood that the terminology or phrases herein are to be interpreted by those skilled in the art in light of the teachings and guidance.

[0084] 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. The specification and examples are considered as illustrative only, and it is intended that the true scope and spirit of the invention be indicated by the following claims.

[0085] Example 1: IL-2 and IL-21 Proliferation in the presence of IL-21 results in fewer differentiated cells and a higher percentage of CAR+CD8+ cells. Purified human T cells were seeded at a concentration of 0.2E6 cells / mL + interleukin (IL)-2 (300 IU / mL) (Peprotech) in AIM-V medium containing 5% human serum, 1% penicillin-streptomycin (Invitrogen), and 1% antibiotic-antimycotic (Invitrogen). T cells were activated with anti-CD3 / CD28 Dynabeads (Invitrogen) according to the manufacturer's protocol. After 24 hours, lentivirus was added to the wells and the plates were centrifuged at 2000 g for 2 hours at 37°C. After centrifugation, the cells were washed and resuspended in fresh medium containing IL-2 (300 IU / mL), IL-21 (10 ng / mL, R&D Systems), IL-10 (10 ng / mL, R&D Systems), and IL-15 (10 ng / mL, R&D Systems) as indicated. The plates were placed in a 37°C, 5% CO2 incubator and the cells were split as needed to maintain a cell density of approximately 1E6 cells / mL. After 10 days, the cells were harvested and analyzed by flow cytometry (Figures 1A–1D).

[0086] The combination of IL-2 and IL-21 results in a better phenotype and long-term cell proliferation in TNT cells collected on day 8. All selected T cells (CD4 and CD8) were seeded at 1.5E6 viable cells / mL in 125 mL shake flasks at a 10% working volume in X-VIVO 15 medium (Lonza) supplemented with 5% CTS Serum Replacement (Thermo Fisher) and stirred at 51 rpm on day 0. ImmunoCult CD3 / CD28 / CD2 T cell activator (Stemcell Technologies) was added to the cell culture at 25 μL / mL to activate the T cells immediately after seeding. After culturing in an incubator at 37 °C and 5% CO2 for 2 days, GPC3 LVV was added to the cell culture at an MOI of 10 and the stirring speed was increased to 169 rpm to enhance LVV transduction. On day 3, 0.9E6 viable cells were transferred and cultured in 100 mL of X-VIVO 15 medium + 5% (v / v) CTS serum replacement supplemented with either 100 IU / mL of IL-2 (Akron) alone, or 10 ng / mL of IL-21 (Akron) alone, or 25 IU / mL of IL-2 and 10 ng / mL of 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, 5E6 viable cells were passaged and cultured in 100 mL of the same medium containing the same concentration of fresh cytokine. 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 the expression of CD3, CD4, CD8, GPC3 CAR, CD45RO, CD45RA, CD62L, and CCR7 was analyzed by flow cytometry (Figures 2A - 2F).

[0087] High concentrations of IL-2 can mask the effects of IL-21. All selected T cells (CD4 and CD8) were seeded at 1.5E6 viable cells / mL in a 125 mL shake flask at a 10% working volume in X-VIVO 15 medium (Lonza) supplemented with 5% CTS Serum Replacement (Thermo Fisher) and stirred at 51 rpm on day 0. ImmunoCult CD3 / CD28 / CD2 T cell activator (Stemcell Technologies) was added to the cell culture at 25 μL / mL to activate the T cells immediately after seeding. After culturing for 2 days in an incubator at 37 °C and 5% CO2, GPC3 LVV was added to the cell culture at an MOI of 10 and the stirring speed was increased to 169 rpm to enhance LVV transduction. On day 3, 0.9E6 viable cells were transferred and cultured in 100 mL of X-VIVO 15 medium + 5% (v / v) CTS serum replacement supplemented with only 100 IU / mL of IL-2 (Akron), or 25 IU / mL of IL-2 and 10 ng / mL of IL-21, or 50 IU / mL of IL-2 and 10 ng / mL of IL-21, or 100 IU / mL of IL-2 and 10 ng / mL of IL-21, or 25 IU / mL of IL-2 and 5 ng / mL of IL-21, or 25 IU / mL of IL-2 and 2 ng / mL of 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, the cells were harvested for cell counting and the expression of CD3, CD4, CD8, GPC3 CAR, CD45RO, CD45RA, CD62L, and CCR7 was analyzed by flow cytometry (LSR Fortessa from BD Biosciences) (Figures 3A - 3F).

[0088] Example 2: SMART 4-day CAR-T cell culture process Biological starting material (BSM) from apheresis patients was received from the clinical site within a specified collection window shipped at 2 - 8°C. The BSM was washed on a Cytiva Sefia S2000, and most of the RBCs and platelets were removed using a Flexcell program. It was then formulated in 1:1 PlasmaLyte A (Baxter) containing 5% (w / v) human serum albumin (HSA): CryoStor® CS10, divided into two 70 mL / CS250 bags (OriGen), and cryopreserved using a controlled-rate freezer before storage in the vapor phase of LN2.

[0089] Day 0: At the start of manufacturing, the frozen half of the leukopak was thawed under controlled conditions using PlasmaTherm (Plasma Therm), and CD4+CD8 T lymphocytes were isolated using GMP anti-CD4 and anti-CD8 CliniMACS microbeads (Miltenyi) on a Miltenyi Prodigy®. After isolation, 1.0E+09 purified CD3 T cells were added to the culture chamber of a Miltenyi Prodigy® tubing set and activated with Miltenyi T cell TransAct™ via CD3 / 28 at v / v = 1:17.5 on the same day, and cultured overnight in 70 mL of complete X-VIVO 15 serum-free medium (Lonza) containing 1% (v / v) recombinant serum substitute (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21.

[0090] Day 1: The next day, the cells were transduced with a lentiviral vector at a predetermined multiplicity of infection. Two hours after the addition of the lentivirus, fresh cell culture medium was added to bring the cell culture volume to 250 mL.

[0091] Days 2 - 4: The cells were continuously cultured and grown on days 2, 3, and 4. 180 mL of the cell culture medium was exchanged every 12 hours with 180 mL of fresh complete medium containing 1% (v / v) recombinant serum substitute (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21.

[0092] Day 4: Cells were washed with cell recovery buffer (PlasmaLyte A (Baxter) containing 5% (w / v) human serum albumin (HSA)) and concentrated by volume reduction to generate drug substance (DS). Samples were taken for analysis.

[0093] Example 3: SMART CAR-T Cell Culture Process (Shaking Flask Scale-Down Model) Day 0: For the scale-down model study, CD4+CD8 T lymphocytes were enriched from cryopreserved biological starting material on a Prodigy or manually using GMP anti-CD4 and anti-CD8 CliniMACS microbeads (Miltenyi). After isolation, 1.0x10 8 Purified CD3 T cells were added to a 125 mL shaking flask and activated on the same day via CD3 / 28 with Miltenyi T cell TransAct™ at v / v = 1:17.5 and cultured overnight in 7 mL of complete X-VIVO 15 serum-free medium (Lonza) containing 1% (v / v) recombinant serum substitute (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21. The shaking flask was placed on an orbital shaker at 50 rpm.

[0094] Day 1: The next day, the cells were transduced with a lentiviral vector at a predetermined multiplicity of infection. Two hours after the addition of lentivirus, fresh cell culture medium was added to bring the cell culture volume to 25 mL. After the volume increase, the agitation speed of the orbital shaker was increased to 65 rpm.

[0095] Day 2: The cell culture was divided into two equal fractions (each approximately 12 mL), and 5 mL of spent medium was removed from the cell culture. To each cell culture fraction in a 125 mL shaking flask, 18 mL (total 25 mL) of complete X-VIVO 15 serum-free medium (Lonza) containing 1% (v / v) recombinant serum substitute (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21 was added.

[0096] Day 3: The cell cultures were exchanged every 24 hours with 18 mL of fresh complete medium containing 1% (v / v) recombinant serum substitute (ITSE-A), 40 IU / mL recombinant human IL-2, and 0.19 U / mL recombinant human IL-21.

[0097] On day 4, the cells were washed with recovery buffer (PlasmaLyte A (Baxter) containing 5% (w / v) human serum albumin (HSA)) and concentrated by volume reduction to generate drug substance (DS). Samples were taken for analysis.

[0098] Using the shake flask process, excellent T cell proliferation was seen for both STEAP2 and GPC3 CAR-T cells with a seeding of 1×10 9 cells. Total viable cell numbers were determined and the expanded T cells also maintained a high viability (Figures 4A–4B and 5A–5B). CAR-T cells grown in IL-10 or IL-21 were shown to be less activated and furthermore, CAR-T cells were concentrated for + CAR + and CD8

[0099] Example 4: Analysis test of SMART 4-day process versus conventional culture process (TNT) The relative purity of SMART process T cells was evaluated. As shown in Figures 8A and 8B, the T cell population was a high purity population of cells with at least 98% overall CD3 positivity for both STEAP2 and GPC3 CAR-T cells. The levels of CAR expression (Figures 6A and 6B) showed a correlation between CAR and TGFβRII expression in STEAP2 (Figures 9A and 9B) and GPC3 (Figure 9B) CAR-T cells. Similarly, high expression levels of STEAP2 CAR were seen when starting with either PBMC from prostate cancer patients (Figure 9A, lane 4C) or PBMC from healthy donors (Figure 9A, lane 4H). Interestingly, for STEAP2 CAR expression, the percentage of CAR+ T cells further increased when cells were harvested on day 6 compared to day 4 (Figure 9A).

[0100] The differentiation profile of viable CAR+ T cells showed a dominant early memory phenotype (Figure 10). As shown in Figure 10, central memory (T CM )(CCR7+CD45RO+) is the dominant phenotype for CAR-positive T cells collected on day 6, while CAR-T cells collected on day 4 showed both stem cell memory (T SCM ) and T CM . Differentiation into T CM cells increased as the proliferation period was extended to 6 days. Importantly, the phenotypes of CAR-positive T cells from cancer patients and healthy donors were equivalent.

[0101] Viable CAR+ T cells also showed a more late-stage activation profile, with less than 1% of the cells being PD1 / LAG3 / TIM3 triple-positive (Figures 11A and 11B). In the activation profile, CAR+ T cells showed more late-stage activation (CD25+). Activation was slightly decreased in CAR-T cells collected on day 6 compared to cells collected on day 4. In the exhaustion profile, overall, the percentage of cells expressing exhaustion markers was very low, less than 4% double-positive and less than 1% triple-positive for PD1 / LAG3 / Tim3. Slight differences were observed in the expression of exhaustion markers of CAR-T cells generated from 4-day vs. 6-day treated cells.

[0102] The functionality of STEAP2 and GPC3 CAR-T cells is shown in Figures 12A - 12B and Figures 11A - 11B. As shown in Figures 12A and 12B, STEAP2 and GPC3 CAR-T cells show target-dependent killing activity over a range of E:T ratios. Cytokine release was observed when cells were co-cultured with target-expressing cell lines shown in Figures 13A and 13B for STEAP2 and GPC3 CAR-T cells at an E:T ratio of 1:2.

[0103] Example 5: Biological Characterization of SMART CAR-T Cells Both GPC3 and STEAP2 SMART CAR-T cells were analyzed to determine the mechanism for their increased activity compared to CAR-T cells produced from conventional processes. The shorter SMART expansion process was hypothesized to produce cells with higher stemness and fitness. The expression of stemness genes from the day 4 and day 12 processes was analyzed for TCF7, CD27, CCR7, FOXO1, CD28, and BCL6. As shown in FIGS. 14A-14E, FIGS. 15A-15D, and FIGS. 16A-16D, day 4 T cells showed higher expression of stemness genes and higher metabolic fitness compared to TNT CAR-T cells. This translated into both better expression of STEAP2 CAR by day 4 (FIGS. 17A and 17B) and a higher proliferation fold of SMART day 4 GPC3 CAR-T cells (FIGS. 18D and 18E) compared to the conventional TNT process in an in vitro serial killing assay. SMART CAR-T cells also increased the antigen-specific secretion of effector cytokines. As shown in FIGS. 18A-18C, SMART CAR-T cells produced higher levels of IFNγ, IL-2, and IL-21 in the serial killing assay. Metabolic fitness was shown by day 4 SMART CAR-T cells having higher OCR (FIGS. 24A-24D, FIGS. 25A-25D) and ECAR than day 11 TNT CAR-T (FIGS. 26A-26D).

[0104] Example 6: In Vivo Efficacy of SMART CAR-T Cells To determine the impact of the in vitro phenotypes of TNT and SMART cells on their activity in an in vivo setting, NSG mice were transplanted with GPC3-positive HUH7 tumors overexpressing human TGFβ. The tumors were 200 mm 3When the average size was reached, the mice were randomized and administered TNT or SMART GPC3 CAR-T cells at the doses shown in FIGS. 19A and 19B via intravenous (IV) injection. Tumor volume and body weight were monitored twice a week throughout the study, revealing superior tumor control by SMART CAR+T cells compared to TNT CAR+T cells at all doses tested (FIGS. 19A and 19B). Blood was drawn from the mice on the indicated days and IFNγ in the serum was analyzed by MSD (FIGS. 19C and 19D). A significant decrease in tumor volume was seen after administration of SMART CAR-T cells, which correlated with higher concentrations of IFNγ in the serum.

[0105] Separately, the same experiment was conducted using STEAP2 CAR-T cells with transplanted STEAP2-positive C4-2 tumors that were exogenously expressing human TGFβ. When the tumors reached 175 mm 3 in average size, the mice were randomized and administered IV with a graduated range of TNT or SMART STEAP2 CAR-T cells as shown in FIG. 19E. Tumor volume and body weight were monitored twice a week throughout the study, revealing superior tumor control by SMART CAR+T cells compared to TNT CAR+T cells at all doses tested.

[0106] The CD4 / CD8 ratio from prostate cancer T cells that had undergone expansion was also analyzed. As shown in FIGS. 20A and 20B, the 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. (FIGS. 21A and 21B).

[0107] STEAP2-positive C4-2 cells that overexpress exogenous human TGFb were transplanted into male NSG mice. When the tumor size reached an average of 175 mm 3 the mice were randomized into treatment groups and administered various amounts of SMART CAR-T cells from two different donors as shown in FIG. 22. A significant decrease in tumor volume was seen after administration of SMART CAR-T cells from both donors.

[0108] Similar experiments were conducted to mitigate the potential contribution of GvHD by transplanting C4-2 TGFb cells into NSG MHC class 1 / class 2 knockout mice. In this study, 6e6 doses of TNT and SMART 40A3 CAR-T cells from the same donor were compared. This comparison revealed superior tumor growth inhibition and more overall complete responders in the SMART CAR-T treatment group. Second donor SMART materials of 1e6 and 3e6 were also effective in this setting, resulting in 2 / 5 and 4 / 5 complete responders, respectively. A comparable reduction in tumor volume was not seen with (TNT) CAR-T cells on day 12 (Figure 23).

Claims

1. A method for increasing a population of T cells, wherein (a) a sample contains CD3 ＋ (b) Isolating T cells and (b) in a culture medium containing human interleukin 21 (IL-21) the CD3 ＋ Culturing T cells and (c) CD3 ＋ (d) Activating T cells and the CD3 ＋ A method comprising: (a) transducing T cells with a vector containing 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 the CAR-T cells in a culture medium; and (f) collecting the CAR-T cells or T cell receptor (TCR) cells.

2. A method for producing a T cell therapeutic agent, wherein (a) CD3 ＋ (b) Obtain a sample containing a population of T cells, and (b) in a culture medium containing human interleukin 21 (IL-21) the CD3 ＋ Culturing T cells and (c) CD3 ＋ (d) Activating T cells and the CD3 ＋ A method comprising: (a) transducing T cells with a vector containing 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 the CAR-T cells or T cell receptor (TCR) cells in a culture medium; and (f) collecting the CAR-T cells or T cell receptor (TCR) cells.

3. A method for expanding a population of T cells, comprising: (a) isolating CD4 ＋ and CD8 ＋ T cells from a sample and forming a population of CD3 ＋ T cells; (b) culturing the CD3 ＋ T cells in a culture medium containing human interleukin 21 (IL-21); (c) activating the CD3 ＋ T cells; (d) transducing the CD3 ＋ T cells with a vector containing 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 the CAR-T cells or T cell receptor (TCR) cells in a 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. Part (d) is the CD3 ＋ The method according to any one of claims 1 to 3, comprising transducing T cells with a vector containing nucleic acid encoding CAR to produce CAR-T cells.

6. Part (d) is the CD3 ＋ The method according to any one of claims 1 to 3, comprising transducing T cells with a vector containing nucleic acid encoding TCR to produce TCR cells.

7. Approximately 1×10 ６ ~Approx. 1×10 ９ 3 CDs ＋ The method according to any one of claims 1 to 3, wherein T cells are cultured in the culture medium in step (b).

8. The method according to any one of claims 1 to 3, wherein the sample is a concentrated apheresis product collected via leukocyte apheresis.

9. The CD3 in step (c) ＋ The method according to any one of claims 1 to 3, wherein T cells are cultured for about one day or about two days.

10. The method according to any one of claims 1 to 3, 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 3, wherein the CD3+ T cells in step (c) are activated by magnetic microbeads.

12. The method according to any one of claims 1 to 3, 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 according to claim 12, wherein the anti-CD3 antibody or its CD3-binding fragment and the anti-CD28 antibody or its CD28-binding fragment are bound to magnetic microbeads.

14. The method according to any one of claims 1 to 3, wherein the CAR-T cells or TCR cells are cultured for about 2 to about 10 days in step (e).

15. The method according to any one of claims 1 to 3, wherein the CAR-T cells or TCR cells are cultured for about 4 to about 6 days in step (e).

16. The method according to claim 15, wherein the CAR-T T cells are cultured for about 4 days in step (e).

17. The method according to claim 15, wherein the CAR-T cells or TCR cells are cultured for about 6 days in step (e).

18. The method according to claim 4, 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 according to any one of claims 1 to 3, wherein the concentration of human IL-21 is approximately 0.19 U / mL.

20. The method according to claim 19, wherein the culture medium further comprises human interleukin-2 (IL-2), and the concentration of human IL-2 is about 40 IU / mL.

21. The CD3 ＋ The method according to any one of claims 1 to 3, wherein the T cells are stirred during step (b).

22. A method for producing a T cell therapeutic agent, comprising (a) a sample from CD4 ＋ and CD8 ＋ T cells were isolated and CD3 ＋ (b) Forming a population of T cells and (b) the CD3 ＋ (c) The T cells are cultured in a culture medium containing human interleukin 2 at a concentration of 40 IU / mL and human interleukin 21 at a concentration of 0.19 U / mL, and (c) the CD3 ＋ (d) Activating T cells with magnetic beads containing an anti-CD3 antibody or its CD3-binding fragment, and an anti-CD28 antibody or its CD28-binding fragment, and (d) the CD3 ＋ A method comprising: (a) transducing T cells with a lentiviral vector virus containing nucleic acid encoding a chimeric antigen receptor (CAR) to produce CAR-T cells; (b) culturing the CAR-T cells in a culture medium for about 4 days; and (f) collecting the CAR-T cells.

23. The CD4 ＋ and CD8 ＋ The method according to claim 3 or 22, wherein T cells are isolated by positive selection.

24. The method according to any one of claims 1 to 3 and 22, wherein the vector is a virus, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), transposon, DNA vector, mRNA, lipid nanoparticles (LNP), or CRISPR-Cas system.

25. The method according to any one of claims 1 to 3 and 22, wherein the vector is a lentivirus.

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

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

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

29. The method according to any one of claims 1 to 3 and 22, wherein the volume of the cell culture medium increases after step (d).

30. The method according to claim 29, wherein the volume of the cell culture medium is increased by at least six times.

31. The method according to any one of claims 1 to 3 and 22, 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 3 and 22, wherein the culture medium in step (e) is replaced every 12 hours.

33. The method according to any one of claims 1 to 3 and 22, wherein the CAR-T cells or TCR cells are proliferated by at least about 1 to about 5 times during step (e).

34. The method according to any one of claims 1 to 3 and 22, wherein the CAR-T cells or TCR cells are proliferated at least about 1 to about 3 times during step (e).

35. The method according to claim 34, wherein the CAR-T cells or TCR cells are proliferated by approximately twofold during step (e).

36. The method according to claim 34, wherein the CAR-T cells or TCR cells are proliferated by approximately three times during step (e).

37. The method according to any one of claims 1 to 3 and 22, wherein the CAR is bound to STEAP2 or glypican-3 (GPC3).

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

46.

39. The method according to 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 with respect to the amino acid sequence described in Sequence ID No.

9.

40. The method according to any one of claims 1 to 3 and 22, wherein the CAR encodes an antigen-binding domain that binds to GPC3, the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises CDR1 containing the amino acid sequence of SEQ ID NO: 112, CDR2 containing the amino acid sequence of SEQ ID NO: 113, and CDR3 containing the amino acid sequence of SEQ ID NO: 114, and the VL comprises CDR1 containing the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 118, CDR2 containing the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 119, and CDR3 containing the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO:

120.

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

111.

42. The method according to any one of claims 1 to 3 and 22, wherein the nucleic acid also encodes a protective molecule.

43. The method according to claim 42, wherein the protective molecule comprises a dominant-negative TGFβ receptor type 2 (TGFβRIIDN).

44. The method according to claim 42, wherein the protective 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 respect to the amino acid sequence described in SEQ ID NO:

105.

45. The method according to claim 44, wherein the protective molecule comprises the amino acid sequence described in SEQ ID NO:

105.

46. The method according to any one of claims 1 to 3 and 22, wherein the CAR-T cells or TCR cells are formulated in an isotonic solution.

47. The method according to claim 46, wherein the isotonic solution comprises a plasma light containing human serum albumin.

48. The isotonic solution is approximately 1 × 10 ６ ~Approx. 1×10 ９ The method according to claim 46, comprising CAR-T cells or TCR cells.

49. The aforementioned isotonic solution is approximately 3.4 × 10 ６ The method according to claim 48, comprising 1 CAR-T cell or TCR cell.

50. The CAR-T cells or TCR cells are T ＣＭ and T ＳＣＭ The method according to any one of claims 1 to 3 and 22, wherein the method is a mixture of cells.

51. Approximately 15% to 50% of the aforementioned CAR-T cells or TCR cells are T ＳＣＭ The method according to claim 50, wherein the cell expresses CD45RA, CCR7, and CD27, but does not express CD45RO.

52. Approximately 20% to 30% of the aforementioned CAR-T cells or TCR cells are T ＳＣＭ The method according to claim 51, wherein the cell expresses CD45RA, CCR7, and CD27, but does not express CD45RO.

53. The method according to any one of claims 1 to 3 and 22, wherein more than 50% of the CAR-T cells or TCR cells express a chimeric antigen receptor or a T cell receptor.

54. The method according to 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 according to any one of claims 1 to 3 and 22, wherein more than 50% of the CAR-T cells or TCR cells express CD8.

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

57. The method according to any one of claims 1 to 3 and 22, wherein the CAR-T cells or TCR cells have an oxygen consumption rate (OCR) of more than 100 pmol / min.

58. The method according to any one of claims 1 to 3 and 22, wherein the CAR-T cells or TCR cells have an OCR of about 50 pmol / min to about 200 pmol / min.

59. The method according to any one of claims 1 to 3 and 22, wherein the CAR-T cells or TCR cells have an extracellular acidification rate (ECAR) of more than 30 mpH / min.

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

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