Engineered immune cells

Abstract

To provide an immune cell expressing a chimeric antigen receptor, wherein the immune cell is modified such that the expression and / or function of lymphocyte-specific protein tyrosine kinase is reduced or eliminated.SOLUTION: An immune cell expressing a chimeric antigen receptor is provided, wherein the chimeric antigen receptor comprises an intracellular signaling domain or a fragment thereof that functions in the absence of lymphocyte-specific protein tyrosine kinase, and the immune cell is modified such that the expression and / or function of lymphocyte-specific protein tyrosine kinase is reduced or eliminated.SELECTED DRAWING: None

Description

Technical Field

[0001] Field The present disclosure generally relates to the field of immunology. In particular, the present disclosure relates to immune cells expressing chimeric antigen receptors (CARs) and methods for treating a subject's disease.

Background Art

[0002] Background Immunotherapy has made unprecedented progress in the treatment of cancer patients in recent years. In adoptive T cell therapy, isolated human T cells are genetically modified to enhance their specificity for a particular tumor antigen, such as by expression of a chimeric antigen receptor. Adoptive T cell therapy that requires chimeric antigen receptor T cells (CAR-T cells) is a major part of the immuno-oncology pipeline. To date, three generations of CAR-T technology have been developed and used in clinical trials for several cancers, including B cell malignancies and multiple myeloma.

[0003] Despite its great utility as a cancer treatment, adoptive immunotherapy with CAR-T cells is partially limited by the expression of endogenous T cell receptors on the cell surface. CAR-T cells expressing endogenous T cell receptors can recognize major and minor histocompatibility antigens after administration to allogeneic patients. This has non-specific effects and can lead to the development of graft-versus-host disease (GVHD) in patients.

[0004] Another limitation of adoptive T cell immunotherapy with CAR-T cells is that after re-infusion into patients, CAR-T cells begin to exhibit a "depletion" phenotype due to the expression of inhibitory receptors such as PD-1, LAG-3, and TIGIT, and T cell effector function is lost. This is a particular problem for solid tumors that often express ligands for these inhibitory receptors (such as PD-L1 and PD-L2 for PD-1), limiting the utility of CAR-T therapy for solid tumors.

[0005] Therefore, it is necessary to overcome or at least mitigate one or more of the problems mentioned above.

SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

[0006] SUMMARY OF THE INVENTION Provided herein are immune cells expressing a chimeric antigen receptor (CAR).

[0007] In one aspect, an immune cell expressing a chimeric antigen receptor (CAR) is provided, wherein the CAR comprises an intracellular signaling domain or fragment that functions in the absence of lymphocyte-specific protein tyrosine kinase (LCK), and the immune cell is modified such that the expression and / or function of LCK is reduced or abolished.

[0008] In one embodiment, the intracellular signaling domain or fragment functions in the presence of a dysfunctional LCK.

[0009] In one aspect, an immune cell expressing a CAR is provided, wherein the immune cell is modified such that the expression or function of the LCK gene is disrupted.

[0010] In one aspect, provided is a method for producing an immune cell as defined herein, the method comprising contacting the immune cell with an inhibitor of LCK for a time and under conditions sufficient to reduce or abolish the expression and / or function of LCK.

[0011] In one aspect, provided is a vector system comprising 1) a vector comprising a nucleic acid sequence encoding an inhibitor of LCK, and 2) a vector comprising a nucleic acid sequence encoding a CAR.

[0012] In one aspect, provided is a vector comprising a nucleic acid sequence encoding an inhibitor of LCK and a nucleic acid sequence encoding a CAR.

[0013] In one aspect, provided is a method for improving the efficacy of CAR-expressing immune cells in cell therapy, the method comprising contacting the CAR-expressing immune cells with an inhibitor of LCK for a time and under conditions sufficient to reduce or abolish the expression and / or function of LCK.

[0014] In one aspect, a method of treating a subject in need thereof is provided, the method comprising administering immune cells as defined herein under time and conditions sufficient to treat the subject.

[0015] In one aspect, immune cells as defined herein are provided for use in the treatment of a subject in need thereof.

[0016] In one aspect, the use of immune cells as defined herein in the manufacture of a medicament for treating a subject in need thereof is provided.

[0017] Brief Description of the Drawings Here, some embodiments of the present invention will be described by way of non-limiting examples only with reference to the accompanying drawings.

Brief Description of the Drawings

[0018]

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Modes for Carrying Out the Invention

[0019] Detailed Description In one aspect, an immune cell expressing a chimeric antigen receptor (CAR) is provided, the CAR comprising an intracellular signaling domain or fragment that functions in the absence of lymphocyte-specific protein tyrosine kinase (LCK), and the immune cell is modified such that the expression and / or function of LCK is reduced or eliminated.

[0020] In one embodiment, an immune cell expressing a chimeric antigen receptor (CAR) is provided, the CAR comprising an intracellular signaling domain or fragment that functions in the absence of LCK or in the presence of a dysfunctional LCK, and the immune cell being modified such that the expression and / or function of LCK is reduced or eliminated.

[0021] Without being bound by theory, the inventors have found that the SRC family kinase LCK, an important signaling kinase in T cell receptor (TCR) signaling, may not be important for CAR signaling. Since LCK is extremely important for signaling via the TCR, it can be confirmed that only antigen recognition by the CAR leads to T cell activation by deleting or inhibiting LCK in CAR-T cells. Thereby, the off-target effect can be reduced and the safety of CAR technology can be improved. This can also help avoid the occurrence of autoimmunity caused by the endogenous TCR and reduce the possibility of graft-versus-host disease of allogeneic CAR-T cells.

[0022] The term "chimeric antigen receptor" or "CAR" can refer to a series of polypeptides that, when within an immune effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation. The CAR can include at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the "intracellular signaling domain") that includes a functional signaling domain derived from the primary signaling domain and / or costimulatory domain defined below. The series of polypeptides can be adjacent to each other. In some embodiments, the series of polypeptides includes a dimerization switch that can bind the polypeptides to each other in the presence of a dimerizing molecule, for example, binding the antigen-binding domain to the intracellular signaling domain.

[0023] In one embodiment, the CAR has a nucleic acid or amino acid sequence as shown in Table 1.

[0024] In one embodiment, an immune cell expressing a chimeric antigen receptor (CAR) is provided, the CAR comprising an intracellular signaling domain or fragment that functions in the absence of LCK, and the immune cell is modified such that the expression and / or function of LCK is knocked out or knocked down.

[0025] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates signals that promote the immune effector functions of CAR-containing cells, such as CAR T cells. Examples of immune effector functions in CAR T cells include cytolytic activity and helper activity, including the secretion of cytokines.

[0026] In one embodiment, an immune cell expressing a chimeric antigen receptor (CAR) is provided, the CAR comprising CD28 or a fragment of CD28 that functions in the absence of LCK, and the immune cell is modified such that the expression and / or function of LCK is knocked out or knocked down.

[0027] An intracellular signaling domain or fragment may be one that functions in the absence of LCK or in the presence of a dysfunctional LCK. An intracellular signaling domain or fragment that functions in the absence of LCK or in the presence of a dysfunctional LCK can interact with FYN and / or be phosphorylated by FYN, and thus can cause LCK-independent signaling. The intracellular signaling domain can include, for example, the signaling domain or fragment of ADD2, BCAR1, c-Raf, CBLC, CD28, CD36, CD44, CDH1, CHRNA7, CTNND1, CBL, CSF1R, DLG4, dystroglycan, EPHA8, FYB, FASLG, GNB2L1, GRIN2A, ITK, Janus kinase 2, KHDRBS1, LKB1, nephrin, PAG1, PIK3R2, PRKCQ, PTK2B, PTK2, PTPRT, UNC119, RICS, SH2D1A, SKAP1, Syk, TNK2, TRPC6, tau protein, TrkB, TYK2, TUBA3C, WAS, or ZAP-70. An intracellular signaling domain or fragment that functions in the absence of LCK or in the presence of a dysfunctional LCK can be CD28 or a fragment of CD28 that can induce LCK-independent signaling. In one embodiment, the intracellular signaling domain includes the signaling domain or fragment of the CD28 protein that functions in the absence of LCK or in the presence of a dysfunctional LCK.

[0028] In one embodiment, the intracellular signaling domain includes the signaling domain or fragment of the CD28 protein having a PYAP motif. In one embodiment, the intracellular signaling domain includes the sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 1).

[0029] In one embodiment, the intracellular signaling domain further includes a primary signaling domain that includes a functional signaling domain of a protein selected from CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ, FcεRIβ, CD79a, CD79b, FcγRIIa, DAP10, or DAP12. The intracellular signaling domain may further include one or more functional signaling domains derived from at least one co-stimulatory domain, as defined below. In one embodiment, the intracellular signaling domain includes a co-stimulatory domain that includes a functional signaling domain of a protein selected from the group consisting of DAP10, CD28, CARD11, SLAMF1, LCK1, LCK3, LAT, OX40, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0030] In one embodiment, the CAR includes an extracellular antigen-binding domain. The antigen-binding domain can be, for example, an antibody or an antibody fragment. The antigen-binding domain can also be an autoantigen that can be recognized by a self-antigen specific B-cell receptor on a B lymphocyte, and thus induces T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. The antigen-binding domain can also be a peptide or protein ligand.

[0031] As used herein, the term "antibody" can refer to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. The antibody can be polyclonal or monoclonal, multichain or single-chain, or intact immunoglobulin, and can be derived from a natural or recombinant source.

[0032] The term "antibody fragment" can refer to at least a part of an antibody that retains the ability to specifically interact with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, bispecific antibodies formed from antibody fragments such as bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv.

[0033] In one embodiment, the "antibody fragment" is an scFV.

[0034] The term "scFv" refers to a fusion protein that includes at least one antibody fragment containing the variable region of the light chain and at least one antibody fragment containing the variable region of the heavy chain, wherein the variable regions of the light and heavy chains are adjacent and linked, for example, via a synthetic linker, such as a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv used herein can have the VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL-linker-VH or VH-linker-VL.

[0035] The CAR can include a target-specific binding element, also referred to as an antigen-binding domain. The selection of the moiety depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on the target cell associated with a particular pathological condition. Thus, examples of cell surface markers that can act as ligands for the antigen moiety domain of the CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0036] The CAR can be engineered to target a desired tumor antigen by engineering a desired antigen-binding domain that specifically binds to an antigen on the tumor cell. In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder such as cancer. The antigens discussed herein are included by way of example only. This list is not intended to be exclusive and additional examples will readily become apparent to those skilled in the art.

[0037] Tumor antigens are proteins produced by tumor cells that induce an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding domain of the present invention depends on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostain, PSMA, Her2 / neu, survivin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, folate receptor (FRa), and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, EGFR, CA-IX, MUC1, HER2, and any combination thereof. In one embodiment, the first CAR comprises an antigen-binding domain that binds to mesothelin, and the second CAR comprises an antigen-binding domain that binds to FRa. In one embodiment, the CAR comprises an antigen-binding domain that binds to HER2.

[0038] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express several proteins that can function as target antigens for an immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP 100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to a group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are tumor fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but the success has been limited.

[0039] The types of tumor antigens referred to in the present invention can also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur in other cells in the body. TAA-related antigens are not unique to tumor cells; rather, they are expressed in normal cells under conditions that cannot induce a state of immune tolerance to the antigen. The expression of the antigen on the tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens that are expressed in normal cells during fetal development when the immune system is immature and unable to respond, or antigens that are normally present at extremely low levels in normal cells but are expressed at much higher levels in tumor cells.

[0040] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, etc.; and viral antigens such as Epstein-Barr virus antigens EBVA, LMP2 (e.g., LMP2A or LMP2B) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4. Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS are included.

[0041] In one embodiment, the scFV is an anti-CD19 scFV domain. The anti-CD19 scFV domain can have, for example, the sequence of (a)

Chemical formula

[0042] In one embodiment, the scFV is an scFVC that can bind to a peptide-MHC complex presenting one of the LMP2A protein peptides. The scFV can be, for example, (a) [Chemical formula] and may have the sequence of.

[0043] In one embodiment, the scFV is an anti-LMP2 scFV domain.

[0044] The "antibody fragment" may include antibody fragment sequences of other antibodies known in the art, depending on the antigen to be targeted.

[0045] In one embodiment, the chimeric antigen receptor further includes a signal peptide. The signal peptide can have, for example, the sequence of MALPVTALLLPLALLLHAARP (SEQ ID NO: 4).

[0046] The CAR can be a CAR having a protein sequence as shown in Table 1.

[0047] In one embodiment, the immune cell is a recombinant immune cell. The term "recombinant" refers to a cell modified by the introduction of a heterologous nucleic acid, or a cell derived from a cell that has been modified in such a way but does not include modification of the cell by natural events such as those that occur without intentional human intervention (e.g., spontaneous mutations, natural transformation, natural transduction, natural translocation). Recombinant immune cells can be cells that do not occur naturally. Recombinant immune cells can also be engineered cells. In one embodiment, the recombinant immune cell is an engineered immune cell such as an engineered T cell or an engineered NK cell. In one embodiment, the recombinant immune cell is an isolated immune cell.

[0048] In one embodiment, the immune cell is a T cell or an NK cell.

[0049] As used herein, the term "immune cell" refers to cells of hematopoietic origin that play a role in the immune response. Immune cells include lymphocytes such as B cells and T cells; natural killer (NK) cells; myeloid cells such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils, and granulocytes.

[0050] In one embodiment, the immune cell is an immune effector cell. As used herein, the term "immune effector cell" refers to a cell involved in promoting an immune response, such as an immune effector response. Examples of immune effector cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0051] As used herein, the term "immune effector function or immune effector response" refers to a function or response that enhances or promotes the immune attack of target cells by, for example, immune effector cells. For example, the function or response of an immune effector refers to the properties of T cells or NK cells that promote the death of target cells or the inhibition of their growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0052] The term "stimulation" refers to the binding of a stimulatory molecule (e.g., TCR / CD3 complex or CAR) to its cognate ligand (or tumor antigen in the case of CAR), thereby mediating a signaling event such as, but not limited to, signal transduction via the TCR / CD3 complex or signal transduction via the signaling domain of an appropriate NK receptor or CAR, which induces a primary response. Stimulation can mediate a change in the expression of specific molecules.

[0053] As used herein, the term "T cell" includes CD4+ T cells and CD8+ T cells. The term T cell also includes both type 1 T helper cells and type 2 T helper cells, as well as Th-IL 17 cells.

[0054] In one embodiment, the immune cells are modified such that the expression and / or function of LCK is reduced or eliminated. The immune cells can be modified, for example, to obtain LCK knockout or knockdown. The term "knockout" can refer to the loss of a gene or gene expression. For example, a gene can be knocked out by either a deletion or addition of a nucleotide sequence that leads to a disruption of the reading frame. As another example, a gene can be knocked out by replacing a part of it with an unrelated sequence. On the other hand, the term "knockdown" can refer to a reduction in the expression of a gene or its gene product. As a result of gene knockdown, the activity or function of a protein can be weakened, or the protein level can be reduced or eliminated.

[0055] In one embodiment, the immune cells contain or are in contact with an inhibitor of LCK.

[0056] The inhibitor of LCK can be a nucleic acid sequence that can down-regulate or eliminate the gene expression of LCK, or modify the function of LCK.

[0057] The nucleic acid can be a nucleic acid that can down-regulate or eliminate the gene expression of LCK, or modify the function of LCK, and is selected from the group consisting of antisense RNA, antagomir RNA, siRNA, shRNA, CRISPR systems, zinc finger nuclease systems, and transcription activator-like effector-based nuclease (TALEN) systems. In one embodiment, the nucleic acid encodes an intracellular antibody that binds to a protease that degrades LCK or indirectly results in the degradation of intracellular LCK.

[0058] It is possible to perform DNA cleavage of the genome of living cells using a site-specific nuclease, and it is known in the art that such DNA cleavage can result in permanent modification of the genome via mutagenic non-homologous end joining (NHEJ) repair or via homologous recombination at a transgenic DNA sequence. NHEJ can cause mutagenesis at the cleavage site, resulting in inactivation of the allele. NHEJ-related mutagenesis can inactivate an allele via the generation of premature stop codons, frameshift mutations that produce abnormal non-functional proteins, or can trigger mechanisms such as nonsense-mediated mRNA decay. The use of nucleases to induce mutagenesis via NHEJ can be utilized to target specific mutations or sequences present in the wild-type allele. The use of nucleases to induce double-strand breaks at a target locus is known to stimulate homologous recombination repair (HDR), particularly transgenic DNA sequences adjacent to sequences homologous to the genomic target. In this way, exogenous nucleic acid sequences can be inserted into the target locus. Such exogenous nucleic acids can encode, for example, chimeric antigen receptors, exogenous TCRs, or any sequence or polypeptide of interest.

[0059] In different embodiments, various different types of nucleases are useful for practicing the present invention. In one embodiment, the present invention can be practiced using a recombinant meganuclease. In another embodiment, the present invention can be practiced using a CRISPR nuclease. Methods for generating CRISPRs that recognize a given DNA site are known in the art. In another embodiment, the present invention can be practiced using TALEN or Compact TALEN. In a further embodiment, the present invention can be practiced using MegaTAL.

[0060] Engineered endonucleases based on the CRISPR / Cas9 system are also known in the art. CRISPR endonucleases comprise two components: (1) a caspase effector nuclease, typically the microbial Cas9; and (2) a short "guide RNA" containing a nucleotide target sequence that directs the nuclease to a desired location within the genome.

[0061] The term "CRISPR" refers to a caspase-based endonuclease that includes a caspase such as Cas9 and a guide RNA that hybridizes to a recognition site in genomic DNA to induce DNA cleavage by the caspase. The guide RNA for knockout can be the guide RNA shown in Table 2. In one embodiment, the guide RNA is used with SEQ ID NO: 43 to knockdown the expression of the LCK gene in cells. In one embodiment, the guide RNA is SEQ ID NO: 10.

[0062] In one embodiment, an inhibitor of LCK is an inhibitor of the LCK protein. An inhibitor of LCK can be an inhibitor of LCK kinase activity. An inhibitor of LCK can be selected from the group consisting of aminokynazoline, A-420983, A770041, dasatinib, saractinib, and masatinib.

[0063] In one embodiment, the immune cells have a reduced expression of PD-1 compared to cells that have not been modified such that the expression and / or function of LCK is reduced or eliminated. Thereby, the tendency of T cells to become exhausted can be reduced. This can also improve the CAR-T cell response against solid tumors.

[0064] In one embodiment, the immune cell comprises a vector comprising a nucleic acid encoding a CAR. In one embodiment, the immune cell comprises a vector comprising a nucleic acid encoding an inhibitor of LCK that can downregulate or ablate the gene expression of LCK. In one embodiment, the immune cell comprises a vector comprising a nucleic acid encoding a CAR and a nucleic acid encoding an inhibitor of LCK that can downregulate or ablate the gene expression of LCK.

[0065] The term "vector" or "expression construct" may refer to a nucleic acid molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of an operably linked coding sequence (e.g., an inserted sequence encoding a product) in a particular cell. The expression vector construct may contain sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vector constructs include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses that incorporate recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0066] "Vector" can also be an "introduction vector" that contains an isolated nucleic acid and refers to a composition that can be used to deliver the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "introduction vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as, for example, polylysine compounds and liposomes. Examples of viral introduction vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors.

[0067] In some embodiments, the CAR sequences are delivered to cells using retroviral or lentiviral vectors. CAR-expressing retroviral and lentiviral vectors can be used to deliver to various types of eukaryotic cells, as well as tissues and whole organisms, using transduced cells as carriers or using encapsulation, conjugation, or cell-free local or systemic delivery of naked vectors. The methods used can be used for any purpose for which stable expression is required or sufficient.

[0068] The term "expression" can refer to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0069] The term "encoding" or "encode" includes reference to nucleotides and / or amino acids that correspond to other nucleotides or amino acids in the sense of transcription and / or translation.

[0070] The term "nucleic acid" includes deoxyribonucleotide or ribonucleotide polymers in either single-stranded or double-stranded form, and includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to natural nucleotides, unless otherwise limited. The terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", and "polynucleotide" are used interchangeably herein unless the context indicates otherwise. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNA, shRNA, RNAi agents, and primers. A polynucleotide can be modified or substituted with one or more bases, sugars, and / or phosphates using any of a variety of modifications or substitutions described herein or known in the art. A polynucleotide can include modified nucleotides such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure can be imparted before or after construction of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation to a label component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.In some contexts, terms such as "nucleic acid" or "polynucleotide" may include any substance that transmits genetic information or performs the function of a nucleic acid or polynucleotide (e.g., can be translated into a protein or act as an RNAi agent), even if not strictly composed of nucleotides (consisting of a sugar, base, and phosphate); such genetic materials can include, by way of non-limiting example, peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2'-fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), and / or unlocked nucleic acid (UNA).

[0071] The terms "protein" and "polypeptide" are used synonymously and can refer to any polymer of amino acids (dipeptide or greater) linked via peptide bonds or modified peptide bonds. Polypeptides of less than about 10 to 20 amino acid residues are generally called "peptides". The polypeptides of the present invention can include non-peptide components such as carbohydrate groups. Carbohydrates and other non-peptide substituents can be added to the polypeptide by the cell in which the polypeptide is produced and vary depending on the cell type. Polypeptides are defined herein with respect to their amino acid backbone structure; substituents such as carbohydrate groups are generally not specified but can still be present.

[0072] Immune cells expressing a CAR are provided herein, and the immune cells are modified such that the LCK gene is disrupted. Immune cells expressing a CAR are provided herein, and the immune cells are modified such that the expression or function of the LCK gene is disrupted. In one embodiment, CART cells in which the LCK gene is disrupted are provided. In one embodiment, CART cells in which the expression or function of the LCK gene is disrupted are provided. In one embodiment, a method of disrupting the LCK gene in CART cells or immune cells expressing a CAR is provided. In one embodiment, a method of disrupting the expression or function of the LCK gene in CART cells or immune cells expressing a CAR is provided.

[0073] The terms "disrupt" and "disrupted" are used interchangeably herein and refer to any genetic modification that reduces or eliminates the expression and / or functional activity of a nucleic acid or its expression product. For example, disruption of a gene includes within its scope any genetic modification that reduces or eliminates the expression of the gene and / or the functional activity of the corresponding gene product (e.g., mRNA and / or protein). Genetic modifications include complete or partial inactivation, suppression, deletion, interruption, blockade, or down-regulation of a nucleic acid (e.g., a gene). Exemplary genetic modifications include, but are not limited to, gene knockout, inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or the use of inhibitory nucleic acids (e.g., inhibitory RNAs such as sense or antisense RNA, siRNA, shRNA, miRNA, etc., molecules that mediate RNA interference), inhibitory polypeptides (e.g., antibodies, polypeptide binding partners, dominant negative polypeptides, enzymes, etc.), or other molecules that inhibit the activity of the LCK gene or the level or functional activity of the expression product of the LCK gene.

[0074] In one aspect, provided is a method for manufacturing (or preparing) immune cells as defined herein, the method comprising contacting the immune cells with an inhibitor of LCK for a time and under conditions sufficient to reduce or abolish the expression and / or function of LCK. The inhibitor of LCK can be a nucleic acid sequence capable of down-regulating or abolishing gene expression or modifying the function of LCK. In one embodiment, the inhibitor of LCK is a CRISPR system. The method can further comprise introducing a nucleic acid encoding a CAR into the immune cells. The method can include a prior step of harvesting immune cells from a patient.

[0075] In one embodiment, provided is a nucleic acid encoding an inhibitor of LCK. In one embodiment, provided is a nucleic acid encoding a CAR. In one embodiment, provided is a nucleic acid comprising a nucleic acid encoding an inhibitor of LCK and a nucleic acid encoding a CAR.

[0076] In one aspect, provided is a vector system comprising 1) a vector comprising a nucleic acid sequence encoding an inhibitor of LCK, and 2) a vector comprising a nucleic acid sequence encoding a CAR.

[0077] In one aspect, provided is a vector comprising a nucleic acid sequence encoding an inhibitor of LCK and a nucleic acid sequence encoding a CAR.

[0078] In one embodiment, the nucleic acid sequence encoding a CAR is also an inhibitor of LCK.

[0079] In one embodiment, the present invention provides a pharmaceutical composition comprising the immune cells or vectors described herein and a pharmaceutically acceptable carrier.

[0080] The term "pharmaceutically acceptable carrier" means a solid or liquid filler, diluent, or encapsulating substance that can be safely used in local or systemic administration to mammals, including animals, preferably humans. Representative pharmaceutically acceptable carriers include all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweetening agents, flavoring agents, dyes, such substances, etc., and combinations thereof (see, e.g., Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Its use in pharmaceutical compositions is contemplated, except where any conventional carrier is incompatible with the active ingredient.

[0081] In one aspect, a method of improving the efficacy of CAR-expressing immune cells in cell therapy is provided, the method comprising contacting the CAR-expressing immune cells with an inhibitor of LCK for a time and under conditions sufficient to reduce or abrogate the expression and / or function of LCK.

[0082] In one embodiment, the CAR-expressing immune cells are CAR T cells.

[0083] In one embodiment, the off-target effect of the CAR-expressing immune cells is reduced. In one embodiment, the exhausted phenotype in the CAR-expressing cells is reduced. In one embodiment, the memory of the CAR-expressing immune cells is improved.

[0084] A method of reducing the off-target effect of CAR-expressing immune cells in cell therapy is disclosed herein, the method comprising contacting the CAR-expressing immune cells with an inhibitor of LCK for a time and under conditions sufficient to reduce or abrogate the expression and / or function of LCK.

[0085] Methods for reducing the exhausted phenotype of CAR-expressing immune cells in cell therapy are disclosed herein, the methods comprising contacting the CAR-expressing immune cells with an inhibitor of LCK for a time and under conditions sufficient to reduce or abolish the expression and / or function of LCK.

[0086] Methods for improving the memory of CAR-expressing immune cells in cell therapy are disclosed herein, the methods comprising contacting the CAR-expressing immune cells with an inhibitor of LCK for a time and under conditions sufficient to reduce or abolish the expression and / or function of LCK.

[0087] In one aspect, a method of treating a subject in need thereof is provided, the method comprising administering an immune cell as defined herein for a time and under conditions sufficient to treat the subject.

[0088] In one embodiment, the subject has a disease associated with the expression of a tumor antigen (e.g., a proliferative disease, a pre-cancerous condition, cancer, and non-cancer related symptoms associated with the expression of a tumor antigen).

[0089] The phrase "diseases associated with the expression of the tumor antigens described herein" includes, but is not limited to, for example, proliferative diseases such as cancer or malignant tumors, or pre-cancerous states such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia; or diseases associated with the expression of the tumor antigens described herein, or conditions associated with cells expressing the tumor antigens described herein, including non-cancer-related symptoms associated with cells expressing the tumor antigens described herein. In one aspect, the cancer associated with the expression of the tumor antigens described herein is a hematological cancer. In one aspect, the cancer associated with the expression of the tumor antigens described herein is a solid cancer. Further diseases associated with the expression of the tumor antigens described herein include, but are not limited to, for example, atypical and / or non-classical cancers, malignant tumors, pre-cancerous states, or proliferative diseases associated with the expression of the tumor antigens described herein. Non-cancer-related symptoms associated with the expression of the tumor antigens described herein include, but are not limited to, for example, autoimmune diseases (such as lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells express the mRNA encoding the tumor antigen, or have expressed it at any time. In one embodiment, the tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In one embodiment, the tumor antigen-expressing cells produce a detectable level of the tumor antigen protein at one point in time and then substantially no longer produce a detectable tumor antigen protein. In one embodiment, the tumor antigen-expressing cells overexpress the tumor antigen protein.

[0090] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in cells of a disease region such as a solid tumor within that tissue or organ, compared to the level of expression in normal cells of a particular tissue or organ of a patient. Patients having a solid tumor or a hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0091] In one embodiment, the subject has an infectious disease. The infectious disease can result in the expression of one or more infection markers (such as bacterial markers or viral markers) on the surface of infected cells that can be targeted by the immune cells of the present invention. The infectious disease can be, for example, an infection caused by Epstein - Barr virus.

[0092] In one embodiment, the subject has an autoimmune disease such as rheumatoid arthritis, psoriasis, or systemic lupus erythematosus.

[0093] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive reaction to self - antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, orchitis, glomerulonephritis, Graves' disease, Guillain - Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

[0094] The terms "cancer" and "cancerous" typically refer to or describe a physiological state in mammals characterized by a partially disordered cell growth. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including cancers at initial and advanced stages. The term "pre-cancerous" typically refers to a state or growth that precedes or develops into cancer. "Non-metastatic" means a cancer that is benign or remains at the primary site and does not invade the lymphatic or vascular system or tissues outside the primary site. Generally, non-metastatic cancers are any cancers that are stage 0, I, or II cancers, and in some cases, stage III cancers. "Early-stage cancer" means a cancer that is neither invasive nor metastatic, or a cancer classified as stage 0, I, or II. The term "advanced cancer" generally refers to stage III or stage IV cancers, but may also refer to stage II cancers or sub-stages of stage II cancers. One of ordinary skill in the art will understand that the classification of a stage II cancer as either an early-stage or advanced-stage cancer depends on the particular type of cancer.

[0095] The term "cancer" includes, but is not limited to, breast cancer, colorectal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or uveal melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vulvar cancer, squamous cell carcinoma, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, CNS tumor, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, myeloma, brainstem glioma, pituitary adenoma, choroidal melanoma (also known as uveal melanoma), testicular cancer, oral cancer, pharyngeal cancer, or combinations thereof.

[0096] Treatable cancers include tumors that are not angiogenesis or not yet substantially angiogenesis, as well as angiogenesis tumors. Cancer can include non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) or solid tumors. The types of cancer to be treated with the CAR of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or malignant lymphomas, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0097] Blood cancer is cancer of the blood or bone marrow. Examples of blood (or hematogenous) cancers include acute leukemia (acute lymphoblastic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, myeloblastic, and promyelocytic, myelomonocytic, monocytic, and erythroleukemia, etc.), chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia, etc.), Burkitt lymphoma, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (indolent and high-grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and leukemia including myelodysplasia.

[0098] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be either benign or malignant. Various types of solid tumors (such as sarcomas, carcinomas, lymphomas, etc.) are named according to the type of cells that form them. Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, malignant lymphoma, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocytoma, cholangiocarcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (such as glioma (such as brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, anaplastic glioma, meningioma, glioblastoma, retinoblastoma, and brain metastases, etc.).

[0099] In one embodiment, a method of immunizing a subject against a disease is provided, the method comprising administering immune cells as defined herein for a time and under conditions sufficient to immunize the subject.

[0100] The term "administering" refers to contacting, applying, injecting, transfusing, or providing the composition of the present invention to a subject.

[0101] As used herein, the term "treating" can refer to (1) preventing or delaying the appearance of one or more symptoms of a disorder; (2) inhibiting the onset of a disorder or one or more symptoms of a disorder; (3) alleviating a disorder, i.e., causing regression of the disorder or causing at least one or more symptoms of the disorder to regress; and / or (4) causing a reduction in the severity of one or more symptoms of a disorder.

[0102] The term "subject" as used throughout this specification is to be understood to mean a human, or can be a domestic animal or pet. While the methods of the present invention are particularly contemplated for the treatment of humans, such methods are also applicable to veterinary medicine, including the treatment of pets such as dogs and cats, as well as domestic animals such as horses, cows, and sheep, or zoo animals such as primates, felines, canines, bovines, ungulates, etc. A "subject" can include a person, patient, or individual, and can be of any age or gender.

[0103] The methods defined herein can include administering to a subject in need thereof an effective amount of immune cells. The term "effective amount" as defined herein means the administration of an amount of an agent that is effective for its induction, treatment, or prevention, as a single administration or as part of a series of administrations, to an individual in need thereof. The effective amount will vary depending on the health and condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. The amount is expected to fall within a relatively broad range that can be determined through routine testing.

[0104] In one aspect, there is provided immune cells as defined herein for use in the treatment of a subject in need thereof.

[0105] In one aspect, there is provided the use of immune cells as defined herein in the manufacture of a medicament for treating a subject in need thereof.

[0106] The immune cells of the present invention can be administered alone or as a pharmaceutical composition in combination with other components such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention can include the target cell populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline and phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0107] The pharmaceutical compositions of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the condition of the patient, the type and severity of the patient's illness, but appropriate dosages may also be determined by clinical trials.

[0108] Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient. In addition to using cell-based vaccines for ex vivo immunization, the methods described herein also include compositions and methods for in vivo immunization that induce an immune response against an antigen of a patient.

[0109] The use of immune cells as defined herein is provided herein.

[0110] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes, individually or collectively, all steps, features, compositions, and compounds referred to or indicated herein, as well as any and all combinations of any two or more of said steps or features.

[0111] As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "agent" includes a plurality of agents including mixtures thereof.

[0112] Throughout this specification and the following claims, unless the context clearly requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," are to be construed in an inclusive sense, that is, as including the specified integers or steps or integers or steps of a group but not excluding other integers or steps or integers or steps of a group.

[0113] Any reference in this specification to any prior publication (or information derived therefrom) or to any known matter is not, and should not be taken as, an admission or acknowledgment or any form of suggestion that that prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0114] Specific embodiments of the invention are for illustrative purposes only and are not intended to limit the generality of the foregoing specification. This will be described with reference to the following examples.

Examples

[0115] Examples Materials and Methods Plasmids and Sequences The lentiviral vector and related packaging plasmids were purchased from Vectorbuilder. Chimeric antigen receptors having CD28 and CD137 co-stimulatory sequences were synthesized and cloned into the lentiviral vector by Vectorbuilder. The human CD8A, CD8B, CD80, CD86, and LCK genes were cloned from the in-house human cDNA library. The scFv constructs of TCR-like antibodies were generated in the laboratory of Paul A. Macary (NUS), with the peptide LMP2A 426-434 (from EBV) and HLA-A * 02:01-specific TCR and the peptide E183-91 (from HBV) with A * The 02:01-specific TCRs were kind gifts from Hans Stauss (University College London) and Antonio Bertoletti (Duke-NUS Medical School), respectively. The single-chain trimer GAG-HLA-A2 was a gift from Keith Gould (Imperial College London). Peptide mutagenesis: from GAG (SLYNTVATL) to LMP2A 426-434 (CLGGLLTMV) (L2), LMP1 125-133 (YLLEMLWRL) (L1), EBNA1 562-570 (FMVFLQTHI) (El), E183-91 (FLLTRILTI) (E183), or deletion of the single-chain trimer GAG-HLA-A2; the CD28 Y170F, P187, 190A, and intracellular domain deletion mutations were all performed using the Q5 mutagenesis kit (New England Biolabs). All molecular cloning work was performed using the In-Fusion HD cloning kit (Clontech), and to generate artificial antigen-presenting CHO cells, the single-chain trimer MHC construct was cloned into pcDNA3-Clover (Addgene plasmid #40259).

[0116]

Table 1

[0117]

Table 2

[0118]

Table 3

[0119]

Table 4

[0120] Cell Lines and Cell Culture The human T cell Jurkat cell line, endogenous TCR, and co-receptor-deficient Jurkat 76 were a kind gift from Dr. Heemskerk MH. Wild-type Jurkat E6-1 (TIB-152), LCK-deficient Jurkat cam1.6 (CRL-2063), and Daudi cells (CCL-213) were obtained from the American Type Culture Collection. Cells were maintained in RPMI-1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine (Gibco), and MEM non-essential amino acids (Gibco) in a humidified 5% CO2 incubator at 37°C. Human embryonic kidney epithelial cells (HEK293) were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine (Gibco), and MEM non-essential amino acids (Gibco). The tetracycline-regulated expression (T-REx) CHO cell line was purchased from Invitrogen and used for the generation of artificial antigen-presenting cell lines. CHO cells were cultured in Ham’s F-12 (Gibco) medium containing 10% fetal bovine serum (Hyclone), 2 mM L-glutamine (Gibco), and MEM non-essential amino acids (Gibco). Transfection of the single-chain trimeric MHC construct into CHO was performed using the polyethyleneimine (PEI) method. After transfection, cells were selected by the addition of a drug (1.0 mg / ml G418 sulfate from Hyclone). Subsequently, single-cell sorting was applied to select appropriate HLA-expressing clones. pMHC complex expression was regularly checked by flow cytometry.

[0121] Antibodies and Chemicals In this study, the following antibodies were used: Myc-tag mouse mAb Alexa Fluor 647 (9B11), anti-pSrc family (pY416), anti-pPLCγ1, anti-p44 / 42 Erk1 / 2, anti-Erk1 / 2 (all from Cell Signaling Technology); rabbit anti-human FYN (FYN-59), anti-human CD28 Alexa 488 (CD28.2), anti-human CD80 PE (2D10), anti-human CD19 FITC, anti-human CD86 Brilliant Violet 421 (BU63), anti-human CD3 APC, anti-human CD279 (PD-1) PE (all from Biolegend); anti-pCD3ζ (pY142), mouse anti-human c-CBL, anti-PLCγ1 (all from Becton Dickinson); anti-human HLA-A2 APC (BB7.2), anti-human CD8A APC, anti-human CD8B PE-Cy7 (all from eBioscience); mouse anti-human LCK (3A5, Santa Cruz Biotechnology); goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor 647 (Thermo Fisher Scientific); specific HLA / A2-L2, HLA / A2-L1. HLA / A2-E1 TCR-like antibodies were generated as described (Sim et al., 2013). For the chemicals used in this study: SRC family kinase (SFK) inhibitor PP2 was purchased from Sigma-Aldrich; specific LCK or FYN inhibitors (A770041 or SU6656, respectively) were obtained from MedChemExpress or SelleckChem, respectively; calcium dye Indo-1, AM was obtained from Thermo Fisher Scientific.

[0122] Production and transduction of lentivirus A total of 6.5×10 per well 5Individual HEK293 cells were seeded onto six-well plates the day before transfection and incubated at 5% CO2 and 37°C. Subsequently, the cells were transfected with the packaging plasmid and lentiviral vector using polyethyleneimine (PEI), and the medium was replaced 12 hours later. The viral supernatant was harvested twice over the next two days. The harvested viral supernatant was titrated, filtered through a 0.45 μm membrane filter (Millipore), and concentrated 100-fold using an ultracentrifuge tube (Millipore). For lentiviral transduction, 1×10 6 Jurkat cells per ml were used, with polybrene and HEPES added at 8 - 10 μg / ml and 10 mM respectively, followed by spinoculation at 2,500 rpm for 2 hours. For CHO cell transduction, the viral solution was added directly to the cells without spinoculation. After 24 hours, the cells and the viral solution were separated, and the cells were cultured in maintenance medium. After an additional 48-hour culture period, flow cytometry analysis was performed to check the expression of the construct.

[0123] Electroporation Electroporation was performed according to the instructions of the Amaxa cell line nucleofector kit (VCA-1003). Briefly, 10 6 cells of each sample were prepared and spun down at 90×g for 10 minutes. The cells were resuspended in 100 μl of nucleofector solution and combined with 2 μg of DNA. Electroporation program X-05 (high efficiency) was applied to the Nucleofector® 2b Device. The expression of the construct was detected by flow cytometer 18 hours later.

[0124] Imaging For total internal reflection fluorescence microscopy (TIRFM), a lipid bilayer containing specific pMHC and other anchor proteins was prepared as described above. Briefly, 0.2 mol% liposomes were prepared, evaporated at 37 °C under N2, and sonicated to obtain a 4 mM lipid stock. A glass 8-well chamber LabTekII chamber slide (Fisher Scientific) was washed with 6 M NaOH for 2 hours and rinsed with ddH2O before adding lipids. The lipids were diluted 10-fold with PBS, added to a clean chamber slide, and incubated for 30 minutes. Excess liposomes were washed away with 12 ml of PBS. The bilayer was blocked with 2 mg / ml BSA for 30 minutes. 5 μg / ml streptavidin was added and incubated for 30 minutes. After washing away the excess streptavidin, biotinylated HLA / A2-L2 monomer, recombinant human ICAM1 protein, hIgG1-Fc.His tag (Thermo Fisher Scientific) were added to the bilayer for 30 minutes. After washing, the bilayer was ready to use. 10 5 CAR-Jurkat or TCR-Jurkat cells containing CD8α-mCherry at 10 cells per 100 μl were added to one well of the chamber at 37 °C for 10 minutes. Then, 100 μl of 8% paraformaldehyde was added to fix the cells and stop the stimulation. TIRF microscopy was performed on an Olympus IX83 inverted microscope equipped with a 4-laser TIRF module later. Images were obtained using a 40x / 1.49 NA oil immersion lens. Fluorescence excited within the 100-nm evanescent field was recorded with a Hamamatsu ORCA Flash 4.0 camera. For regular fluorescence imaging, 10 5 CAR-Jurkat or TCR-Jurkat cells containing CD8α-mCherry at 10 cells per 100 μl were mixed with 10 5 cell-specific CHO-APC in 100 μl of RPMI medium in one well of the chamber for 10 minutes. Subsequently, 100 μl of 8% paraformaldehyde was added. The mobilization of CD8 was detected later on an Olympus IX83 inverted microscope with a normal module. Images were obtained using a 20x - 40x / 1.49 NA oil immersion lens.

[0125] Calcium efflux assay Cell samples were diluted in phosphate-buffered saline (PBS) at 10 × 10 per ml. 6 Cells were suspended at a cell density of 1000x and loaded with 2 μM Indo-1 AM for 30 min in a 37 °C incubator, followed by washing twice with culture RPMI. Cells were pre-warmed to 37 °C for 10 min before analysis and maintained at 37 °C in culture RPMI during event collection. For cell stimulation, HLA / A2-L2 monomers were pre-refolded, biotinylated, and cross-linked with streptavidin Alexa 647 (Thermo Fisher) to form antigen tetramers. Cells were then stimulated with the tetramers. The mean fluorescence ratio of Indo-1 high (BUV395) / Indo-1 low (DAPI) was calculated using FlowJo by a kinetics program.

[0126] T cell stimulation assay Artificial antigen-presenting CHO cells (APC-CHO) were added at 2–3 × 10 per well. 4 Cells were seeded one day prior in 96-well plates at 10 cells / ml. For APC-CHO requiring peptide pulsing, peptide was added at 4 μM for 3 hours and then washed out before addition of CAR-T or TCR-T cells. Each CAR-T or TCR T cell sample was counted and cultured in RPMI medium at 10 cells / ml. 6 Cells were suspended at a concentration of 100 μl per well. Then, 200 μl of cell suspension was added per well to the APC-CHO pre-seeded plates. For inhibitor experiments, inhibitors were added to the cell mixture accordingly. All experiments were performed in technical triplicates. Cells were incubated at 37° C., 5% CO2 for 18 hours. After incubation, supernatants were collected for human IL-2 ELISA assay, which was performed according to the manufacturer's protocol (Invitrogen). T cell pellets were either resuspended for surface staining or used for another round of stimulation by repeating the above method.

[0127] Western blotting Total 10 6Individual cell samples were lysed with NP-40 lysis buffer. Cell debris was pelleted, the supernatant was collected, and heated with reducing protein loading buffer (Thermo Fisher Scientific). To detect phosphorylation after stimulation, 10 5 individual APC-CHO cells were seeded into 24-well plates one day before stimulation. 10 6 individual CAR-T or TCR-T cells were added to each well and incubated at 37 °C, 5% CO2 for the specified period. The stimulated CAR-T or TCR-T cells were collected and prepared as described above. Samples were loaded onto a 4–12% Bis-Tris gradient gel (NuPAGE, Invitrogen) and transferred to a PVDF membrane (Immobilon-FL Transfer Membrane, Millipore). The membrane was then blocked with blocking buffer (Odyssey, LI-COR) for 1 hour at room temperature. Subsequently, the membrane was probed with various primary antibodies. The secondary antibodies used were IRDye 800CW goat anti-mouse IgG2b (Cat# 926-32352, LI-COR) and IRDye 680LT goat anti-rabbit (Cat#926-68021). Blotting was quantified using a LI-COR Odyssey infrared imaging system.

[0128] CRISPR-Cas9 gene editing The Cas9 plasmid was obtained from Addgene (#52961). The gRNA sequences for FYN and LCK were searched at http: / / chopchop.cbu.uib.no / and are shown in Table 2. The Cas9 sequence was linked to mTagBFP by a P2A cleavage linker and then cloned into a lentiviral vector together with the gRNA sequences. After transduction into Jurkat cells, an mTagBFP fluorescent marker was used after single cell sorting. Intracellular staining and Western blotting were performed for screening of clones.

[0129]

Table 5

[0130] Flow Cytometry and Cell Sorting Flow cytometry experiments were performed using a BD LSR Fortessa X-20 (Becton Dickinson). Cell sorting was performed by the Flow Cytometry Laboratory, Immunology Programme, National University of Singapore, using either a Mo-flo XDP (Beckman Coulter, Inc.) or a SY3200 (Sony Biotechnology Inc.). Data analysis was performed using FlowJo.

[0131] Statistical Information and Data Analysis Two-sided Student's t-tests or two-way ANOVA analyses were performed for column data or curve data, respectively, using GraphPad Prism 7. The data meet the preconditions for the tests. The variances are similar among the groups being compared.

[0132] Example 1 Artificial antigen-presenting CHO cells provide antigens for CAR and TCR T cell stimulation To compare the molecular recognition between CAR and TCR in detail, Jurkat T cells expressing CAR and TCR with the same peptide-MHC specificity were generated. The CAR construct was generated based on a previous report in which the CD28 co-stimulatory domain was described (Figure 1A). The scFv on the CAR was constructed from a TCR-like antibody that recognizes a peptide epitope of the latent membrane protein 2A (LMP2A) protein from Epstein-Barr virus (EBV) presented by HLA-A2. The term CAR can refer to this second-generation CAR that uses the CD28 transmembrane and cytoplasmic domains. Then, lentivirus was applied to deliver the CAR into Jurkat 76T cells that lacked endogenous TCRα and β chains but contained a full set of CD3 subunits. Lentiviral transduction was efficient with both the CAR or a TCR specific for the same peptide-MHC complex as the CAR (Figure 1B). CAR- or TCR-expressing Jurkat cells were sorted to generate stable CAR- or TCR-Jurkat cells. There are various human Jurkat T cell lines available for T cell signaling studies. Here, three Jurkat-derived cell lines, Jurkat 76, JE6.1, and Jcam1.6 were used. Jurkat 76 was used for most experiments and is referred to as Jurkat unless otherwise stated. Only Jurkat 76 did not express CD3 on the surface, while JE6.1 and Jcam1.6 expressed different amounts of CD3 (Figure 1C). None of these three cell lines expressed CD8 or CD4 (Figure 11A). In addition, an artificial antigen presentation system based on the expression of single-chain form of human MHC class I in heterologous CHO cells was also engineered. This antigen-presenting cell (APC) system was constructed in two types: a monopeptide system in which the selected peptide covalently binds to β2-microglobulin and the heavy chain (Figure 1D), and a multipeptide system in which the peptide groove of HLA-A2 is open and multiple peptides can be pulsed into the cells to bind to MHC-I molecules (Figure 1E). Both systems efficiently presented peptides, as observed by the fact that specific peptide presentation was significantly detected by specific TCR-like antibodies, in contrast to non-related peptide presentation, respectively (Figure 1D, Figure 1E).Nevertheless, as seen in FIGS. 1D and 1E, CAR-T cells responded differently to stimulation by these systems. LMP2A (L2) peptide-specific CAR-Ts specifically respond to CHO-L2 but not to unrelated CHO-GAG. However, TCR-like CAR-T cells showed some non-specific responses to non-pulsed CHO cells expressing HLA-A2 (FIG. 1E). This non-specific activation was also seen with TCR-like CARs having other specificities such as the EBNA1 peptide-targeted E1-CAR or the LMP1 peptide-targeted L1-CAR. As detected by anti-HLA-A2 (BB7.2) at different peptide concentrations, the HLA-A2 construct is expressed without intentionally pulsing the peptide onto CHO-APCs (FIG. 11C). Thus, various unknown peptides from CHO cells are presented to the multi-peptide system. Some peptide sequences are similar to the specific peptide and may trigger non-specific triggers.

[0133] Activation of TCR-like CARs is not enhanced by CD8 and can transmit T cell signaling without LCK Since this TCR-like CAR has the same specificity for peptide MHC as TCR, it is very interesting to clarify the contribution of the CD8 co-receptor to T cell activation. It was first shown that the CD8 co-receptor can be mobilized by CAR-T cells as well as TCR-T cells using total internal reflection fluorescence microscopy (TIRFM) used to detect events at the contact surface (Figure 2A). CD8α was labeled as a chimera with the fluorescent protein mCherry. Both CAR-T and TCR-T expressed CD8α-mCherry on the surface (Figure 7A). In the case of TIRFM, a supported lipid bilayer was prepared on a glass plate, and the integrin ligand ICAM-1 was added to immobilize CAR-T or TCR-T on the detection surface. CAR-T or TCR-T cells expressing CD8α-mCherry were added to bilayers containing or not containing specific pMHC for 10 minutes, and then the mean fluorescence intensity (MFI) significantly increased in both groups to which pMHC was added. Clustering of CD8α-mCherry within the contact surface between CAR-T and the bilayer was evident. The MFI ratio between the inside and outside of the immunological synapse was calculated using a conventional wide-field fluorescence microscope (Figure 7B). No significant difference was detected between CAR-T and TCR-T using CD8α-mCherry, and both MFI ratios exceeded the defined cut-off ratio of 1.5 for immunological synapse formation by the inventors, demonstrating that CD8α-mCherry was mobilized in both CAR-T and TCR-T. Next, on the premise that CD8 can be mobilized, it was required to clarify whether CD8 functionally contributes to CAR-T. After co-transducing both CAR-Jurkat cells and TCR-Jurkat cells with CD8α and CD8β, it was shown that the expression levels of CAR or TCR were similar (Figures 7C, 7D), and as expected, it was found that the reactivity of TCR-Jurkat carrying the CD8αβ co-receptor significantly increased. TCR-T with CD8 showed faster and stronger calcium efflux than TCR-T without CD8 (Figure 2B). IL-2 produced by TCR-T with the CD8 co-receptor was almost three times higher than that without the CD8 co-receptor (Figure 2C).However, the reactivity of CAR-Jurkat cells bearing CD8 was not enhanced compared to cells lacking CD8 (Figures 2B and 2C). The calcium efflux of CAR-T cells with CD8 and those without CD8 was equivalent, and no significant increase in IL-2 production was detected in CAR-T cells with CD8. CD8 is thought to be important in enhancing TCR signaling by introducing LCK to the immune synapse. Subsequently, a hypothesis was put forward that LCK may not be so important for CAR signaling, at least with regard to CD8-bound LCK. Surprisingly, CAR-T cells were able to activate TCR signaling even in the absence of LCK, as observed when using the LCK-deficient Jurkat cell line Jcam1.6 (Figures 2D and 2E). CAR-Jcam cells were able to produce IL-2 and efflux calcium normally, while TCR-Jcam cells were unable to produce IL-2 upon antigen stimulation.

[0134] Example 2 LCK-independent CAR signaling requires the CD28 costimulatory domain To identify which domain triggers non-canonical T cell signaling of CAR in the absence of LCK, systematic domain substitutions were performed on second-generation CARs carrying CD28. First, it was tested whether LCK-independent signaling is due to the antigen specificity of the extracellular domain. The TCR-like scFv was replaced with CD19-scFv, and the CD19-expressing Daudi cell line was used as the target cell (Figure 8A). CD19-specific CAR-T cells were activated by CD19-expressing Daudi cells in the presence or absence of LCK, suggesting that LCK-independent CAR signaling is independent of the antigen specificity of the extracellular CAR domain (Figure 3A). To determine the importance of the CD3ζ domain, deletion of the CD3ζ intracellular domain resulted in no detectable production of IL-2, demonstrating the indispensability of the CD3ζ ITAM in CAR-T signaling (Figure 3B). Then, an attempt was made to determine the role of the co-stimulatory signaling domain in mediating LCK-independent CAR triggering by 1) deleting the co-stimulatory CD28 domain to generate a first-generation CAR, 2) replacing CD28 with the CD137 (4-1BB) intracellular domain, or adding the CD137 domain to generate a third-generation CAR (Figure 3C). First-generation CAR-1 without a co-stimulatory domain behaved like a TCR, and signaling stopped when CAR-1 was introduced into LCK-deficient Jcam1.6 cells. CD137-CAR was constructed. The results showed that, in the case of the second-generation CAR, signaling occurred LCK-independently only with the design containing the CD28 co-stimulatory domain. Nevertheless, in the third-generation CAR containing both the CD28 domain and the CD137 domain, activation of CAR-T in response to the antigen was observed under LCK-deficient conditions, but in the absence of LCK, it was not as strong as that of the CAR containing only the CD28 intracellular domain (Figure 3C). The involvement of the CD28 signaling pathway was demonstrated by mutation of the functional binding motif of the CD28 intracellular domain. The PI3K binding motif and the proline-rich region were tested because they have been reported to be crucial for CD28 signaling (Figure 3D).Three mutations were introduced into the CAR construct; deletion of the intracellular domain; mutation of the PI3K-binding motif YMNM to FMNM; replacement of the prolines in the proline-rich region PYAP with alanines. Then, all three of these mutations were transduced into the Jcam1.6 cell line. Production of IL-2 was completely suppressed without the CD28 intracellular domain. A decrease in IL-2 production was observed with both the FMNM and AYAA mutants, and the AYAA mutant decreased activation more than the FMNM mutant. To further test the effect of CD28 signaling on LCK-independent signaling, CD80 and CD86 were co-transduced into CHO-L2 to activate CD28 signaling. Expression of endogenous CD28 was confirmed in both Jurkat and Jcam1.6 (Figure 8B, Figure 8C). Surprisingly, CAR-1-Jcam and TCR-Jcam cells showed a recovery of IL-2 production when stimulated with CD80 and CD86-expressing CHO-L2 cells. These results indicate that LCK-independent CAR signaling requires a CD28 co-stimulatory signal that is at least partially mediated via the YMNM and PYAP motifs from either the CD28 intracellular domain present in second-generation CARs or the endogenous CD28 molecule when using first-generation CARs or TCRs.

[0135] CD28-CAR transmits downstream signaling depending on FYN Next, the inventors sought to identify the kinases involved in CAR phosphorylation during signaling in the absence of LCK. The production of IL-2 in CAR-Jurkat or CAR-Jcam was completely suppressed after the addition of the SRC family kinase (SFK) inhibitor PP2 (Figures 4A, 9A), indicating that CAR signaling is highly dependent on SFK. Considering that LCK and FYN are the most prominent and relevant SFKs in T cells, specific inhibitors A770041 and SU6656 targeting LCK and FYN respectively were used to test the relative roles of these two SFKs in CAR and TCR signaling. As shown in Figure 4B, TCR-Jurkat was more sensitive to the LCK inhibitor than CAR-Jurkat. Conversely, CAR-Jurkat was more sensitive to the FYN inhibitor than TCR-Jurkat (Figure 4B). The IC50 of the inhibitor for CAR-Jurkat or TCR-Jurkat further demonstrated completely different sensitivities; the IC50 of the LCK inhibitor was 6.6 nM in TCR-Jurkat but 47 nM in CAR-Jurkat. In the case of the FYN inhibitor, the IC50 in CAR-Jurkat (3765 nM) was lower than that for TCR-Jurkat (5583 nM) (Figure 9B). Different from the LCK-dependent TCR, this tendency of CAR to preferentially use FYN suggested that FYN could be the kinase activating downstream signaling from CAR. To further test the involvement of FYN activation in LCK-independent signaling, Western blotting was performed. The results of FYN phosphorylation of CAR-Jcam at different time points showed that the activation site Y420 of FYN was phosphorylated after specific APC binding of CAR-Jcam but not TCR-Jcam (Figure 4C), and the FYN Y420 phosphorylation increased nearly two-fold after 30 minutes. The phosphorylation intensity was calculated by the intensity of pY420 relative to the intensity of total FYN. The activation of important downstream signaling molecules was also examined in LCK-deficient CAR-Jcam (Figure 4D). The phosphorylation of PLCγ1, Erk, and CD3ζ was detected after the activation of CAR-Jcam, but only Erk was phosphorylated in CAR1-Jcam.The differences in downstream signal activation between CAR-Jcam and CAR1-Jcam were also reflected in the calcium efflux experiments (Figure 9C). CAR-Jcam cells, but not CAR1-Jcam cells, showed calcium influx in response to the cognate antigen. Different kinetic patterns after activation were also seen between CAR-Jcam and TCR-Jurkat. Phosphorylation of PLCγ1, Erk, and CD3ζ was more stable after CAR signaling than after TCR phosphorylation. TCR activation was a relatively short pulse, as phosphorylation of each molecule increased at 30 minutes and then waned.

[0136] To more appropriately verify the roles of LCK and FYN in CAR and TCR signaling, the CRISPR-Cas9 gene editing system was introduced to knock out LCK or FYN. After gRNA screening (Table 2) and single clone sorting (Figure 9D), LCK knockout clone 20 and FYN knockout clone 8 were selected as the LCK or FYN knockout systems for further experiments (Figure 4E). Before the experiments, CAR- or TCR-transduced LCK or FYN knockout T cells were sorted so that the expression of TCR and CAR was equivalent. In LCK knockout Jurkat cells, TCR was unable to activate signaling to produce IL-2, but CAR still functioned, consistent with previous results obtained using the Lck-deficient Jcam1.6 cell line (Figure 4F). However, in FYN knockout Jurkat cells, TCR produced a slightly higher amount of IL-2 than wild-type TCR-Jurkat. However, IL-2 production in CAR-Jurkat FYN KO cells was dramatically reduced compared to wild-type CAR-Jurkat (Figure 4F). The expression of CAR or TCR was equivalent in FYN KO Jurkat cells, and the absence of FYN was confirmed in CAR- or TCR-Jurkat FYN KO by Western blotting (Figure 9E, Figure 9F).

[0137] Example 3 The LCK-deficient CAR-T resets the activation threshold and selectively enables only CAR triggering in T cells expressing both CAR and TCR Next, based on these findings, we sought to investigate the potential functional consequences of the LCK-independent trigger mechanism and whether LCK-deficient CAR-T cells could actually be applied. We first tested the effect of the presence or absence of LCK on the CAR activation threshold. Some of the non-specificities of the multi-peptide presentation system (Figure 1E) have already been noted. This non-specific activation of the TCR-like CAR has been reported previously, and the specificity can decrease when the affinity of the TCR-like antibody exceeds a certain threshold. After sorting TCR-like CAR-T cells for different amounts of CAR expression, it was found that the increase in the amount of IL-2 secreted from low-expression to high-expression CARs against non-pulsed HLA-A2 or specifically peptide-pulsed HLA-A2 was completely different (Figure 5A). This difference indicates that the TCR-like CAR may have different affinities for non-specific and specific binding, and the affinity of specific binding is higher than that of non-specific binding. Considering that the activating kinases are different in each system, the hypothesis was put forward that the activation threshold is likely to be different between LCK-deficient CAR-T and LCK-sufficient CAR-T. Therefore, the responses to specific and non-specific antigens can be distinguished in LCK-deficient CAR-T. As seen in the comparison of CAR-Jcam, CAR-Jurkat 76, and CAR-JE6-1, the production of IL-2 by CAR-Jcam was as low as that of the negative control, showing the non-related peptide GAG, while CAR-JE6-1 showed the same non-specific activation as CAR-Jurkat 76 (Figure 5B). In particular, JE6-1 expresses the endogenous TCR-like Jcam1.6 (Figure 1C), thus excluding the contribution of the endogenous TCR to the non-specific activation by the CAR. To further verify the role of LCK in mediating the activation of TCR-like CAR-T cells against low-affinity non-specific antigens, LCK was transduced into Jcam1.6 cells to generate LCK-positive CAR-Jcam cells. Again, non-pulsed HLA-A2 APCs induced strong IL-2 production in LCK-positive CAR-Jcam cells rather than LCK-negative CAR-Jcam cells (Figure 5C). It was also observed that the production of IL-2 against antigenic CHO-L2 was lower in LCK-positive CAR-Jcam than in LCK-negative CAR-Jcam cells (Figure 10A).Furthermore, LCK deficiency was predicted to enable selective triggering of CAR rather than TCR in T cells expressing both CAR and TCR. To test this hypothesis, a L2 peptide-specific CAR and a TCR with specificity for the hepatitis B virus (HBV)-derived E183-91 (FLLTRILTI) epitope (thus called E183-TCR) were co-transduced into Jurkat cells or LCK knockout Jurkat cells (Figure 10B). Jurkat-TCR+CAR was induced to produce IL-2 by CHO-E183 or CHO-L2, indicating that the activation of CAR and TCR was not impaired in the dual CAR+TCR system. However, CHO-L2 rather than CHO-E183 induced IL-2 production in LCK-deficient Jurkat T cells co-expressing CAR and TCR, indicating that LCK deficiency enables the reconstitution of the TCR signaling pathway for selective triggering of CAR rather than TCR.

[0138] LCK-deficient CAR-T cells express low levels of PD-1 and show reduced down-regulation of CAR after stimulation Many changes in gene expression occur after TCR signaling. One important change is the upregulation of the co-inhibitory molecule PD-1, an important marker of exhaustion in cancer immunotherapy (Figure 6A). PD-1 was not expressed on the cell surface before stimulation in either type of CAR-T cell. Nevertheless, after stimulation of different CAR-T cells, the difference was obvious. As seen in Figure 6A, CAR-Jcam transduced with LCK upregulated PD-1 more strongly than LCK-deficient CAR-Jcam. In addition, the expression of PD-1 was lower in CAR-Jurkat LCK KO than in CAR-Jurkat cells. However, TCR-Jurkat expressed the largest amount of PD-1 among these groups. These differences in PD-1 upregulation were even more prominent after several stimulations (Figure 6B). CAR-Jcam+LCK upregulated PD-1 almost three times higher than CAR-Jcam after three stimulations. The same upregulation trend was observed in the CAR-Jurkat cell samples. The expression of PD-1 in CAR-Jurkat LCK KO was the lowest among the groups, and TCR-Jurkat upregulated PD-1 the most strongly. Furthermore, as shown in previous studies, CAR or TCR was downregulated after antigen stimulation in the presence of LCK. CAR showed a decrease in antigen-dependent receptor downregulation in LCK-deficient cells such as Jcam or Jurkat LCK KO (Figure 6A, Figure 6D). Compared with the amount of CAR or TCR before stimulation, both were downregulated in CAR-Jurkat and TCR-Jurkat, and TCR was dramatically downregulated. However, the amount of CAR was more stable after antigen stimulation as long as LCK was absent, as seen in CAR-Jcam or CAR-Jurkat LCK KO (Figure 12). This decrease in PD-1 upregulation and CAR downregulation suggested that LCK-deficient CAR-T may be resistant to inhibitory signaling via PD-1 and may be less "exhausted" compared to LCK-sufficient CAR-T.

[0139] Example 3 Method CD8 + Activation and culture of T cells A blood sample was collected from a volunteer, and naive CD8 + T cells were isolated using RosetteSep™ Human CD8 + T Cell Enrichment Cocktail (Stemcell) and Ficoll (GE Healthcare Life Sciences) gradient centrifugation. Subsequently, naive CD8 + T cells were stimulated with anti-CD3 / CD28 beads (ThermoFisher) in Biotarget medium (Biological Industry) supplemented with 4% human platelet lysate (Ultra-GRO™-Advanced, AventaCell) containing 100 U / ml IL-2 (R&D System) to produce mature cytotoxic CD8 + T cells. After 48 hours of activation, mature CD8 + T cells were cultured in a medium containing 100 U / ml IL-2, 10 ng / ml IL-15, and 10 ng / ml IL-7 (R&D System). The medium was changed every two days, and the cells were replated at 10 6 cells per ml. T cells were restimulated with feeder cells that were peripheral blood mononuclear cells (PBMCs) from the donor. PBMCs were freshly isolated from the blood by gradient centrifugation and irradiated at 30 Gy. PBMCs and T cells were resuspended in the same medium at a ratio of 2:1. IL-2 at a final concentration of 100 U / ml, IL-7 at a final concentration of 10 ng / ml, and IL-15 at a final concentration of 10 ng / ml were added to the culture. Lectin from Phaseolus vulgaris (Sigma-Aldrich) was added to the culture at a concentration of 1.5 μg / ml. Blood was collected from healthy volunteers under a protocol approved by the NUS IRB. Informed consent was obtained from all donors.

[0140] CRISPR-Cas9 gene editing Homologous recombination repair (HDR) targeting LCK has already been described. LCK gRNA2, GCCGGGAAAAGTGATTCGAG (SEQ ID NO: 10), was selected and chemically modified. Briefly, the complete RNA sequence was [Chem.] . The asterisk ( * ) represents 2'-O-methyl 3'-phosphorothioate. Cas9-NLS protein (New England Biology) and LCK gRNA2 were incubated at a 1:2 molecular ratio at 37 °C for 30 minutes to form a ribonucleoprotein (RNP) complex. The double-stranded DNA donor was designed such that the lkb homologous arms flanked the CAR construct on both sides. 120 pmol of RNP and 2 μg of dsDNA were electroporated into 1 million activated CD8 + T cells by an Amaxa 4D electroporation system (Lonza) via program EH115.

[0141] Cytotoxicity assay 40,000 Daudi or Raji cells per well were seeded in a U-bottom 96-well plate, followed by the addition of 4,000 - 400,000 CAR-T or LCK locus CAR-T cells at an effector to target (E:T) ratio of 0.1:1 to 10:1 per well. The cell mixture was incubated at 37 °C, 5% CO2 for 18 hours. Then, the supernatant was collected and the release of LDH from dead cells was detected using the CytoTox 96® non-radioactive cytotoxicity assay (Promega). The cell pellet was resuspended and stained with antibody conjugates to detect the expression of CD62L, PD-1, TIM-3, and LAG-3 after contact with the target cells.

[0142] Mouse model and in vivo analysis 6 - 8-week-old NOD / MrkBomT ac-Prkdc scidFemale mice (Taconic) were used under a protocol approved by the NUS Institutional Animal Care and Use Committee. CAR-T cells were restimulated 3 days prior and expanded by feeder cells. Raji cells were administered by tail vein injection at 4 million cells per mouse. Raji cells generated a very uniform tumor burden and no mice were excluded prior to treatment. 5×10 6 、2.5×10 6 、or 1×10 6 expanded CAR-T cells were administered via the tail vein on day 4 after Raji cell administration. Mice were continuously monitored and euthanized when paralysis was observed. For CAR-T cell phenotypic examination after in vivo administration, bone marrow from each mouse was extracted on days 11 and 18. Memory and exhaustion surface markers were detected and analyzed by FACS.

[0143] Flow Cytometry and Cell Sorting Flow cytometry experiments were performed on a BD LSR Fortessa X-20 (Becton Dickinson). Cell sorting was performed by either Mo-Flo XDP (Beckman Coulter, Inc.) or SY3200 (Sony Biotechnology Inc.) at the Flow Cytometry Laboratory, Immunology Programme, National University of Singapore. Data analysis was performed using FlowJo.

[0144] Statistics and Data Analysis GraphPad Prism 7 was used to perform two-sided Student's t-test or two-way ANOVA analysis on column data or curve data, respectively. The data meet the test prerequisites. The variances are similar between the groups being compared.

[0145] Results The inventors then +An attempt was made to summarize the findings from Jurkat cells in T cells. For this purpose, the CRISPR / Cas9 system was utilized to perform homologous recombination repair (HDR), and the CD28-CAR construct was introduced into the LCK locus and inserted at the position of the 196th amino acid of LCK (Figure 13A). After HDR, CAR + CD8 + A distinct population of T cells was detected (Figure 14A). Next, CAR + CD8 + T cells were sorted and the expression of LCK protein was measured. As shown in Figure 14B, the expression of LCK was dramatically decreased compared to conventional CAR-T cells. Since a P2A cleavage sequence was added to the N-terminus of CD28-CAR, cleaved LCK was also observed (Figure 14B). Considering that amino acid 196 is located at the end of the SH2 domain of LCK and not part of the catalytic domain, this cleaved LCK is a dysfunctional mutant. Genotyping at the insertion site also confirmed that the CAR construct was inserted into the LCK locus (Figure 14C). Two CD19-expressing cell lines, Daudi and Raji, were used to test the cytotoxicity of T cells. Both conventional CAR-T and LCK locus CAR-T had comparable cytotoxicity, but also showed lower cytotoxicity against Raji cells than Daudi cells. This lower cytotoxicity against Raji cells could be caused by the resistance mechanism of Raji cells to T cell killing (Figure 13B). Control CD8 +T cells also exhibited some cytotoxicity against these cancer cells at a high E:T ratio. Since LCK locus CAR-T cells could only kill CD19-expressing Daudi cells but not CD19-negative Jurkat cells, the specificity of LCK locus CAR-T cells was retained as shown in Figure 14D. The differences between conventional CAR-T and LCK locus CAR-T were tested by immunotyping of exhaustion molecules, PD-1, TIM-3, LAG-3, and the memory marker CD62L. At rest, conventional CAR-T cells had higher expression of TIM-3 (25%) and lower expression of CD62L (49%) than LCK locus CAR-T cells. The expression of LCK locus CAR-T cells was 14% for TIM-3 and 85% for CD62L (Figure 13C). After contact with target cells at different E:T ratios, the expression of exhaustion molecules and memory molecules changed (Figure 14E). At a low E:T ratio, higher expression of exhaustion molecules and lower expression of CD62L were observed. When using a radar chart to summarize the expression of surface markers and compare between different groups, the differences between these two CAR-T cells became more obvious (Figure 13D). Conventional CAR-T cells tended to express more exhaustion molecules and reduce the expression of CD62L as the E:T decreased. However, LCK locus CAR-T cells were more persistent and did not tend to upregulate exhaustion markers as much as conventional CAR-T cells, which had high expression of PD-1, TIM-3, and LAG-3 but low expression of CD62L.

[0146] A more sustained phenotype of LCK locus CAR-T cells, i.e., more memory phenotypes and fewer exhausted phenotypes, suggested that they might exhibit superior in vivo anti-cancer efficacy compared to conventional CAR-T cells. Then, when conventional CAR-T cells and LCK locus CAR-T cells were exposed to tackle Raji cells in a mouse model, Raji was more resistant to CAR-T cell toxicity than Daudi cells in vitro (Figure 13B). This also suggested that they might be more resistant in vivo. Using the NOD / SCID immunodeficient mouse strain, CAR-T cells were intravenously administered at different doses, and Raji cells were administered 4 days later (Figure 13E). Consistent with the inventors' expectations, conventional CAR-T cells did not show a significant improvement in in vivo efficacy compared to CD8 + T cells. However, LCK locus CAR-T cells showed significantly enhanced in vivo performance at all three doses of 1 million to 5 million cells (Figure 13F). At the end of the experiment, one mouse in the group treated with 5 million LCK locus CAR-T cells was still alive. No cancer cells were found in multiple organs extracted from this mouse (Figure 14F). Raji cells and T cells were further analyzed from the mouse bone marrow on days 11 and 18 after Raji cell injection to confirm their numbers, as well as the memory and exhaustion states of T cells. There was no significant difference in the number of T cells and Raji cells from conventional CAR-T cells on days 11 and 18 and the number of T cells and Raji cells from the LCK locus CAR-T cell group (Figure 14G). However, as shown in Figure 13G, LCK locus CAR-T cells showed significantly higher expression of both the memory marker CD45RO on days 11 and 18 compared to conventional CAR-T cells. In addition, conventional CAR-T cells upregulated the exhaustion markers PD-1, TIM-3, and LAG3 more than LCK locus CAR-T cells. This was particularly prominent in cells co-expressing all three of these exhaustion markers (Figure 13H). Compared to conventional CAR-T cells, LCK locus CAR-T cells had more memory phenotypes and fewer exhausted phenotypes, which supported their enhanced in vivo efficacy.

Claims

**Claim 1** An immune cell expressing a chimeric antigen receptor (CAR), wherein the CAR comprises an intracellular signaling domain or fragment that functions in the absence of lymphocyte-specific protein tyrosine kinase (LCK), the immune cell is modified such that the expression and / or function of LCK is reduced or absent. **Claim 2** The immune cell according to claim 1, wherein the intracellular signaling domain comprises a signaling domain or fragment of a CD28 protein that functions in the absence of LCK. **Claim 3** The immune cell according to claim 1, wherein the immune cell is a T cell or an NK cell. **Claim 4** The immune cell according to claim 1, wherein the immune cell comprises an inhibitor of LCK or is contacted with the inhibitor of LCK. **Claim 5** The immune cell according to claim 4, wherein the inhibitor of LCK is a nucleic acid sequence capable of down-regulating or abolishing gene expression or modifying the function of LCK. **Claim 6** The immune cell according to claim 5, wherein the nucleic acid capable of down-regulating the gene expression of LCK is selected from the group consisting of antisense RNA, antagomir RNA, siRNA, shRNA, CRISPR system, zinc finger nuclease system, and transcription activator-like effector-based nuclease (TALEN) system. **Claim 7** The immune cell according to claim 4, wherein the inhibitor of LCK is an inhibitor of the LCK protein. **Claim 8** The immune cell according to claim 7, wherein the inhibitor of LCK is selected from the group consisting of aminokynazoline, A-420983, A770041, dasatinib, saracatinib, and masatinib. **Claim 9** The immune cell according to claim 1, wherein the immune cell comprises a vector containing a nucleic acid encoding the CAR. **Claim 10** The immune cell according to claim 1, wherein the immune cell comprises a vector containing a nucleic acid encoding an inhibitor of LCK capable of down-regulating or abolishing gene expression or modifying the function of LCK. **Claim 11** The immune cell according to claim 1, wherein the immune cell comprises a vector containing a nucleic acid encoding the CAR and a nucleic acid encoding an inhibitor of LCK capable of down-regulating or abolishing the gene expression of LCK. **Claim 12** The immune cell according to claim 1, wherein the CAR comprises an extracellular antigen-binding domain. **Claim 13** The immune cell according to claim 1, wherein the intracellular signaling domain further comprises a primary signaling domain comprising a functional signaling domain of a protein selected from CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcεRIβ, CD79a, CD79b, FcγRIIa, DAP10, or DAP12.

14. The immune cell according to claim 1, wherein the intracellular signaling domain further comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of DAP10, CD28, CARD11, SLAMF1, LCK1, LCK3, LAT, OX40, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

15. The immune cell according to claim 1, wherein the expression of PD-1 is decreased as compared to a cell not modified such that the expression and / or function of LCK is decreased or abolished.

16. An immune cell expressing a CAR, which is modified such that the expression or function of the LCK gene is disrupted.

17. A method for producing the immune cell according to claim 1, comprising contacting the immune cell with an inhibitor of LCK for a time and under conditions sufficient to decrease or abolish the expression and / or function of LCK.

18. The method according to claim 17, wherein the inhibitor of LCK is a nucleic acid sequence capable of downregulating the gene expression of LCK.

19. The method according to claim 18, wherein the inhibitor of LCK is a CRISPR system.

20. The method according to claim 17, further comprising introducing a nucleic acid encoding a CAR into the immune cell.

21. A vector system comprising 1) a vector comprising a nucleic acid sequence encoding an inhibitor of LCK, and 2) a vector comprising a nucleic acid sequence encoding a CAR.

22. A vector comprising a nucleic acid sequence encoding an inhibitor of LCK and a nucleic acid sequence encoding a CAR.

23. The vector according to claim 22, wherein the nucleic acid sequence encoding the CAR is an inhibitor of LCK.

24. A method for improving the efficacy of a CAR-expressing immune cell in cell therapy, comprising contacting the CAR-expressing immune cell with an inhibitor of LCK for a time and under conditions sufficient to decrease or abolish the expression and / or function of LCK.

25. The method according to claim 24, wherein the CAR-expressing immune cell is a CAR T cell.

26. The method according to claim 24, wherein the off-target effect of the CAR-expressing immune cell is reduced.

27. The method according to claim 24, wherein the exhaustion phenotype in the CAR-expressing cell is reduced.

28. The method according to claim 24, wherein the memory of the CAR-expressing immune cell is improved.

29. A method of treating a subject in need thereof, comprising administering the immune cell according to any one of claims 1 to 16 under conditions and for a time sufficient to treat the subject.

30. The method according to claim 29, wherein the subject has a disease associated with the expression of a tumor antigen (for example, a proliferative disease, a pre-cancerous condition, cancer, and non-cancer-related symptoms associated with the expression of a tumor antigen).

31. The immune cell according to any one of claims 1 to 16 for use in the treatment of a subject in need thereof.

32. Use of the immune cell according to any one of claims 1 to 16 in the manufacture of a medicament for treating a subject in need thereof.

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