How to generate more efficient CAR-T cells

Abstract

The present invention describes a novel method for obtaining more effective CAR-T cells. The present inventors have long been studying the function of FOXO1 in T lymphocyte physiology. Recently, using pharmacological FOXO1 inhibitors, the present inventors have demonstrated that acute blockade of this transcription factor induces dramatic metabolic modifications in resting T cells, particularly allowing their infection by lentiviruses (Roux et al., 2019). The tolerance induced by FOXO1 inhibition led the present inventors to investigate the possibility of generating CAR-T cells lacking the exhaustion properties of conventional CAR-T cells through simple pharmacological treatment in ex vivo culture. The inventors have shown for the first time that inhibition of FOXO1 enhances the ability to induce target cell lysis by increasing the expression of TNF-α and other inflammatory cytokines, induces spontaneous cell polarization comparable to that obtained by stimulation with chemokines, thus improving cell motility and inducing the proliferation of memory T cells, making it possible to obtain CAR-T cells that are more efficient for treating solid tumors than conventional CAR-T cells obtained by known protocols. Accordingly, the present invention relates to an ex vivo method for obtaining improved CAR-T cells, comprising: i) culturing T cells obtained from a subject with a FOXO1 inhibitor for 2 to 10 days; and ii) transforming the T cells into CAR-T cells by known methods.

Description

[Technical Field]

[0001] The present invention relates to an ex vivo method for obtaining improved CAR-T cells, comprising: i) culturing T cells obtained from a subject with a FOXO1 inhibitor for 2 to 10 days; and ii) transforming the T cells into CAR-T cells by known methods. [Background technology]

[0002] Chimeric antigen receptor T cells (CAR-T cells) are a promising therapeutic approach for cancer treatment. CARs are synthetic immune receptors that combine an antigen-binding domain, typically a single-chain variable fragment (scFv), with a T-cell signaling domain to confer MHC-unrestricted specificity to defined cell surface antigens to T cells. Clinical trials have demonstrated the excellent activity of CD19 CAR-T cells against B-cell malignancies, and the US Food and Drug Administration and European Medicines Agency recently approved CAR-T cell therapy for the treatment of patients with B-cell precursor ALL, diffuse large B-cell lymphoma, and primary mediastinal large B-cell lymphoma (Schuster et al., 2019; Neelapu et al., 2017). Although this treatment has shown remarkable success in some hematological cancers, relapse occurs in 30% of patients.

[0003] Furthermore, the efficacy of this treatment for solid tumors remains limited (Majzner and Mackall, 2019). Numerous studies have demonstrated that the ex vivo expansion step required for T cell transduction with lentiviral particles induces low engraftment potential of CAR-T cells, leading to a loss of antitumor activity (Klebanoff AC et al., 2017; Ghassemi et al., 2018). This adverse effect could be mitigated by inhibiting signaling pathways recruited during the expansion step required for CAR-T cell transduction and expansion (Bowers et al., 2017; Crompton et al., 2015; Klebanoff et al., 2017; Urak et al., 2017; van der Waart et al., 2014). An alternative approach would be to induce CAR expression in resting T cells in the absence of any stimuli, with the aim of reducing the loss of antitumor activity associated with ex vivo T cell activation and expansion, as shown by Ghassemi et al., 2022. Summary of the Invention

[0004] The present inventors have long been studying the function of FOXO1 in T lymphocyte physiology. Using a FOXO1 pharmacological inhibitor, the present inventors recently described how acute blockade of this transcription factor induces dramatic metabolic modifications in quiescent cells, particularly enabling their infection by lentiviruses (Roux et al., 2019). In this context, they recently used AS1842856 as a tool to investigate FOXO1 regulation of the expression of some of its less well-studied targets in human T lymphocytes. During their experiments, they observed that AS1842856 treatment of human T cells purified from healthy donors, in the absence of any growth factors (e.g., cytokines), after several days of culture, substantially increased their metabolic activity, which correlated with the acquisition of activated / memory T cell phenotypic and functional characteristics. More specifically, they found that AS1842856 significantly increased the expression of CD8 T cells. + Not only within T cells but also CD4 +It induces increased intracellular production of granzyme B, resulting in the production of human CD8 + It was found to result in enhanced cytotoxic activity of primary T lymphocytes.

[0005] The tolerance induced by FOXO1 inhibition led us to investigate the possibility of generating CAR-T cells lacking the exhaustion properties of conventional CAR-T cells through simple pharmacological treatment in vitro. We found that inhibition of FOXO1 with the pharmacological agent AS1842856 enabled the acquisition of phenotypic and functional properties that led to not only resting T cell infection but also significantly enhanced CAR-T cell antitumor activity. Specifically, we demonstrated for the first time that FOXO1 inhibition enhanced the ability to induce target cell lysis by increasing the expression of TNF-α and other inflammatory cytokines, induced spontaneous cell polarization comparable to that achieved by chemokine stimulation, and thus improved cell motility and induced memory T cell proliferation, enabling the generation of more efficient CAR-T cells for treating solid tumors compared to conventional CAR-T cells obtained by known protocols.

[0006] Accordingly, the present invention relates to an in vitro method for obtaining improved CAR-T cells, comprising the steps of: i) culturing T cells obtained from a subject with a FOXO1 inhibitor for 2 to 10 days; and ii) transforming the T cells into CAR-T cells by known methods. In particular, the present invention is defined by its claims.

[0007] [Detailed Description of the Invention] FOXO1 inhibitors may be an invaluable tool for use in therapies requiring ex vivo antitumor T cell protocols to obtain CAR-T cells. In particular, FOXO1 inhibitors may be very useful in protocols for obtaining CAR-T cells, eliminating the need for T cell activation and proliferation steps, meaning that cellular TCR activators, such as antibodies directed against CD3 and CD28 molecules, are not required. The inventors have demonstrated that FOXO1 inhibition not only enables the infection of resting T cells but also allows them to acquire phenotypic and functional characteristics (particularly fewer exhaustion markers) that lead to a significant increase in the antitumor activity of CAR-T cells. The inventors have also shown that T cells obtained using our protocol continue to proliferate homeostatically after in vivo injection to induce CAR expression. In other words, our novel protocol allows for faster CAR-T cell generation, as CAR expression can be induced by proliferation and no proliferation or activation steps are required, and as explained above, allows for more efficient CAR-T cell generation.

[0008] Thus, in a first aspect, the present invention provides an in vitro method for obtaining improved CAR-T cells, comprising: i) culturing T cells obtained from the subject with a FOXO1 inhibitor for 2 to 10 days; ii) transforming the T cells into CAR-T cells by known methods; The present invention relates to a method comprising:

[0009] Thus, in certain embodiments, the present invention provides an ex vivo method for obtaining improved CAR-T cells, comprising: i) providing T cells from a subject; ii) culturing the T cells with a FOXO1 inhibitor for 2 to 10 days; iii) transforming the T cells into CAR-T cells by known methods; Contains Regarding the method.

[0010] Following the FOXO1 inhibitor protocol, the resulting CAR-T cells can be injected into a subject in need thereof.

[0011] Thus, in the methods of the present invention, the transformation of T cells into CAR-T cells is carried out without prior activation.

[0012] Thus, in the methods of the present invention, transformation of T cells into CAR-T cells is performed without prior ex vivo activation (e.g., activation with anti-CD3 and / or anti-CD28 antibodies) and expansion, thus preventing activation-induced differentiation of T cells.

[0013] In certain embodiments, the T cells were harvested from a subject prior to the methods of the invention. In certain embodiments, the T cells were harvested from a subject and stored (frozen, i.e., prepared in cryopreservation medium) prior to the methods of the invention.

[0014] In certain embodiments, the method comprises the additional step of adding IL-7 and / or IL-15 after using the inhibitor of FOXO1.

[0015] The present invention provides an in vitro method for obtaining improved CAR-T cells, comprising: i) providing T cells from a subject; ii) culturing the T cells in culture with a FOXO1 inhibitor for 2 to 10 days; iii) adding IL-7 and / or IL-15 to the culture medium; iv) transforming the T cells into CAR-T cells by known methods; The present invention also relates to a method comprising:

[0016] The methods described above allow these methods to be carried out in vitro.

[0017] In certain embodiments, IL-7 and / or IL-15 are administered simultaneously with a FOXO1 inhibitor.

[0018] As used herein, the term T cell refers to, for example, CD3 + T cells, CD4 + T cells, CD8 + By this is meant T cells, TILs T cells (tumor infiltrating lymphocyte T cells), NKT cells, which can be isolated from peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs), or from a biopsy if the cells are, for example, TILs.

[0019] In certain embodiments, the T cells are resting T cells (i.e., T cells that have not been activated via the T cell receptor (TCR) or co-receptors, such as CD3 and / or CD28). In certain embodiments, the T cells are resting cells.

[0020] As used herein, the term "resting T cells" has its general meaning in the art and refers to non-proliferating, non-dividing, or resting T cells that are in the G0 stage of the cell cycle. T cells may be naturally resting.

[0021] In certain embodiments, the medium is a medium suitable for culturing T cells, i.e., a medium optimized for culturing T cells. Media suitable for culturing T cells are commercially available and include, but are not limited to, RPMI 1640 basal medium supplemented with or without 10% human serum, particularly human AB serum.

[0022] According to the present invention, the T cells may be in contact with the FOXO1 inhibitor for 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0023] After the protocol is completed and the CAR-T cells are obtained, these cells are injected into a subject in need thereof.

[0024] According to the present invention, the concentration of the FOXO1 inhibitor is 50-1000 nM. In particular, the concentration is 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nM. In particular, the concentration is 500 nM.

[0025] According to the present invention, the number of T cells collected in the body of a subject in need and used in the method of the present invention is 10 per kg. 4 ~10 9 In particular, the density of T cells used in the protocol is 5×10 6 There are individuals.

[0026] In other words, to obtain an adequate number of T cells, the amount of blood collected from a subject in need is between 5 and 100 ml. Particularly, the amount of blood collected is 5, 10, 20, 30, 40 or 50 ml.

[0027] As used herein, "FOXO1" belongs to the forkhead box class O transcription factors, which are known to be key molecules for regulating and maintaining cellular quiescence in various cell types. In unstimulated cells, these transcription factors reside in the cell nucleus in an unphosphorylated and active state, thus maintaining the transcription of many genes. These transcription factors act as master regulators for linking signals delivered by growth factors to molecular events leading to cell growth and cell division. FOXO1 corresponds to the most abundant FOXO molecule present in T cells (Entrez Gene ID No.: 2308).

[0028] As used herein, "FOXO1 inhibitor" refers to an inhibitor that induces a transition from the quiescent G0 to G1 stage of the cell cycle. For example, the AS1842856 compound exerts its effect by inhibiting FOXO1 binding to DNA. Without altering the phosphorylation status or expression of FOXO1, AS1842856 appears to preserve the role of FOXO1 in chromatin remodeling. Use of the inhibitors of the present invention induces a stem cell memory phenotype (TSCM) along with elevated granzyme B expression and increased tumor necrosis factor alpha secretion. After treatment of T cells with a FOXO1 inhibitor, the cells have enhanced proliferation capacity, improved cytotoxic potential, improved migratory properties, and improved tumor eradication efficiency in vivo.

[0029] [CAR-T cells] As used herein, the term "chimeric antigen receptor (CAR)" refers to an artificial T cell receptor, chimeric T cell receptor, or chimeric immune receptor, and encompasses engineered receptors that graft artificial specificity onto specific immune effector cells, i.e., T cells of the present invention.

[0030] CARs typically comprise an ectodomain (extracellular domain) and an endodomain (cytoplasmic domain) joined by a transmembrane domain. The ectodomain, which is expressed on the surface of a cell, contains an antigen-binding or receptor domain and, optionally, a spacer (or hinge) region that connects the antigen-binding domain to the transmembrane domain. The transmembrane domain is typically a hydrophobic alpha helix that spans the lipid bilayer of the cell membrane. The endodomain of a CAR consists of an intracellular signaling module that triggers cell activation upon antigen binding. The endodomain may contain several signaling domains, as described below.

[0031] [Antigen-binding domain] The extracellular domain of the CAR comprises an antigen-binding domain that specifically binds or recognizes a target antigen.

[0032] As used herein, "bind" or "binding" refers to peptides, polypeptides, proteins, fusion proteins, and antibodies (including antibody fragments) that recognize and contact an antigen. Preferably, it refers to an antigen-antibody type interaction. "Specific binding" means that the antigen-binding domain of a CAR recognizes a particular antigen but does not substantially recognize or bind to other molecules in a given sample. "Specific binding" is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope). As used herein, the term "specific binding" refers to contact between the antigen-binding domain of a CAR and an antigen with a binding affinity of at least 10 M. In some embodiments, the antigen-binding domain of a CAR binds with an affinity of at least about 10 M, and preferably 10 M, 10 M, 10 M, or 10 M. Binding affinity can be measured by any method available to those skilled in the art, particularly surface plasmon resonance (SPR).

[0033] In one embodiment, such an antigen-binding domain is an antibody, preferably a single-chain antibody. Preferably, the antibody is a humanized antibody. In particular, such an antigen-binding domain is an antibody fragment selected from a fragment antigen-binding (Fab) fragment, a F(ab')2 fragment, a Fab' fragment, an Fv fragment, a recombinant IgG (rIgG) fragment, a single-chain antibody fragment, a single-chain variable fragment (scFv), a single-domain antibody (e.g., sdAb, sdFv, nanobody) fragment, a bispecific antibody, and a multispecific antibody formed from antibody fragments. In a specific embodiment, the antibody is a single-chain antibody fragment, such as an scFv, comprising a variable heavy chain region and / or a variable light chain region. In particular, such an antigen-binding domain is selected from Fab and scFv.

[0034] In embodiments in which the antigen-targeting domain is an scFv, the scFv can be derived from the variable heavy (VH) and variable light (VL) regions of an antigen-specific mAb linked by a flexible linker. The scFv retains the same specificity and similar affinity as the intact antibody from which it is derived. The peptide linker connecting the scFv VH and VL domains joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising VH-VL pairing and the fidelity of the antigen-binding site. The length of the peptide linker can range from 10 to 30 amino acids. In one embodiment, the scFv peptide linker is a Gly / Ser linker and contains one or more repeats of these amino acids.

[0035] The extracellular domain of the CAR may comprise one or more antigen-binding domains.

[0036] In certain embodiments, the CAR specifically binds to a tumor-associated antigen (TAA). In particular, the CAR specifically binds to any TAA expressed on the surface of tumor cells, particularly CD19, GD2, EGFR, CD20, CD22, CD33, CD138, CD52, CD30, ROR1, HER2, EpCAM, MUC-1, MUC5AC, BCMA, CD38, SLAMF7 / CS1, CD123, IL-13Ra2, LeY, MUC16, PSMA, and more preferably, the TAA is CD19, CD20, CD22, CD33, CD138, BCMA, CD38, SLAMF7 / CS1, IL-13Ra2, HER2, or EGFR.

[0037] In another specific embodiment, the CAR targets an intracellular oncoprotein or intracellular tumor-associated antigen, in particular WT-1, NY-ESO-1, MAGE, PRAME, RAS, mesothelin, c-Met, CEA, CSPG-4, EBNA3C, CA-125, or GPA7. In particular, said intracellular oncoprotein or tumor-associated antigen is expressed on the cell surface as a peptide that is processed and bound to a histocompatibility (HLA) molecule.

[0038] The terms "tumor-associated antigen," "TAA," "tumor antigen," and "cancer cell antigen" are used interchangeably herein. In each instance, these terms refer to a peptide, protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0039] The term "antigen" as used herein has its general meaning in the art and generally refers to a substance or fragment thereof that is recognized and selectively bound by an antibody or T-cell antigen receptor, resulting in the induction of an immune response. Antigens according to the present invention are typically, but not exclusively, peptides and proteins. Antigens may be natural or synthetic and generally induce an immune response that is specific to that antigen.

[0040] The term "HLA-A2" as used herein has its general meaning in the art and refers to an HLA serotype that falls within the HLA-A'A' serotype group, and * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:05, HLA-A * 02:06, HLA-A * 02:07, and HLA-A * 02:11HLA-A including gene products * HLA-A2 is encoded by the 02 allele group. HLA-A2 is very common in the Caucasian population (40-50%), and HLA-A2 + It provides an ideal cellular target for the first portion, as it is suitable for use in a high percentage of donor and HLA-A2-recipient combinations.

[0041] As used herein, the terms "antibody" and "immunoglobulin" have the same meaning and are used equivalently in the present invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds an antigen. Thus, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes), namely, IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of antibody molecules. Each chain contains distinct sequence domains. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain three (α, δ, γ) to five (μ, ε) domains: one variable domain (VH) and three to four constant domains (CH1, CH2, CH3, and CH4, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine antigen binding recognition and specificity. The constant domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region. Antibody specificity resides in the structural complementarity between the antibody combining site and antigenic determinants. The antibody combining site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FR) can participate in antibody binding or influence the overall domain structure and thus the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.The light and heavy chains of an immunoglobulin each have three CDRs designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, comprising the CDR sets from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequences interposed between the CDRs. Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al., as described in Kabat et al., 1987, "Sequences of Proteins of Immunological Interest," US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering system is used herein. Kabat residue designations do not always directly correspond to the linear numbering of amino acid residues within a sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than strictly indicated by Kabat numbering, corresponding to the shortening of, or insertion into, a structural component of the basic variable domain structure, whether framework or complementarity-determining region (CDR). The correct Kabat residue numbering may be determined for a given antibody by aligning homologous residues within the antibody's sequence with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0042] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0043] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0044] As used herein, the term "chimeric antibody" refers to an antibody comprising the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In some embodiments, a "chimeric antibody" is an antibody molecule in which (a) the constant regions (i.e., heavy and / or light chains), or portions thereof, have been modified, substituted, or exchanged such that the antigen-binding site (variable region) is linked to constant regions of a different or modified class, effector function, and / or species, or to entirely different molecules, such as enzymes, toxins, hormones, growth factors, drugs, etc., that confer new properties to the chimeric antibody; or (b) the variable regions, or portions thereof, have been modified, substituted, or exchanged with variable regions having different or modified antigen specificities. Chimeric antibodies also include primatized, particularly humanized, antibodies. Furthermore, chimeric antibodies may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). (See U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0045] As used herein, the term "humanized antibody" refers to an antibody having variable region framework and constant regions derived from a human antibody while retaining the CDRs of the original non-human antibody. In some embodiments, humanized antibodies contain minimal sequence derived from a non-human immunoglobulin. Humanized antibodies and antibody fragments may largely consist of a human immunoglobulin (recipient antibody or antibody fragment) in which residues from the recipient's complementarity-determining region (CDR) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding region is derived, while avoiding an immune response against the non-human antibody. These modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0046] As used herein, the term "antibody fragment" refers to at least a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody, that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). A "fragment" includes a portion of an intact antibody, generally the antigen-binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; bispecific antibodies; any antibody fragment that is a polypeptide having a primary structure consisting of a single uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including, but not limited to, (1) a single-chain Fv molecule; (2) a single-chain polypeptide comprising only a light-chain variable domain, or a fragment thereof comprising the three CDRs of a light-chain variable domain, without the associated heavy-chain portion; and (3) a single-chain polypeptide comprising only a heavy-chain variable region, or a fragment thereof comprising the three CDRs of a heavy-chain variable region, without the associated light-chain portion; and multispecific antibodies formed from antibody fragments. Such antibody fragments can be obtained using standard methods.

[0047] As used herein, the term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv as used herein may have the VL and VH variable regions in either order; for example, in relation to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or VH-linker-VL.

[0048] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind a target molecule (e.g., an epitope presented on an antigen) while having relatively little detectable reactivity with other target molecules. Specificity can be relatively determined by binding or competitive binding assays, for example, using a Biacore instrument, as described elsewhere herein. Specificity can be demonstrated by an affinity / avidity ratio between binding to a particular antigen and nonspecific binding to other unrelated molecules, for example, of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1, or more.

[0049] As used herein, the term "affinity" refers to the strength of binding of an antibody to a target molecule (e.g., an epitope). The affinity of a binding protein is given by the dissociation constant, Kd. For antibodies, Kd is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant, Ka, is defined as 1 / Kd. Preferred methods for determining the affinity of a binding protein can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. One preferred standard method well known in the art for determining the affinity of a binding protein is the use of a Biacore instrument.

[0050] The term "binding" as used herein refers to a direct association between two molecules resulting from covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including, for example, salt and water bridges. In particular, the term "binding" as used herein in the context of antibody binding to a predetermined target molecule (e.g., an antigen or epitope) typically refers to binding with an affinity corresponding to a KD of about 10 M or less, e.g., about 10 M or less, e.g., about 10 M or less, about 10 M or less, or even 10 M or less.

[0051] [Spacer or hinge domain] The CAR optionally comprises a spacer or hinge domain that links the antigen binding domain to the transmembrane domain.

[0052] In some embodiments, the CAR comprises a hinge sequence between the antigen-binding domain and the transmembrane domain and / or between the transmembrane domain and the cytoplasmic domain. Those skilled in the art will recognize that a hinge sequence is a short amino acid sequence that facilitates flexibility.

[0053] In particular, the spacer or hinge domain that links the antigen-binding domain to the transmembrane domain is designed to have sufficient flexibility to allow the antigen-binding domain to be oriented in a manner that allows antigen recognition.

[0054] The hinge may be derived from or comprise at least a portion of an immunoglobulin Fc region, such as, for example, an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In some embodiments, the hinge domain comprises at least a portion of an IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA immunoglobulin Fc region that falls within the CH2 and CH3 domains.

[0055] Exemplary hinges include, but are not limited to, a CD8a hinge, a CD28 hinge, an IgG1 / IgG4 (hinge-Fc portion) sequence, an IgG4 hinge alone, an IgG4 hinge linked to a CH2 and CH3 domain, or an IgG4 hinge linked to a CH3 domain, as described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, WO 2014 / 031687, U.S. Pat. No. 8,822,647, or U.S. Pat. App. Pub. No. 2014 / 0271635. The present invention relates to the use of all or a portion of residues 118-178 of CD8a (GenBank Accession No. NP_001759.3), residues 135-195 of CD8 (GenBank Accession No. AAA35664), residues 315-396 of CD4 (GenBank Accession No. NP_000607.1), or residues 137-152 of CD28 (GenBank Accession No. NP_006130.1) as the hinge domain. Furthermore, a portion of the antibody H-chain or L-chain constant region (CHI region or CL region) can be used as the spacer domain. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0056] For example, in some embodiments, the hinge sequence is derived from the CD8 alpha molecule or the CD28 molecule.

[0057] [Transmembrane domain] The transmembrane domain of the CAR functions to anchor the receptor on the cell surface. The choice of transmembrane domain can depend on adjacent spacer and intracellular sequences.

[0058] In some embodiments, the transmembrane domain is derived from either natural or synthetic sources. If the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., at least the transmembrane regions of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, synthetic transmembrane domains primarily comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. The transmembrane domain is thermodynamically stable in the membrane. It can be a single alpha helix, a transmembrane beta barrel, a gramicidin A beta helix, or any other structure.

[0059] Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine doublet may provide a suitable linker.

[0060] [Intracellular domain] The terms "intracellular domain," "cytoplasmic domain," and "intracellular signaling domain" are used interchangeably herein. The role of the intracellular domain of a CAR is to generate an activation signal to the T cell immediately upon antigen recognition by the extracellular domain.

[0061] Examples of intracellular domain sequences that are particularly useful in the present invention include those derived from the intracellular signaling domains of lymphocyte receptor chains, TCR / CD3 complex proteins, Fc receptor subunits, IL-2 receptor subunits, CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD278 (ICOS), FcsRI, DAP10, and DAP12. It is highly preferred that the intracellular domain in the CAR comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0062] The intracellular domain of the CAR can be designed to include a signaling domain (e.g., a CD3ζ signaling domain) alone or in combination with a costimulatory domain. Costimulatory molecules can be defined as cell surface molecules required for efficient lymphocyte responses to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD244 (2B4), ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. The intracellular signaling domains of the costimulatory domains listed above can be used alone or in combination with other costimulatory domains. In particular, a CAR can comprise any combination of two or more costimulatory domains from the group consisting of CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD244 (2B4), ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.

[0063] Thus, for example, a CAR can be designed to include a signaling domain such as a CD3ζ signaling domain and two costimulatory signaling domains selected from CD28 and CD40, CD28 and 4-1BB (CD137), CD28 and OX40 (CD134), and CD28 and LFA-1.

[0064] A "first-generation CAR" contains a single signaling domain. A CAR containing one signaling domain together with one additional costimulatory domain is called a "second-generation CAR," while a CAR containing one signaling domain together with two additional costimulatory domains is listed as a "third-generation CAR." For example, a first-generation CAR contains only the CD3ζ chain as a single signaling domain. Second- and third-generation CARs are each composed of one or two additional costimulatory signaling domains, such as CD28, CD27, OX-40 (CD134), and 4-1BB (CD137). For example, a second-generation CAR may contain CD3ζ and CD28 signaling domains, while a third-generation CAR may contain CD3ζ, CD28, and OX40 (CD134) or 4-1BB (CD137).

[0065] The CAR of the present invention may be a first-, second-, or third-generation CAR as described above. Preferably, the CAR-T cell is a second- or third-generation CAR.

[0066] "TRUCK" refers to a recently developed "fourth generation" CAR. TRUCK (T cells redirected for universal cytokine killing) is a CAR-redirected T cell used as a vehicle to produce and release a transgenic product that accumulates in targeted tissues. The product, e.g., a pro-inflammatory cytokine, can be constitutively produced or induced once the T cell is activated by the CAR. Other substances, such as enzymes or immunomodulatory molecules, may also be produced and deposited by the CAR-redirected T cell within the target lesion. This strategy involves two separate transgenes expressing a cell activation-responsive promoter linked to, for example, (i) the CAR-T cell and (ii) a cytokine such as IL-12. As a result, an immunostimulatory cytokine such as IL-12 is secreted upon CAR engagement.

[0067] In certain embodiments, the CAR-T cells are fourth generation CAR-T cells as defined above.

[0068] [Methods for obtaining CAR-T cells] Methods and protocols for obtaining CAR-T cells are well known in the art. To obtain CAR-T cells from T cells, transfection, transposon systems such as the sleeping beauty method, or lentiviral infection can be used (see, for example, Martinez Marina et al., 2019).

[0069] Methods for obtaining CAR-T cells using lentiviruses capable of transducing T cells are well known. For example, as shown herein, a lentivirus stock with an MOI of 2 can be used. Protocols for obtaining CAR-T cells are well known in the art (see, for example, Okuma Atsushi, 2021. Generation of CAR-T Cells by Lentiviral Transduction).

[0070] Another method for deriving CAR-T cells from T cells is called sleeping beauty, which uses DNA transposons to transfect cells (see, e.g., Izsvak et al., 2010).

[0071] According to the present invention, the CAR-T cells may be CAR-T cells of first, second, third or fourth generation origin.

[0072] According to the present invention, the methods and protocols used to obtain CAR-T cells do not involve any activation step (e.g., activation with anti-CD3 and / or anti-CD28 antibodies) prior to transformation of the T cells.

[0073] In certain embodiments, a polynucleotide encoding a CAR is introduced into a T cell (via transfection or transduction) to obtain a CAR-T cell.

[0074] As used herein, the terms "transformation," "transfection," or "transduction" refer to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a T cell, causing the T cell to express the introduced gene or sequence and produce the desired substance, i.e., the CAR encoded by the introduced gene or sequence. A T cell that receives and expresses the introduced DNA or RNA is already "transformed."

[0075] Thus, in certain embodiments, the present invention provides an in vitro method for obtaining improved CAR-T cells, comprising: i) culturing T cells obtained from a subject in a medium containing a FOXO1 inhibitor for 2 to 10 days; ii) introducing a polynucleotide encoding a CAR into a T cell to obtain a CAR-T cell; The present invention relates to a method comprising:

[0076] In certain embodiments, the method comprises the additional step of adding IL-7 and / or IL-15 into the culture medium after using the inhibitor of FOXO1.

[0077] It is contemplated that the polynucleotide encoding the CAR can be introduced into the T cell as naked nucleic acid (DNA or RNA) or in a suitable vector.

[0078] Naked DNA generally refers to DNA contained within a plasmid expression vector in the proper orientation for expression. Physical methods for introducing polynucleotide constructs into T cells include particle bombardment, nucleofection, colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0079] In some embodiments, the polynucleotide encoding the CAR is introduced into T cells by a viral vector, such as an adeno-associated virus (AVV), retrovirus, lentivirus, bovine papilloma virus, adenovirus vector, vaccinia virus, polyoma virus, or infectious virus. In certain embodiments, the vector is retroviral. Retroviruses can be selected as gene delivery vectors due to their ability to integrate their genes into the host genome to deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines. To construct a retroviral vector, the polynucleotide of interest is inserted into the viral genome in place of certain viral sequences to produce a replication-deficient virus. To produce virions, a packaging cell line is constructed that contains the gag, pol, and / or env genes but lacks the LTR and / or packaging components. When a recombinant plasmid containing a cDNA with retroviral LTRs and packaging sequences is introduced into this cell line (e.g., by calcium phosphate precipitation), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium. The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a variety of cell types. Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows the virus to modulate its life cycle, as well as during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV1, HIV2) and simian immunodeficiency viruses (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV virulence genes; for example, genes env, vif, vpr, vpu, and nef have been deleted to render the vector biologically safe. Lentiviral vectors are known in the art.See, for example, U.S. Patent Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. Generally, vectors are plasmid- or virus-based and are configured to carry the necessary sequences for incorporating a foreign polynucleotide, selecting the polynucleotide, and introducing the polynucleotide into a host cell. The gag, pol, and env genes of the vector of interest are also known in the art. Thus, the relevant genes are cloned into the selected vector, which is then used to transform the target cell of interest. This illustrates a first vector capable of providing polynucleotides encoding the viral gag and pol genes for producing packaging cells, and another vector capable of providing a polynucleotide encoding the viral env. Introducing a vector providing a heterologous gene into the packaging cells generates producer cells that release infectious viral particles carrying the foreign gene of interest. The env is preferably an amphotropic envelope protein, allowing for transduction of cells of human and other species. More preferably, the env is from the vesicular stomatitis virus (VSV-G). In some embodiments, the vector is a lentivirus.

[0080] Typically, a suitable vector of the present invention will contain "control sequences," which collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, etc., which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences will always be present, so long as the selected coding sequence is capable of being replicated, transcribed, and translated in T cells. Another polynucleotide sequence is a "promoter" sequence, which is used herein in its conventional sense to refer to a nucleotide region comprising DNA regulatory sequences, where the regulatory sequences are derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. The polynucleotide encoding the CAR of the present invention may be operably linked to an inducible promoter or a retroviral long terminal repeat (LTR), a cytomegalovirus (CMV) promoter, a murine stem cell virus (MSCV) U3 promoter, a phosphoglycerate kinase (PGK) promoter, a beta-actin promoter, a ubiquitin promoter, and a simian virus 40 (SV40) / CD43 composite promoter; an elongation factor (EF)-1α promoter; a myeloproliferative sarcoma virus enhancer, a negative control region deletion, a d1587rev primer binding site substitution (MND) promoter; or a spleen focus forming virus (SFFV) promoter. The novel method of the present invention makes it possible to obtain CAR-T cells when CAR expression is inducible by proliferation. Therefore, preferably, the polynucleotide encoding the CAR of the present invention is not operably linked to an inducible promoter.

[0081] In some embodiments, the sequence of the polynucleotide encoding CAR is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery of the frequency deviations of synonymous codons (i.e., codons that encode the same amino acid) in coding DNA among different species. This codon degeneracy allows the same polypeptide to be coded for by a variety of nucleotide sequences. Various codon optimization methods are known in the art, including at least those disclosed in U.S. Patent Nos. 5,786,464 and 6,114,148.

[0082] As used herein, the term "encoding" refers to the inherent property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties that result therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. The phrase "polynucleotide encoding a CAR" can also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0083] In certain embodiments, the polynucleotide encoding the CAR is introduced into the T cell by a DNA transposon (the "sleeping beauty transposon system").

[0084] The term "DNA transposon" as used herein has its general meaning in the art and refers to a DNA transfer vehicle capable of efficient genome insertion. DNA transposons translocate from one DNA site to another in a simple cut-and-paste manner. The Sleeping Beauty transposon system, as disclosed in Izsvak et al., 2010, is composed of Sleeping Beauty (SB) transposase and a transposon designed to insert a specific DNA sequence into the genome of a vertebrate.

[0085] [FOXO1 inhibitor] In a particular embodiment, the FOXO1 inhibitor is AS1842856.

[0086] The term "AS1842856" refers to a cell-permeable inhibitor that blocks the transcriptional activity of FOXO1 and is specific for FOXO1.

[0087] In another embodiment, the FOXO1 inhibitor can be tanzawaic acid D, hymenidine, cribrostatin 6, barbamide, and compound 10 (Sun Yingjia et al., 2016 and Lee et al., 2021).

[0088] In one embodiment, the inhibitors of the present invention may be low molecular weight compounds, such as small organic molecules (natural or unnatural).

[0089] The term "small organic molecule" refers to a molecule (natural or non-natural) of a size comparable to organic molecules commonly used in pharmaceuticals. This term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). The size of certain small organic molecules ranges from up to about 10,000 Da, more particularly up to 5,000 Da, more particularly up to 2,000 Da, and even more particularly up to about 1,000 Da.

[0090] The present invention provides isolated single domain antibodies that inhibit FOXO1.

[0091] The term "single-domain antibody" as used herein has its general meaning in the art and refers to a single heavy-chain variable region of an antibody of the type found in camelids, which naturally lack light chains. Such single-domain antibodies are also called VHHs or "nanobodies." For a general description of (single) domain antibodies, reference is made to the prior art cited above as well as to EP 0 368 684, Ward et al. (Nature 1989, October 12;341(6242):544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490, and WO 2006 / 030220 and WO 2006 / 003388. Nanobodies have a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of their small size is the ability of camelid nanobodies to bind to antigenic sites functionally inaccessible to larger antibody proteins. Thus, camelid nanobodies are useful as reagents for detecting antigens difficult to detect using conventional immunological techniques and as potential therapeutic agents. Thus, another consequence of their small size is that nanobodies can bind to specific sites in grooves or narrow clefts of target proteins, thereby serving in a capacity that more closely resembles the function of traditional low-molecular-weight drugs compared to traditional antibodies. Their small molecular weight and compact size make nanobodies highly thermally stable, stable to extremes of pH and proteolytic degradation, and less antigenic. Another consequence is that nanobodies can easily move from the circulatory system into tissues and even cross the blood-brain barrier, allowing them to treat disorders affecting nervous tissue. Nanobodies can also facilitate drug transport across the blood-brain barrier. See US Patent Application Publication No. 2004 / 0161738, published August 19, 2004. These characteristics, combined with low antigenicity to humans, represent great therapeutic potential.The amino acid sequence and structure of a single domain antibody can be considered to be composed of four framework regions or "FRs," referred to in the art and herein as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework 4" or "FR4," respectively; these framework regions are interrupted by three complementarity-determining regions or "CDRs," referred to in the art as "complementarity-determining region 1," abbreviated "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. A single domain antibody can therefore be defined as an amino acid sequence with the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity-determining regions 1-3. In the context of the present invention, the amino acid residues of single domain antibodies are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system amino acid numbering (http: / / imgt.cines.fr / ).

[0092] Camel Ig can be genetically engineered to generate small proteins with high affinity for targets, resulting in low molecular weight antibody-derived proteins known as "nanobodies" or "VHHs." See U.S. Patent No. 5,759,808, issued June 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279:1256-1261; Dumoulin, M. et al., 2003 Nature 424:783-788; Pleschberger, M. et al., 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al., 2002 Int J Cancer 89:456-62; and Lauwereys, M. et al., 1998 EMBO J 17:3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. In some embodiments herein, camelid antibodies or nanobodies are naturally produced in camelid animals, i.e., produced by camelids after immunization with an [antigen] or a peptide fragment thereof, using techniques described herein for other antibodies. Alternatively, [antigen]-binding camelid nanobodies are engineered, i.e., produced using panning techniques targeting FOXO1, for example, by selection from a library of phage displaying appropriately mutagenized camelid nanobody proteins.

[0093] In some embodiments, the single domain antibody is a "humanized" single domain antibody.

[0094] The term "humanized" as used herein refers to a single domain antibody of the present invention, wherein the amino acid sequence corresponding to that of a naturally occurring VHH domain has been "humanized," i.e., by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (particularly in the framework sequences) with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional chain antibody of human origin. Methods for humanizing single domain antibodies are well known in the art. Typically, humanization alternatives should be selected such that the resulting humanized single domain antibody still retains the advantageous properties of the single domain antibody of the present invention. Those skilled in the art will be able to determine and select suitable humanization alternatives or suitable combinations of humanization alternatives. For example, the single domain antibody of the present invention may be suitably humanized at any framework residue such that the single domain antibody remains soluble and does not significantly lose its affinity for FOXO1.

[0095] In another embodiment, a FOXO1 inhibitor of the present invention is an inhibitor of foxo1 gene expression.

[0096] Small inhibitory RNAs (siRNAs) can also function as inhibitors of foxo1 expression for use in the present invention. DHODH or Chk1 gene expression can be reduced by contacting a subject or cell with small double-stranded RNA (dsRNA) or a vector or construct that causes the production of small double-stranded RNA, such that foxo1 gene expression is specifically inhibited (i.e., RNA interference or RNAi). Methods for selecting appropriate dsRNA or dsRNA-encoding vectors are well known in the art for genes whose sequence is known (see, e.g., Tuschl, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, ​​TR. et al. (2002); U.S. Patent Nos. 6,573,099 and 6,506,559, and International Patent Publication Nos. 01 / 036646, 99 / 032619, and 01 / 068836).

[0097] Ribozymes can also function as inhibitors of foxo1 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Thus, within the scope of the present invention, engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of DHODH or CHk1 mRNA sequences are useful. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the sequences GUA, GUU, and GUC. Once identified, short RNA sequences of approximately 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, eg, ribonuclease protection assays.

[0098] Both antisense oligonucleotides and ribozymes useful as inhibitors of foxo1 gene expression can be prepared by known methods. These methods include techniques for chemical synthesis, such as solid-phase phosphoramidite chemical synthesis. Alternatively, antisense RNA molecules can be produced by in vitro or in vivo transcription of DNA sequences encoding the RNA molecules. Such DNA sequences can be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as the T7 or SP6 polymerase promoter. Various modifications to the oligonucleotides of the present invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0099] The antisense oligonucleotide siRNA and ribozyme of the present invention can be delivered alone or in combination with vector in vivo.In its broadest sense, " vector " refers to any vehicle that can facilitate the introduction of antisense oligonucleotide siRNA or ribozyme nucleic acid into cells, particularly the cells that express FOXO1.In particular, vector transports nucleic acid to cells with a lower level of degradation than would result in the absence of vector.Generally, the vector that is advantageous in the present invention includes but is not limited to plasmid, phagemid, virus, virus that is engineered by inserting or incorporating antisense oligonucleotide siRNA or ribozyme nucleic acid sequence or other vehicle derived from bacterial source. Viral vectors are a particular type of vector and include, but are not limited to, nucleic acid sequences derived from viruses such as retroviruses; e.g., Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, Rous sarcoma virus; adenovirus, adeno-associated virus, SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; poliovirus; and RNA viruses, such as retroviruses. Other vectors not listed herein but known to those of skill in the art are readily available.

[0100] Certain viral vectors are based on non-cytopathic eukaryotic viruses, in which nonessential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy clinical trials. The most useful retroviruses are replication-deficient (i.e., capable of directing the synthesis of desired proteins but unable to manufacture infectious particles). Such genetically modified retroviral expression vectors are generally useful for highly efficient gene transduction in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retrovirus by the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles) are provided in Kriegler 1990 and Murry, 1991.

[0101] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., 1989. For the past several years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes to cells in vivo. These vectors are highly advantageous for this purpose because they do not have the same safety concerns as many viral vectors. However, these plasmids contain promoters compatible with the host cell and can express peptides from genes operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. Furthermore, plasmids can be custom designed to remove or add specific fragments of DNA using restriction enzymes and ligation reactions. Plasmids can be delivered via a variety of parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intraocularly, intradermally, subcutaneously, or by other routes. It may also be administered via nasal spray or drops, rectal suppositories, and orally. It may also be administered into the epidermis or mucosal surfaces using a gene gun. The plasmid may be provided in the form of an aqueous solution, dried onto gold particles, or in conjunction with another DNA delivery system, including, but not limited to, liposomes, dendrimers, cochleates, and microencapsulation.

[0102] In certain embodiments, the nucleic acid sequence of antisense oligonucleotide, siRNA, shRNA or ribozyme is under the control of a heterologous regulatory region, for example, a heterologous promoter.The promoter can be specific to Müller glia cells, microglia cells, endothelial cells, pericytes and astrocytes.For example, the specific expression in Müller glia cells can be obtained through the promoter of glutamine synthetase gene.The promoter can also be a viral promoter, for example, CMV promoter or any synthetic promoter.

[0103] In certain embodiments, endonucleases can be used to disable expression of the FOXO1 gene, transcript or protein variant.

[0104] Indeed, novel technologies offer a means to manipulate genomes as an alternative to more conventional approaches such as cDNA overexpression or RNA interference-mediated silencing. Indeed, natural and engineered nuclease enzymes have attracted tremendous interest in recent years. The mechanisms behind endonuclease-based genome inactivation generally require a first step: DNA single- or double-strand breaks, which can then trigger two different cellular mechanisms for DNA repair, namely, error-prone non-homologous end joining (NHEJ) and high-fidelity homology-directed repair (HDR), which can be exploited for DNA inactivation.

[0105] In certain embodiments, the endonuclease is CRISPR-cas.The term "CRISPR-cas" as used herein has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats, which is a fragment of prokaryotic DNA that contains short repeats of base sequence.

[0106] In some embodiments, the endonuclease is CRISPR-cas9 from Streptococcus pyogenes. The CRISPR / Cas9 system is described in U.S. Patent No. 8,697,359 and U.S. Patent Application Publication No. 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has recently been engineered into a new, powerful tool for genome editing. This has already been demonstrated in humans (Mali et al., 2013, Science, Vol. 339: 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8: e2671), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8: e68708), nematodes (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11), and bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8: e2671.), plants (Mali et al., 2013, Science, Vol. 339: 823-826.), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141: 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41: 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi:10.1534 / genetics.113.160713.), monkeys (Niu et al., 2014, Cell, Vol. 156: 836-843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6: 97-99.), and pigs (Hai It has been successfully used to target important genes in many cell lines and organisms, including rats (Ma et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11), rats (Ma et al., 2014, Cell Res., Vol. 24:122-125), and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56:122-129). Multiple groups are now utilizing this method to introduce single point mutations (deletions or insertions) within specific target genes via a single gRNA.Pairs of gRNA-directed Cas9 nucleases can alternatively be used to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent and exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genomic loci.

[0107] In some embodiments, the endonuclease is CRISPR-Cpf1, a more recently characterized CRISPR derived from Provotella and Francisella 1 (Cpf1) in Zetsche et al. ("Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0108] As used herein, the terms "treatment" or "treating" refer to prophylactic (both prophylactic and preventative) treatment, including curative or disease-modifying treatment, and the prevention of disease recurrence, including treatment of subjects at risk of or suspected of having a disease, as well as subjects who are ill or have been diagnosed with a disease or medical condition. Treatment may be administered to a subject with or who may ultimately acquire a medical disorder with the intent to prevent, cure, or delay the onset of the disorder or recurrent disorder, reduce its severity, or ameliorate one or more symptoms thereof, or to extend the subject's lifespan beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment for a disease (e.g., a dosing pattern used during therapy). A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the subject during the initial period of the treatment regimen. The induction regimen may (partially or entirely) use a "loading regimen", which may include administering a larger dose of drug than the one a physician would use during a maintenance regimen, administering a drug at a higher frequency than the one a physician would use during a maintenance regimen, or both. The term "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a subject during disease treatment, for example, to keep the subject in remission for a long period of time (months or years). A maintenance regimen can be continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, or yearly), or intermittent therapy (e.g., interrupted therapy, intermittent therapy, treatment upon relapse, or treatment upon reaching a specific predetermined criterion (e.g., symptom of disease)).

[0109] As used herein, the term "subject" refers to mammals, such as rodents, felines, canines, and primates. In particular, subjects according to the present invention are humans.

[0110] [Use of the CAR-T cells of the present invention] The CAR-T cells obtained by the methods of the present invention can be used to improve immune responses and therefore can be used to treat diseases that require a boost in the immune system, such as cancer and infectious diseases.

[0111] Thus, a second aspect of the present invention relates to CAR-T cells obtained (or produced) by the method of the present invention for improving immune responses.

[0112] In particular, the present invention relates to CAR-T cells obtained by the method of the present invention for use in the treatment of cancer or infectious diseases.

[0113] The present invention also relates to a method for improving the immune system using CAR-T cells obtained by the method of the present invention.

[0114] The present invention also relates to a method for treating cancer or an infectious disease using the CAR-T cells obtained by the method of the present invention.

[0115] In other words, therefore, the present invention relates to a method for treating cancer or an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CAR-T cells obtained by the method of the present invention.

[0116] In certain embodiments, the CAR-T cells improve the subject's immune system.

[0117] Furthermore, the population of CAR-T cells prepared as described above can be utilized in methods and compositions for adaptive immunotherapy according to known techniques or variations thereof that will be apparent to those skilled in the art based on this disclosure. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; see also U.S. Patent No. 4,690,915 to Rosenberg et al. Adaptive cancer immunotherapy refers to a therapeutic approach in which immune cells with anti-tumor activity are administered to a tumor-bearing host with the goal that the cells directly or indirectly mediate the regression of established tumors. Infusion of lymphocytes, particularly T lymphocytes, falls into this category.

[0118] According to the present invention, the cancer may be a liquid or solid cancer.

[0119] In one embodiment, the cancer is adrenocortical carcinoma, anal cancer, bile duct cancer (e.g., perihepatic carcinoma, distal bile duct carcinoma, intrahepatic cholangiocarcinoma), bladder cancer, bone cancer (e.g., osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma), brain and central nervous system cancer (e.g., meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, germ cell tumor, tumor, craniopharyngioma), breast cancer (e.g., ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular adenocarcinoma, lobular carcinoma in situ, gynecomastia), Castleman's disease (e.g., giant lymph node hyperplasia, angiocapillary lymphadenopathy), cervical cancer, colorectal cancer, endometrial cancer (endometrial adenocarcinoma, adenomatous cell carcinoma, papillary serous adenocarcinoma, clear cell carcinoma), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g., choriocarcinoma, destructive villous adenocarcinoma) tumor), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g., renal cell carcinoma), laryngeal and hypopharyngeal cancer, liver cancer (e.g., hemangioma, hepatocellular adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung carcinoma, non-small cell lung carcinoma), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g., olfactory neuroblastoma, midline granuloma), nasopharyngeal carcinoma, neuroblastoma, oral and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer The cancer may be selected from the group consisting of retinoblastoma, rhabdomyosarcoma (e.g., embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), gastric cancer, testicular cancer (e.g., seminoma, non-seminomatous germ cell carcinoma), thymus cancer, thyroid cancer (e.g., follicular carcinoma, undifferentiated carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g., uterine leiomyosarcoma).

[0120] According to the present invention, an infectious disease may be caused by a pathogen such as a virus, bacterium, protozoan, prion, viroid or fungus.

[0121] According to the present invention, the bacterium may be selected from the group consisting of Streptococcus pneumoniae; Staphylococcus aureus; Haemophilus influenza, Myoplasma species, Moraxella catarrhalis, Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella enterica serovar Typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, Campylobacter, Mycobacterium tuberculosis, and Streptomyce.

[0122] According to the present invention, the fungus may be selected from the group consisting of Aspergillus, Candida albicans and Cryptococcus neoformans.

[0123] More particularly, infectious diseases are caused by respiratory viruses.

[0124] In particular, the respiratory virus may be an influenza virus, such as influenza A virus (IAV) or influenza B virus (IAB), adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), human rhinovirus (HRV), human respiratory syncytial virus (HRSV), or coronavirus.

[0125] As used herein, the term "coronavirus" has its common meaning in the art and refers to any member of the family Coronaviridae. Coronaviruses are viruses with genomes that are positive-stranded RNA, ranging in length from about 27 kb to about 33 kb, depending on the particular virus. The virion RNA has a cap at the 5' end and a poly(A) tail at the 3' end. The length of the RNA makes coronaviruses the largest RNA virus genome. Specifically, coronavirus RNA encodes: (1) an RNA-dependent RNA polymerase; (2) an N-protein; (3) three envelope glycoproteins; and (4) three nonstructural proteins. Specifically, coronavirus particles contain at least four standard structural proteins: E (envelope protein), M (membrane protein), N (nucleocapsid protein), and S (spike protein). The S protein cleaves into three chains: spike protein S1, spike protein S2, and spike protein S2'. Replicase protein production is initiated by translation of ORF1a and ORF1ab via a ribosomal frameshifting mechanism. This mechanism produces two large viral polyproteins, pp1a and pp1ab, which are further processed by two virus-encoded cysteine ​​proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), sometimes called main protease (Mpro). Coronaviruses infect a variety of mammals and birds. Coronaviruses cause respiratory infections (commonly), enteric infections (mainly in children over 12 months of age), and possibly neurological syndromes. Coronaviruses are transmitted via aerosols of respiratory secretions. Coronaviruses are exemplified by, but not limited to, human enteric coV (ATCC Accession No. VR-1475), human coV229E (ATCC Accession No. VR-740), human coV OC43 (ATCC Accession No. VR-920), Middle East Respiratory Syndrome-associated coronavirus (MERS-Cov), and SARS coronavirus (Centers for Disease Control), particularly SARS-Cov1 and SARS-Cov2.

[0126] According to the present invention, the coronavirus may be MERS-CoV, SARS-CoV, SARS-CoV-2 or any future novel member of this lineage.

[0127] In particular, therefore, the present invention also relates to CAR-T cells obtained by the method of the present invention for use in the treatment of infectious diseases caused by the above-mentioned pathogens in a subject in need thereof.

[0128] [Therapeutic composition] In a third aspect, the present invention relates to a therapeutic composition comprising CAR-T cells obtained by the method of the present invention for improving immune responses.

[0129] In another embodiment, the present invention relates to a therapeutic composition comprising the CAR-T cells obtained by the method of the present invention for use in the treatment of cancer or an infectious disease.

[0130] According to the present invention, the CAR-T cells are administered in a therapeutically effective amount.

[0131] Any of the therapeutic agents of the present invention can be combined with pharmaceutically acceptable excipients, and optionally with a sustained release matrix, such as a biodegradable polymer, to form a therapeutic composition.

[0132] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount that is effective, at the necessary dosage and for the necessary period of time, to achieve a desired therapeutic result. The therapeutically effective amount of the CAR-T cells of the present invention can vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the CAR-T cells of the present invention to elicit a desired response in the individual's body. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the CAR-T cells of the present invention are outweighed by the therapeutically beneficial effects. The effective dosage and dosage regimen for the CAR-T cell combination of the present invention will depend on the disease or condition being treated and can be determined by one of skill in the art. A physician of ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start the dosage of the oligomer of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, a suitable dose of the composition of the present invention will be the amount of compound that is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such effective doses generally depend on the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize disease progression. Typically, the ability of the CAR-T cells of the present invention may be evaluated in an animal model system that predicts efficacy for treating, for example, cancer or infectious diseases. Alternatively, this property of the composition may be evaluated by examining the compound's ability to induce cytotoxicity using in vitro assays known to skilled practitioners. A therapeutically effective amount of a therapeutic compound may reduce the potential reservoir or ameliorate the subject's symptoms. One skilled in the art would be able to determine such amounts based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.An exemplary non-limiting range for a therapeutically effective amount of the CAR-T cells of the present invention is about 0.1 to 100 mg / kg, such as about 0.1 to 50 mg / kg, for example, about 0.1 to 20 mg / kg, for example, about 0.1 to 10 mg / kg, for example, about 0.5, for example, about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. An exemplary non-limiting range for a therapeutically effective amount of the CAR-T cells of the present invention is 0.02 to 100 mg / kg, for example, 0.02 to 30 mg / kg, for example, about 0.05 to 10 mg / kg or 0.1 to 3 mg / kg, for example, about 0.5 to 2 mg / kg.

[0133] In other words, the quantity of CAR-T cells administered to a subject in need is 10 cells per kg. 4 ~10 9 In particular, the number of cells injected is 10 cells per kg. 6 pieces or 10 7 In particular, the unit for use of the CAR-T cells of the present invention will most advantageously be the number of cells per kg (as indicated above).

[0134] Administration may be intravenous, intramuscular, intraperitoneal, intratumoral, or subcutaneous, for example, administered proximal to the target site. The dosage regimen in the above-described methods and uses of treatment is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered, or the dose may be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of treatment is monitored during therapy, for example, at predefined time points. In some embodiments, efficacy can be monitored by visualization of disease areas or other diagnostic methods further described herein, for example, by performing one or more PET-CT scans. If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six, or more divided doses, optionally administered separately at appropriate intervals throughout the day in unit dosage forms. In some embodiments, the oligomers of the present invention are administered by continuous slow infusion over a prolonged period, such as more than 24 hours, to minimize undesirable side effects. An effective dose of the CAR-T cells of the present invention can also be administered using a weekly, biweekly, or triweekly dosing period. The dosing period can be limited to, for example, 8 weeks, 12 weeks, or until clinical progression is documented.By way of non-limiting example, treatment according to the present invention can be administered using a single dose or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, for at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after initiation of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after initiation of treatment. The CAR-T cells of the present invention may be provided as a daily dosage in an amount of about 0.1 to 100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day during at least one of weeks 15, 16, 17, 18, 19, or 20, or any combination thereof.

[0135] In other words, the number of CAR-T cells administered to a subject in need is 10 cells per kg. 4 ~10 9 In particular, the number of cells injected is 10 cells per kg. 6 or 10 7 The CAR-T cells of the present invention can be administered 1, 2, 3, 4, or 5 times to a subject in need.

[0136] The CAR-T cells of the present invention may be used alone or in combination with any suitable agent.

[0137] Any of the therapeutic agents of the present invention can be combined with pharmaceutically acceptable excipients, and optionally with a sustained release matrix, such as a biodegradable polymer, to form a therapeutic composition.

[0138] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered as needed to mammals, especially humans. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0139] The pharmaceutical compositions of the invention may comprise a further therapeutically active agent.The invention also relates to a kit comprising an inhibitor according to the invention and a further therapeutically active agent.

[0140] For example, an anti-cancer drug may be added to the pharmaceutical composition, as described below.

[0141] The anti-cancer drug may be melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), and bendamustine (Treande).

[0142] Other anticancer drugs include, for example, cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbu These include cyclosporine, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustard, BCNU, nitrosoureas such as carmustine and lomustine, vinca alkaloids such as vinblastine, vincristine, and vinorelbine, imatinib mesylate, hexamethylmelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, herbimycin A, genistein, erbstatin, and lavendustin A. In one embodiment, the additional anticancer agent includes, but is not limited to, alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, antifolates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormone therapy, retinoids, photosensitizers or photodynamic therapy, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, multidrug resistance inhibitors, and Ca + The therapeutic agents may be selected from one or a combination of drug classes such as ATPase inhibitors.

[0143] The additional anti-cancer agent may be selected from, but is not limited to, cytokines, chemokines, growth factors, growth inhibitors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, monospecific, bispecific or multispecific antibodies, monobodies, polybodies.

[0144] Additional anti-cancer agents are selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

[0145] In the method for treating cancer, the additional therapeutic active agent can be an antiemetic.Suitable antiemetics include but are not limited to metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxypemdoyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thioproperazine and tropisetron.In a preferred embodiment, the antiemetic is granisetron or ondansetron.

[0146] In another embodiment, the additional therapeutically active agent may be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim, and epoetin alfa.

[0147] In yet another embodiment, the other therapeutically active agent may be an opioid or non-opioid analgesic. Suitable opioid analgesics include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, ethobupine, buprenorphine, meperidine, loperamide, anilidine, ethoheptazine, pimididine, betaprozin, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazone, pemazocine, cyclazocine, methadone, isomethadone, and propoxyphene. Suitable non-opioid analgesics include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofenac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, piroxicam, and sulindac.

[0148] In yet another embodiment, the additional therapeutically active agent may be an anxiolytic. Suitable anxiolytics include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazepam, clorazepate, clonazepam, chlordiazepoxide, and alprazolam.

[0149] In yet another embodiment, the additional therapeutically active agent may be a checkpoint blockade cancer immunotherapeutic agent.

[0150] Typically, checkpoint blockade cancer immunotherapeutics are therapeutically active agents that block immunoinhibitory receptors expressed by activated T lymphocytes, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, such as various members of the killer cell immunoglobulin-like receptor (KIR) family, or the primary ligands for these receptors, such as the PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

[0151] Typically, checkpoint blockade cancer immunotherapeutics are antibodies.

[0152] In some embodiments, the checkpoint blockade cancer immunotherapeutic is an antibody selected from the group consisting of an anti-CTLA4 antibody, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-PDL2 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, an anti-IDO1 antibody, an anti-TIGIT antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-BTLA antibody, and an anti-B7H6 antibody.

[0153] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will naturally depend on the physical condition to be treated, the severity of the condition, age, weight and sex of the subject.

[0154] The pharmaceutical compositions of the present invention may be formulated for topical, oral, nasal, parenteral, intraocular, intravenous, intramuscular, or subcutaneous administration, and the like.

[0155] In particular, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These vehicles may be in isotonic, sterile saline (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a mixture of such salts) or may be a dried, particularly lyophilized, composition. In particular, these vehicles may be in an organic solvent such as DMSO or ethanol, which, when added with sterile water or saline, allows the formation of an injectable solution.

[0156] Additionally, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; sustained release capsules and any other form currently in use.

[0157] In each embodiment of the methods of treatment described herein, the CAR-T cells of the invention are delivered in a manner consistent with conventional methods associated with the management of the disease or disorder for which treatment is sought, and an effective amount of the CAR-T cells of the invention is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder, according to the disclosure herein.

[0158] Nanocapsules generally can encapsulate compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (sized around 0.1 μm) are generally designed using polymers that are biodegradable. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily prepared.

[0159] Liposomes are formed from phospholipids dispersed in an aqueous medium that spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å that contain aqueous solution within their cores. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0160] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0161] [Figure 1A] FOXO1 inhibition was able to increase the number of cytotoxic T cells and their activity. Human primary T lymphocytes from healthy donors (PBT) were treated with AS1842856 (500 nM) or DMSO equivalent for 7 days. Cells were surface-labeled with anti-CD4 and anti-CD8, then fixed and permeabilized, and then labeled with an antibody against Granzyme B. Graphs present values ​​corresponding to the mean ± SEM obtained from three independent donors. Significance was assessed using a paired Student's t-test. [Figure 1B] FOXO1 inhibition was able to increase the number of cytotoxic T cells and their activity. Human primary T lymphocytes from healthy donors (PBT) were treated with AS1842856 (500 nM) or a DMSO equivalent for 7 days. At the end of the culture period, cells were stimulated with PMA and ionomycin for 3 hours. After CD4 and CD8 labeling, intracellular detection of TNF-α was performed using an antibody against TNF-α. Values ​​correspond to the mean ± SEM obtained from three independent donors. Significance was assessed using a paired Student's t-test. [Figure 1C] FOXO1 inhibition was able to increase the number of cytotoxic T cells and enhance their activity. Human primary T lymphocytes from healthy donors (PBT) were treated with AS1842856 (500 nM) or DMSO equivalent for 7 days. Purified CD8+ T cells from vehicle- or treated cells were cocultured with P815 target cells pre-coated with anti-CD3 mAb, and the percentage of lysis was assessed by 51Cr release assay at different effector:target ratios. Values ​​correspond to the mean ± SEM obtained from three independent donors. Significance was assessed using two-way ANOVA. [Figure 2A]FOXO1 inhibition promotes T cell motility. PBT were treated with AS1842856 (500 nM) or its DMSO equivalent for 7 days. At the end of the culture, cells were stimulated or not with 100 ng / ml CCL19 for 8 minutes, fixed, stained for F-actin, and analyzed for shape deformation using ImageStream. Graphs present values ​​corresponding to the mean ± SEM of results obtained from three independent donors. Significance was assessed using two-way ANOVA. [Figure 2B] FOXO1 inhibition promotes T cell motility. PBT were treated with AS1842856 (500 nM) or the equivalent of DMSO for 7 days. Migration of T cells treated or not with AS1842856 in vibratome sections of viable Capan-2 tumors. The graph shows the mean velocity and displacement ± SEM of T cells treated or not with AS1842856 in Capan-2 tumor slices from three independent experiments. A minimum of 100 cells were analyzed in each experiment. Significance was assessed using a paired Student's t-test. [Figure 3A] AS1842856 primes human T lymphocytes and induces the acquisition of a memory phenotype. PBTs were treated with 500 nM AS1842856 or vehicle alone for 7 days. Memory subset composition in vehicle- or AS1842856-treated CD4 (upper panel) and CD8 (lower panel) cells. TN is CD45RA+, CD27+, CD95-; TSCM is CD45RA+, CD27+, CD95+; TCM is CD45RA-, CD27+; TEM is CD45RA-, CD27-; and Teff is CD45RA+, CD27-. Graphs show the mean percentages ± SEM of different subsets in vehicle or AS1842856 conditions on CD4 (upper panel) and CD8 (lower panel) T cells from five independent donors. Significance was assessed using one-way ANOVA. [Figure 3B]AS1842856 primes human T lymphocytes and induces the acquisition of a memory phenotype. PBT were treated with 500 nM AS1842856 or vehicle alone for 7 days. CTLA-4, PD-1, and TIGIT expression on CD4 (upper panel) and CD8 (lower panel) T cells was measured by FACS. Graphs show MFI ± SEM obtained from five independent donors. Significance was assessed using a paired Student's t-test. [Figure 4] FOXO1 inhibition increases cell viability. PBTs were treated with AS1842856 (500 nM) or vehicle alone. Every 7 days, several cells from the two conditions were collected and labeled with propidium iodide (PI). The graph shows the mean percentage of viable cells (mean PI-) in the vehicle or AS1842856 condition ± SEM, considering the results for four different donors. Significance was assessed using two-way ANOVA. [Figure 5A] CAR-TAS cells are more potent at eradicating tumors than conventional CAR-T cells. Capan-2LUC tumors were implanted into the right flank of NSG mice on day 7. PBS, 2 x 10 CAR-T cells, or CAR-TAS cells were injected intravenously on day 0. Bioluminescence imaging was performed on the indicated days after CAR-T cell injection by intraperitoneal injection of luciferin. Graphs show median tumor signal intensity ± SEM. Statistical analysis was performed using a two-way ANOVA, and p values ​​correspond to comparisons with the PBS condition. [Figure 5B] CAR-TAS cells are more potent at eradicating tumors than conventional CAR-T cells. Capan-2LUC tumors were implanted into the right flank of NSG mice on day 7. On day 0, PBS, 2 x 10 CAR-T cells, or CAR-TAS cells were intravenously injected. Tumor measurements were performed by ultrasound scanning on day 40. Graphs show median values ​​± SEM from six animals per group. Statistical analysis was performed using Student's t-test. [Figure 5C]CAR-TAS cells are more potent at eradicating tumors than conventional CAR-T cells. Capan-2LUC tumors were implanted into the right flank of NSG mice on day 7. On day 0, PBS, 2 x 10 CAR-T cells, or CAR-TAS cells were intravenously injected. On day 41, tumors were weighed after euthanasia. Graphs show median values ​​± SEM obtained from six animals per group. Statistical analysis was performed using a Student's t-test. [Figure 6A] CAR-TAS cells generated from patient cells show increased proliferative capacity. CAR-T cells and CAR-TAS cells were generated from patients as described above. FACS analysis of memory subset distribution. [Figure 6B] CAR-TAS cells generated from patient cells show increased proliferative capacity. CAR-T cells and CAR-TAS cells were generated from patients as described above. Mean percentages of identified populations ± SEM Tim3+ cells and LAG-3+ cells in patient CAR-T cells and patient CAR-TAS cells. [Figure 6C] CAR-TAS cells generated from patient cells show increased proliferation potential. CAR-T cells and CAR-TAS cells were generated from patients as described above. Comparison of cell proliferation of CAR-T cells or CAR-TAS cells generated from patients after TransAct stimulation alone. The graph shows the fold increase in cell counts each day between days 10 and 28 relative to day 0. CAR-T cells are in black, and CAR-TAS cells are in red. [Example]

[0162] [material and method] Study Approval: Human studies were conducted in accordance with French law on biomedical research and the principles outlined in the 1975 Declaration of Helsinki and its amendments. Approval was obtained from the Institutional Review Board (CPP Ile de France II, #00001072, August 27, 2012). Animal studies were approved by the Animal Experimentation Ethics Committee of Paris Descartes University (CEEA34, 17-039) and the French Ministry of Higher Education and Research (APAFiS#19762).

[0163] Cells: T lymphocytes were purified from blood of healthy donors from the Etablissement Français du Sang (EFS, Paris, France) by Ficoll density gradient centrifugation followed by negative selection with the Easy Sep™ Human T Cell Isolation Kit (Stem Cell, #17951) and cultured in RPMI 1640 GlutaMAX (Gibco, cat #61870-010) medium supplemented with 10% human AB serum (Biowest, #S4190-100), penicillin and streptomycin (50 U / ml and 50 μg / ml, respectively; penicillin-streptomycin was from Thermo Fisher Scientific; cat #15140122), and 1 mM sodium pyruvate (Gibco, cat #11360-039). TAS cells were obtained by treatment with 500 nM AS1842856 (EMD Millipore, #344355) for 7 days. Because AS1842856 was dissolved in DMSO, untreated cells were obtained by culturing for 7 days in a DMSO volume corresponding to the AS1842856 dilution. P815 (ATCC® TIB-64), HEK293T (ATCC® CRL-11268), and Capan-2 (ATCC® HTB-84) cells were maintained in complete DMEM GlutaMAX (Gibco, cat#31966-021) containing 10% FBS, penicillin, and streptomycin (50 U / ml and 50 μg / ml, respectively). The MT4R5 cell line (Amara et al., 2003) was maintained in culture in complete RPMI 1640 GlutaMAX medium containing 10% FBS, penicillin and streptomycin (50 U / ml and 50 μg / ml, respectively), and 1 mM sodium pyruvate. Each cell line was thawed from laboratory frozen stocks generated from early-passage cells and used within 4 weeks of culture for each experiment. Cell lines were tested for mycoplasma monthly using kit Lonza #LT07-118 to ensure that all experiments using the cell lines herein were mycoplasma-free.

[0164] Cell transfection: Cells were transfected by nucleofection using Human T Cell Nucleofector solution (Lonza, VPA-1002) and program U-014 in the Nucleofector AMAXA. For CRISPR, 2 × 10 6 5 × 10 cells were nucleofected with 75 pmol of Cas9 protein (Thermo Fisher, #A36499) at a 1:1 molar ratio and one RNA guide targeting the FOXO1 gene (Thermo Fisher, #sg RNA CRISPR 889854_SGM Foxo1). To induce FOXO1 expression, 5 × 10 cells were nucleofected with 5 μg of DNA from pEGFP-Nl (clontech) or pEGFP-FOXO1-T24A-S256A-S319A-H215R plasmid (Nagashima et al., 2010) as a control. 6 Jurkat T cells (5 × 10 in RPMI 1640 medium) were transfected at 1000 μF and 260 V in a 4 mm polycarbonate cuvette (Eurogentec) with 5 μg of GFP, triple mutant T24A / S256A / S319A FOXO1-GFP (FOXO1-TM-GFP), or FOXO1-TM-GFP DNA-binding mutant plasmid (FOXO1-TM-H215R-GFP) (Fabre et al., 2008). 6 (100 cells / ml) were electroporated. After 4 hours, AS1842856 was added or not added to the cultures. At 48 hours, CD62L expression was analyzed by cytometry.

[0165] Flow cytometry: Cells were washed and stained with antibodies diluted in PBS (Thermo Fisher Scientific; cat#10010001) supplemented with 0.1% BSA (Sigma-Aldrich, cat#A7030-500G) for 20 min at 4°C. After washing with PBS, cells were immediately analyzed by flow cytometry. For intracellular staining, cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, permeabilized with a buffer containing 0.1% Saponin (Sigma-Aldrich, 84510) in PBS, and stained after two PBS washes with antibodies diluted in the same saponin buffer for 30 min at 4°C. After two washes, cells were run on a BD LSRII (BD Biosciences) and analyzed using FlowJo software.

[0166] Viability assay: Cells were harvested and propidium iodide was added (10 μg / ml Invitrogen). Samples were run on a FACS Calibur (Becton Dickinson) and analyzed using FlowJo software.

[0167] Cytotoxicity Assay: CD8 + T cells were isolated using EasySep™ Human CD8 + T cells were isolated by negative selection using the T Cell Isolation Kit (Stem Cell, #17953). AS or purified from naive T cells. 51 Cytotoxicity was measured by Cr release assay. The effector (E):target (T) ratios were 30:1, 10:1, 3:1, and 1:1, with 3000 target cells per well. Percent specific cytotoxicity was calculated as previously described (Echchakir et al., 2000). FcR-positive P815 mouse cells were incubated with OKT3 antibody for 1 hour at 37°C and used as targets in the redirected cytotoxicity assay.

[0168] Proliferation assay: In vitro proliferation assay of primary T cells was assessed by dilution of Cell Trace Fluorescent Green kit (CFSE, Invitrogen, cat#C34554). After two washes in RPMI 1640 GlutaMX medium, 4 x 10 cells per ml were cultured in 5 μM Cell Trace Fluorescent Green solution. 6 The cells were resuspended in 10 ml of RPMI medium. The cells were incubated for 20 minutes at 37°C in the dark. After loading, the cells were washed with a volume of hot RPMI medium supplemented with 10% human AB serum, penicillin, and streptomycin (50 U / ml and 50 μg / ml, respectively) corresponding to 5 times the loading volume. The cell suspension was collected and plated in a 96-well round-bottom plate (2 x 10 cells per well). 5 The cells were delivered to a 500-well plate (200 ml) and stimulated with anti-CD3 / CD28 coated Peas Dynakeads (Invitrogen, #11131D) for 3 days. Samples were run on a BD LSR Fortessa and analyzed using FlowJo software. For the in vivo proliferation assay, T cells were differentiated by dilutions of CFSE as in the in vitro proliferation assay described above. AS Cells were labeled. Then, cells from immunodeficient NSG mice (NOD.Cg-Prkdc) were resuspended in 100 μL of PBS supplemented with 10% FBS. scid Il2rg tm1WjI / SzJ mice, cat#JAX:005557, Charles River) 10 per mouse 7 CFSE + Cells were injected intravenously into the retro-orbital vein. Five, nine, or twelve days after injection, spleens were collected, minced, and filtered over a 40 μm cell strainer (Thermo Fisher #22363547). Cell suspensions were run on a FACS Calibur. A portion of CFSE +Cells were maintained in culture from the day of injection until the end of the experiment to serve as controls. Five, nine, or 12 days after the initiation of culture, cell suspensions were run on a FACS Calibur (Becton Dickinson) simultaneously with spleen-derived cells. Data were analyzed using FlowJo software.

[0169] ImageStream flow cytometry: Cells were washed once in cold PBS and fixed on ice for 20 minutes in cytofix / cytoperm solution (Invitrogen #00-5523-00). Cells were then stained with phalloidin-TRITC (Life Technologies, #R415) for 30 minutes at room temperature. Flow cytometry was performed on an ImageStreamX MKII high-speed imaging flow cytometer (Amnis Corporation). Shape deformation was assessed by aspect ratio (minor axis divided by major axis) using IDEAS Aralysis Software (Amnis Corporation).

[0170] Motility assay: Immunodeficient NSG mice were injected subcutaneously into the flanks with 10 7 Human pancreatic Capan-2 tumors were established by transplantation of 1000 cells. After 2 weeks, mice were euthanized, and tumors were isolated and embedded in 8% low-gelling-temperature agarose (Sigma-Aldrich; cat#A0701-25G) prepared in PBS. 400 μm-thick tumor slices were cut using a vibratome (Leica VT1200S vibratome, RRID:SCR_018453) in an ice-cold PBS bath. Slices were stained with anti-human EpCAM and anti-mouse podoplanin antibodies (Table. Sup1) using 0.4 μm organotypic culture inserts (Merck Millipore; cat#PICM03050) at 37°C for 20 minutes. Slices were then washed in PBS. 5 × 10 cells per treatment condition (vehicle or AS1842856) were added. 6T cells were washed twice with PBS and then labeled with 100 nM Calcein Green (Invitrogen, cat#C34852) or Calcein Red-Orange (Invitrogen, cat#C34851) in PBS for 20 min at 37°C. After one wash in motility medium consisting of RPMI 1640 GlutaMAX supplemented with 0.5% BSA, 1 mM sodium pyruvate, and 10 mM HEPES (Gibco, cat#15630-056), cells from the two treatment conditions were mixed at a 1:1 ratio and resuspended together in 100 μl of motility medium. 30 μL of the mixture was placed on each slice and incubated for 20 min at 37°C using 0.4 μm organotypic culture inserts. After washing in PBS, slices were transferred to 35 mm Petri dishes (containing the motility medium described above) and dynamic imaging of T cells on tumor slices was performed (10x objective, binning = 2, 20 time points every 30 seconds, 15 μm z-stacks) as previously described (Kantari-Mimoun et al., 2021). T cell migration on the slices was measured using the TrackMate plugin in ImageJ software. Software parameters were set as follows: LoG detector (estimated object diameter 10 μm, threshold 50,000), no filter on spots, simple LAP tracker (maximum ligation distance 20 μm, maximum gap closure distance 20 μm, maximum gap closure frame gap 3). Only tracks containing five or more spots were considered for analysis.

[0171] Calcium measurements: T cells were incubated with 1.5 μM Fura-2 / AM (Molecular Probes, F1225) for 20 min at 37°C. Experiments were performed in mammalian saline buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl, 1 mM MgCl, 20 mM HEPES, 11 mM glucose) at 37°C. Calcium measurements by fluorescence spectroscopy were performed as previously described (Conche et al., 2009) using a Cary Eclipse fluorescence spectrophotometer (Varian) (excitation: 340 and 380 nm; emission: 510 nm).

[0172] Western blot analysis: Protein expression levels were analyzed by Western blot as described (Froehlich et al., 2016). Blotting with primary antibodies was followed by goat anti-mouse or goat anti-rabbit-HRP (Jackson ImmunoResearch) incubation and ECL development (GE Healthcare, #RPN2106).

[0173] Lentivirus production: Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped retroviral vectors were generated by transfecting HEK 293T cells as previously described (Berger et al., 2011). The plasmids used were pVSV-G (Plasmid #8454, Addgene), the lentiviral packaging plasmid pCMVR8.74 (Plasmid #22036, Addgene) encoding HIV GAG / POL / REV, and the lentiviral transfer vector plasmid shown in Figure Sup1. The CAR sequence consists of a signal peptide derived from the nimotuzumab sequence and an EGFR-directed scFv (IMGT / 2Dstructure-DB INN 8545H, 8545L), a CD8 hinge and transmembrane domain (194-248 Aa, GenBank: AAH25715.1), a 4-1BB costimulatory domain (214-255 Aa, GenBank: AAX42660.1), and a CD3z signaling domain (52-163 Aa, GenBank: NP_000725.1), linked to GFP via an IRES or P2A sequence. A range of lentiviruses were produced, with 8 × 10 4 MT4R5 cells were infected, and 50% of GFP was detected after 3 days of infection. + All virus stocks were titrated by analyzing the volume required to obtain cells.

[0174] Generation of CAR-T cells: In 24-well plates, 2 × 10 cells were cultured in TEXMACS medium (Miltenyi, #130-097-19) supplemented with 10 ng / ml human IL-7 (Miltenyi, #130-095-362) and 10 ng / ml human IL-15 (Miltenyi, #130-095-764). 6 T cells were cultured and activated with TransAct (Miltenyi, #130-111-16) at a dilution of 1 / 100. Activated T cells were transduced 48 hours after stimulation with an MOI of 2 of lentiviral stock. GFP expression was analyzed by flow cytometry 3 days after transduction. Transduction efficiency with the EGFR CAR ranged from 50% to 85% of positive cells.

[0175] CAR-T AS Cell generation: 3 × 10 per ml in the presence of 500 nM AS1842856 6 T lymphocytes from healthy donors were cultured in complete RPMI containing 10% human AB serum, penicillin and streptomycin (50 U / ml and 50 μg / ml, respectively) at a concentration of 10 cells / ml. On day 7, an MOI of 2 of lentiviral stock was added to T AS Cells were transduced and analyzed for GFP expression by flow cytometry.

[0176] In vivo assay: For the in vivo assay, 6- to 8-week-old NSG mice were used. Each mouse was inoculated with 5 × 10 6 Luciferase-expressing Capan-2 cells (Capan-2 luc ) was subcutaneously injected. Seven days after injection, CAR-T cells were intravenously injected into the retro-orbital vein. Tumor growth was assessed weekly using a PHOTON IMAGER RT (Biospacelab) or, where specified, by bioluminescence detection with a caliper. Forty days after CAR-T cell injection, tumors were observed by ultrasound scanning using a VEVO 2100 device (Visualsonics). At the end of the experiment, mice were euthanized, and tumors were collected and weighed.

[0177] Statistical analysis: Statistical tests used for sample comparisons are specified within the figure legends ( * p<0.05; ** p<0.01; *** p<0.001; ns: not significant). These were performed using Graph Pad Prism software.

[0178] [result] [FOXO1 inhibition enhances the activity of cytotoxic T cells] The basis of CAR-T cell immunotherapy is the ability of these cells to kill tumor cells. Therefore, their cytotoxic activity is essential. Therefore, we focused on the consequences of FOXO1 inhibition with AS1842856 on T cell cytotoxic activity. As previously published, we found that granzyme B, a key molecule that triggers target cell lysis, was upregulated in the presence of FOXO1 inhibitors in the CD8 subset (Jeng et al., 2018; Roux et al., 2019). Surprisingly, this was also found to be true for CD4 T cells (Figure 1A). In addition to granzyme B, increased cytotoxic activity may also result from the production of pro-inflammatory cytokines such as TNF-α. Therefore, we compared the percentage of cells expressing TNF-α by intracellular labeling in T cells treated with or without AS1842856. We found that inhibition of FOXO1 significantly increased TNF-α expression (Figure 1B). These results were confirmed by using an MSD assay to measure the amount of TNF-α secreted into the supernatant of T cells treated with AS1842856 (data not shown). Notably, we also found that other inflammatory cytokines, such as IL-10 and GM-CSF, were also upregulated in T cells. AS We found that increased amounts of IFN-α, but not IFN-α, were secreted by CD8 T cells (data not shown). These characteristics were consistent with the increased secretion of IFN-α by CD8 T cells compared to naive T cells measured in redirected lysis experiments. ASThis correlated with stronger cytotoxic activity against P815-derived target cells (Figure 1C). Thus, inhibition of FOXO1 enhances the ability of CAR-T cells to kill tumor targets, a fundamental function of CAR-T cells.

[0179] To confirm the role of FOXO1 in AS1842856-mediated increased cytotoxicity, we analyzed the consequences of CRISPR-mediated Foxo1 gene inactivation on granzyme B expression. We first observed that Foxo1 inactivation via AS1842856 treatment led to a decrease in CD62L expression, due to the fact that it is a transcriptional target of FOXO1 (Fabre et al., 2008). Under the same conditions, AS1842856 induced a more than two-fold increase in granzyme B expression, while Foxo1 inactivation did not induce any change in granzyme B (data not shown). Therefore, it appears that the increase in granzyme expression levels induced by AS1842856 cannot be reproduced in the absence of FOXO1. Because AS1842856 leads to an increase in FOXO1 that lacks the ability to interfere with its specific DNA-binding site, we analyzed the consequences of overexpressing the FOXO1 H215R mutant. This mutant form of FOXO1 encodes a constitutive nuclear form of FOXO1 that is unable to bind to its DNA-binding site, thus mimicking AS1842856 treatment. Overexpression of this FOXO1 mutant induced not only a clear reduction in CD62L but also a significant increase in granzyme B expression (data not shown). We also observed that after transfection of Jurkat T cell lines, the FOXO1 H215R mutant induced a similar downregulation of CD62L expression, which could not be further reduced by AS1842856 (data not shown). Thus, AS1842856 allows dissociation of the distinct mechanisms of FOXO1 transcriptional regulation by inhibiting only its bona fide transcription factor activity resulting from interaction with the DNA-binding site.

[0180] [FOXO1 inhibition promotes T cell motility] Another limitation of CAR-T cell therapy, particularly in the context of solid tumors, is the ability of these cells to penetrate the tumor bed and migrate within the tumor (Majzner and Mackall, 2019). Since we have shown that several FOXO1 target genes regulate T cell motility (Fabre et al., 2008; Megrelis et al., 2018; Rougerie et al., 2013), we next analyzed whether inhibition of FOXO1 could affect T cell migration. We first investigated the effect of FOXO1 on T AS We observed that the cells spontaneously adopted a strong shape modification typical of polarized cells, as seen through a decrease in their aspect ratio, which was clearly inferior when compared to that of untreated T cells (Figure 2A). Because this T cell deformation is usually dependent on a chemokine response, we compared cell polarization upon CCL19 stimulation for AS1842856-treated or untreated T cells. We found that upon chemokine stimulation, T cells and T AS We observed that the cells displayed comparable aspect ratios. Overall, these results demonstrate that FOXO1 inhibition induces spontaneous cell polarization comparable to that achieved by chemokine stimulation.

[0181] Because T cell differentiation is often associated with a major reprogramming of chemokine receptor expression, we analyzed the effects of AS1842856 treatment on several of them by flow cytometry. We show that modulation of membrane expression by AS1842856 is specific to each chemokine receptor. Thus, while there is a significant decrease in CXCR4, inhibition of FOXO1 induces an increase in CCR4, CX3CR1, and CCR6, three receptors that have been described to promote T cell migration within the tumor microenvironment (data not shown).

[0182] Because polarity establishment is linked to migration ability, we next sought to test the consequences of FOXO1 inhibition on T cell migration. We used a device developed by Asperti-Boursin et al. to monitor motile behavior within live tumor slices (Asperti-Boursin et al., 2007). For these experiments, NSG mice were subcutaneously implanted with Capan-2 cells. Two weeks later, tumors were sliced ​​with a vibratome, and T cells were plated onto fresh slices. As shown in Figure 2C, FOXO1 inhibition conferred greater ability on T cells to migrate within the tumor microenvironment, as exemplified by increased stroke speed and length. Increased T cell motility was also observed in an orthotopic tumor model resulting from intravenous injection of the lung cancer-derived A549 cell line (data not shown). Thus, FOXO1 inhibition induces T cell polarization comparable to that initiated by chemokines as well as increased T cell motility within tumors.

[0183] [FOXO1 inhibition induces acquisition of a stem cell memory phenotype] We previously showed that FOXO1 inhibition induces increased metabolic activity, a phenotype known to be associated with the emergence of memory features (Jeng et al., 2017). Therefore, we analyzed by flow cytometry different T cell subsets obtained after in vitro treatment of human primary T cells with AS1842856 for 48 hours. Since FoxO1 is involved in the differentiation of multiple CD4 subsets, we found that Tfh (CXCR5) + , BcL-6 + ), Th1 (T-bet + ), Th2(GATA3 + ) and Treg (FOXP3 +We investigated the consequences of AS1842856 treatment on the expression of markers linked to the CD4+ / CD8+ subsets and found no significant changes, except for Tfh, which exhibited a slight increase after AS1842856 treatment (data not shown). Using the gating strategy shown in Figure 3A, we quantified the relative proportions of naive (TN), stem cell memory (TSCM), central memory (TCM), and effector memory (TEM) T cells. We found that for both CD4+ and CD8+ T cells, the naive subset (CD45RA + / CD27 + / CD95 - ) and a sharp decrease in the TSCM subset (CD45RA + / CD27 + / CD95 + We observed that AS1842856 induced a profound modification of the differentiation state of T cells, with a significant increase in CD8 expression (data not shown). Coincidentally, through this phenotypic analysis, we observed that AS1842856 induced a decrease in CD8 expression (data not shown), confirming the antigenic stimulation of T cells induced by FOXO1 inhibition, as previously reported for antigen-stimulated CD8 T cells in both humans and mice (Erard et al., 1993; Kambayashi et al., 2001; Maile et al., 2001). In T lymphocytes, differentiation can be linked to an increase in the expression of exhaustion markers, also known as immune checkpoints, which negatively regulate T cell effector function. As shown in Figure 3B, we observed a significant decrease in CD8 expression, with 10% or fewer cells expressing this marker. + and CD4 + We observed a small but significant increase in PD1 expression in both IL-1 and IL-2 cells (left panel). We did not detect any significant modulation of CTLA-4 (middle panel) or CD8 + We detected a consistent increase in TIGIT that reached significance only in T cells. Overall, this phenotypic analysis demonstrated that the most notable event induced by FOXO1 inhibition in T cells was the CD4 + and CD8 +We showed that both the rapid decline in naive T cell subsets correlated with a rapid increase in memory T cells, particularly TSCM.

[0184] [T AS Cells display functional characteristics of memory T cells. We next sought to assess whether the acquisition of this memory phenotype was accompanied by the acquisition of functional properties traditionally associated with memory T cells. We first compared the in vitro survival of primary T cells with and without FOXO1 inhibition. AS cells were observed (Figure 4), with a T of 30% + / - 4% after 28 days of culture, compared to only 2% + / - 4% in untreated cells. ASThe cells were still viable. Because T cells do not proliferate under these culture conditions, this result corresponds to an increased survival rate (data not shown). Therefore, AS1842856 not only exhibits no toxicity but also promotes T cell viability. Memory T cells have been described as having a specific signaling signature upon TCR stimulation. In particular, calcium influx has been described as being lower in memory T cells than in naive T cells (Adachi and Davis, 2011; Hall et al., 1999; Tanchot et al., 1998). We observed that calcium influx induced upon TCR stimulation was slower and smaller in AS1842856-treated T cells than in untreated cells (data not shown). In addition to calcium responses, phosphorylation of ERK and AKT signaling proteins upon TCR stimulation has also been described as different between naive and memory T cell populations (Adachi and Davis, 2011; N. Jones et al., 2019; Kalland et al., 2011). Western blot comparison of the kinetics of ERK and AKT phosphorylation upon TCR stimulation in AS1842856-treated and untreated cells revealed hypophosphorylation of AKT and hyperphosphorylation of ERK after TCR stimulation, including at steady state (data not shown). Thus, analysis of both calcium responses and phosphorylation of ERK and AKT proteins revealed that AS1842856-treated T cells behaved as memory cells. Finally, because it has been described that incompletely differentiated memory cells, such as TSCM cells, have greater proliferative capacity (Gattinoni et al., 2011), we evaluated the consequences of FOXO1 inhibition on the proliferative potential of T cells. To this end, T cells treated or not with AS1842856 were stained with CFSE and then stimulated with anti-CD3 / CD28 beads. Cell proliferation was monitored for 60 hours. We found that T cells treated with AS1842856 proliferated as early as 40 hours after stimulation, whereas untreated cells required an additional 8 hours of stimulation (data not shown).This proliferative advantage is maintained over time, as a greater number of CFSE dilution peaks were observed in AS1842856-treated T cells even after 60 hours of stimulation (data not shown). Dose-response curves show that the percentage of proliferating cells after 48 hours of stimulation was significantly higher in control and T cells. AS The bead / cell ratios for each cell type were identical (data not shown). Thus, the growth benefit of FOXO1 inhibition was observed in T AS Altogether, these results demonstrate that AS1842856 induces a distinct phenotypic and functional transition from naive to memory T cells.

[0185] [CAR transduction in quiescent cells by FOXO1 inhibition] In CAR-T cell production protocols, T cells are first stimulated in vitro to allow CAR expression within the T cells. However, this ex vivo stimulation is likely at the cost of cellular exhaustion (Ghassemi et al., 2018). Therefore, we have previously shown that FOXO1 inhibition confers tolerance to T cells against infection, and therefore we propose a novel approach to the treatment of T AS We explored the possibility of introducing a CAR-encoding vector into cells (Roux et al., 2019). Since the CAR sequence is linked to the GFP sequence by an Internal Ribosome Entry Site (IRES), we monitored the expression of the chimeric receptor by analyzing GFP expression. In a first series of experiments, we found that, unlike the virus used as a control, the CAR-encoding virus was T AS We observed that the two types of viruses allowed very low transduction of preactivated T cells. Preactivated T cells were transduced comparablely with the two types of viruses, demonstrating the efficacy of the CAR-encoding viruses for T cell transduction. ASThe absence of productive transduction of cells was not a result of viral infectivity (data not shown). To characterize the absence of productive transduction, we explored the possibility that the absence of expression could be a result of latent infection, as previously described (Brooks et al., 2003; Novis et al., 2013). Therefore, we used a CAR-encoding virus to transfect T cells. AS After 3 days of culture, the transduced T AS Cells were stimulated with anti-3 / 28 beads. AS Although cells only weakly express CAR immediately after transduction, activation induces a significant increase in CAR expression, demonstrating T cell proliferation with CAR-encoding particles. AS This suggests that cell transduction results in asymptomatic infection. The inventors first hypothesized that this lack of productive transduction was due to the promoter used. Indeed, in the use of retroviral vectors, the CAR was under the control of the EF1α promoter. This promoter has previously been described as being weakly active in resting cells (Ho et al., 2021; S. Jones et al., 2009). Therefore, the inventors created novel vectors in which the CAR was under the control of the MND or CMV promoter, which has been described as being active in resting T cells (Ho et al., 2021; S. Jones et al., 2009). The inventors hypothesized that regardless of the promoter used, the CAR was under the control of the MND or CMV promoter, which has been described as being active in resting T cells (Ho et al., 2021; S. Jones et al., 2009). AS We found that transduction of cells induced low CAR expression, while these induced expression comparable to viral controls in preactivated T cells. We then demonstrated that low expression of GFP downstream of the IRES sequence resulted in T AS We explored the possibility that this lack of productive transduction of cells might be responsible. Therefore, we constructed a retroviral vector in which CAR was linked to GFP via a P2A linker. Again, we found that the CAR virus transduced T AS From all these data, we found that T ASAlthough we were unable to establish the cause of this low productive transduction efficacy of the cells, we concluded that we observed that it was significantly increased after T cell stimulation.

[0186] In clinical protocols, CAR-T cell injection is preceded by an immune depletion step (Majzner and Mackall, 2019), and it has been shown that when T cells are injected into an immunocompromised host, a phenomenon called homeostatic proliferation occurs (Zwang and Turka, 2014). Thus, following T cell activation and proliferation within the host, T AS It is conceivable that intracellular CAR expression could occur. To test this hypothesis, we first investigated the T cell proliferation rate after injection in immunodeficient mice, which mimics the aplastic anemia condition in patients. AS Cell proliferation was analyzed. CFSE-labeled T AS The cells were injected into mice or maintained in vitro, and their proliferation was compared (data not shown). We observed T cells in vivo as early as 9 days after injection. AS They observed that cells proliferated in vitro, whereas the same cells did not proliferate in vitro. AS We demonstrate that CAR-T cells undergo homeostatic proliferation after in vivo injection. To further investigate whether this proliferation correlates with CAR expression, we performed CAR-T AS The cells were injected into the spleen, and the spleen was harvested 9 days after injection. The results showed that homeostatic proliferation of CAR-T cells in vivo was AS These data allow us to conclude that we have established a protocol in which CAR expression can be induced by proliferation.

[0187] [CAR-T AS The cells are more efficient than conventional CAR-T cells at eradicating tumors in vivo. CAR-T cells (CAR-T ASTo investigate the potential benefits of this new protocol for the production of CAR-T cells, we used a conventional model based on the regression of tumors formed by subcutaneous injection of human tumor cells followed by CAR-T cell injection in immunodeficient mice. We used the Capan-2 cell line, known to express abundant epidermal growth factor receptor (EGFR) and a target for EGFR-targeted CARs (Guedan et al., 2018). Specifically (Figure 5A), we transfected 5 × 10 cells into NSG mice. 6 Luciferase-expressing Capan-2 cells (Capan-2 LUC ) was subcutaneously injected. Seven days later, we injected EGFR-targeted CART cells into the orbital venous plexus and tracked tumor growth by bioluminescence. In this experimental model, 10 7 5 x 10 conventional CAR-T cells are required 5 CAR-T cells only moderately controlled tumors (data not shown). AS To ensure that the cells were not exacerbating the alloimmune response, we first compared tumor growth of T cells treated or not with AS1842856. 6 T's AS Injection of 2×10 cells did not lead to tumor regression. 5 conventional CAR-T cells or 2 × 10 5 CAR-T AS We compared tumor growth after injection of the cells. As shown in Figure 5, we found that in this setting, conventional CAR-T cells were unable to control tumor growth, while CAR-T AS We observed that the cells induced sustainable tumor regression, a result confirmed by tumor surface dimensions measured by ultrasound scan on day 40 (Figure 5B) and tumor weight at the end of the experiment (Figure 5C). 6 T's AS Injection of the cells did not result in any tumor regression or weight loss in the mice, suggesting that T cells against Capan-2 tumors ASIt should be noted that a cellular alloimmune response can be excluded in the observed effects (data not shown). AS To expand the applicability of the cells, we investigated the efficacy of CAR-T cells and CAR-T cells after intravenous injection of A549 cells derived from human lung cancer tumors. AS The antitumor efficacy of CAR-T cells was compared. This injection route (iv) allows for orthotopic localization, as tumor engraftment occurs within the lung (data not shown). Again, in this model, CAR-T AS The cells demonstrated a much stronger effect than conventional CAR-T cells. Therefore, the inventors AS They concluded that the cells were more efficient than conventional CAR-T cells in treating solid tumors.

[0188] [CAR-T cells generated from patients' cells AS grow faster than conventional CAR-T cells] While these results were promising, they were performed using T cells derived from healthy donors. However, we now know that a patient's immune system is compromised not only by repeated therapies preceding CAR-T cell therapy but also by the pathology itself. It has been widely publicized that T cells derived from cancer patients are not equivalent to T cells derived from healthy donors, and this is true across multiple cancer diseases (Hoffmann et al., 2017; Metelo et al., 2022). Therefore, to validate the benefits of CAR-T cells in a clinical setting, we conducted a series of experiments on T cells collected from patients included in a CAR-T cell therapy protocol. Two patients had primary mediastinal lymphoma refractory to second-line therapy, one patient had severe large B-cell lymphoma refractory to third-line therapy, and two patients had mantle cell lymphoma refractory to three lines of therapy, each of which had relapsed after three lines of therapy, each of which included one Bruton's tyrosine kinase inhibitor and autologous stem cell transplantation. We compared the phenotypic and functional properties of conventional CAR-T cells and CAR-TAS cells. We first verified the feasibility of obtaining CAR-TAS and CAR-T cells derived from patient cells with comparable efficiency (data not shown). We then observed in Figure 6A that the conventional protocol for obtaining CAR-T cells enabled the generation of more cells displaying a TSCM-specific phenotype than the AS1842856 protocol for all three patients tested, even though patient 1 had more differentiated T cells overall in the CAR-T cells than in the CAR-TAS cells. However, because TCR stimulation induces the expression of memory markers, we could not state that the TSCM phenotype observed for conventional CAR-T cells corresponds to memory cells per se. Furthermore, immune checkpoint analysis revealed that CAR-TAS cells displayed a less exhausted phenotype than CAR-T cells, with reduced expression of Tim3 and LAG-3 (Figure 6B).One favorable prognostic parameter for successful therapy is the ability of cells to rapidly proliferate in the patient's body after infusion (Majzner et al., 2019; Fraietta et al., 2018). Therefore, to mimic this proliferation, we stimulated different CAR-T cells in vitro with TransAct at the end of each manufacturing protocol and counted the cells every two days. While CAR-TAS cells derived from all patients exhibited superior proliferation capabilities compared to conventional CAR-T cells, CAR-TAS cells derived from patient 2 exhibited a delay compared to CAR-T cells (Figure 6C). These results demonstrate that CAR-TAS cells can be easily obtained from CAR-eligible patients and retain superior proliferation properties compared to conventional CAR-T cells.

[0189] [Conclusion] In this study, we showed that AS1842856, as an inhibitor of FOXO1, induced a substantial increase in the metabolic activity of human T cells purified from healthy donors after several days of culture in the absence of any growth factors (e.g., cytokines), which correlated with the acquisition of phenotypic and functional characteristics of activated / memory T cells. More specifically, we showed that AS1842856 significantly increased the metabolic activity of CD8 + Not only within T cells but also CD4 + It also induces increased production of granzyme B in T cells, resulting in the production of human CD8 +We found that FOXO1 inhibition resulted in enhanced cytotoxic activity of primary T lymphocytes. Through tolerance induced by FOXO1 inhibition, we demonstrated that simple pharmacological treatment in ex vivo culture could generate CAR-T cells lacking the exhausted properties of conventional CAR-T cells. We observed that inhibition of FOXO1 with the pharmacological agent AS1842856 not only enabled the infection of resting T cells, but also allowed CAR-T cells to acquire phenotypic and functional characteristics that led to significantly increased antitumor activity. Specifically, we demonstrated for the first time that FOXO1 inhibition enhanced the ability to induce target cell lysis by increasing the expression of TNF-α and other inflammatory cytokines, induced spontaneous cell polarization comparable to that achieved by stimulation with chemokines, thus improving cell motility, inducing a rapid expansion of memory T cells, improving T cell proliferation, and enabling the generation of CAR-T cells that are more efficient in treating solid tumors than conventional CAR-T cells obtained by known protocols. We also demonstrate that T cells obtained using our protocol undergo homeostatic proliferation after in vivo injection to induce CAR expression. The results obtained with patient-derived T cells enable the generation of CAR-T cells with high proliferation potential, which may improve current clinical therapies, which remain limited to a subset of tumors.

[0190] [References] Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure. Adachi, K., & Davis, M. M. (2011). T-cell receptor ligation induces distinct signaling pathways in naive vs. antigen-experienced T cells. Proceedings of the National Academy of Sciences of the United States of America, 108(4), 1549-1554. https: / / doi.org / 10.1073 / pnas.1017340108 Amara A., Vidy A., Boulla G., Mollier K, Garcia-Perez J, Alcami J, Blanpain C, Parmentier M, Virelizier JL, Charneau P, Arenzana-Seisdedos F. (2003). G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates. Journal of Virology, 77(4):2550-8. doi: 10.1128 / jvi.77.4.2550-2558.2003.Asperti-Boursin, F., Real, E., Bismuth, G., Trautmann, A., & Donnadieu, E. (2007). 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Claims

1. In an in vitro method for obtaining improved CAR-T cells, i. culturing T cells obtained from the subject with a FOXO1 inhibitor for 2-10 days; ii. transforming the T cells into CAR-T cells by known methods; A method comprising:

2. In an in vitro method for obtaining improved CAR-T cells, i. providing T cells from a subject; ii. culturing the T cells with a FOXO1 inhibitor for 2-10 days; iii. Transforming the T cells into CAR-T cells by known methods; 10. The method of claim 1, comprising:

3. 3. An in vitro method for obtaining improved CAR-T cells according to claim 1 or 2, comprising a further step of adding IL-7 and / or IL-15 after using an inhibitor of FOXO1.

4. The in vitro method for obtaining improved CAR-T cells according to any one of claims 1 to 3, wherein the FOXO1 inhibitor is AS1842856.

5. CAR-T cells obtained by the method according to any one of claims 1 to 4 for use in improving immune responses.

6. CAR-T cells obtained by the method of any one of claims 1 to 4 for use in the treatment of cancer or infectious diseases.

7. A method for improving the immune system using CAR-T cells obtained by the method according to any one of claims 1 to 4.

8. A therapeutic composition comprising CAR-T cells obtained by the method according to any one of claims 1 to 4, for use in improving immune responses.

9. A therapeutic composition comprising CAR-T cells obtained by the method of any one of claims 1 to 4, for use in the treatment of cancer and infectious diseases.

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