Anti-dinitrophenol chimeric antigen receptor

Abstract

Embodiments provided herein include methods and compositions comprising an anti-dinitrophenol chimeric antigen receptor (CAR). Some embodiments include nucleic acids encoding the CAR, polypeptides encoded by the nucleic acids, cells containing the nucleic acids or polypeptides, and methods utilizing the cells. Some embodiments further include the use of dinitrophenol (DNP) and its derivatives.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 969,931, “Anti-Dinitrophenol Chimeric Antigen Receptor,” filed on 4 February 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence listing reference This application was filed together with an electronic sequence listing. This sequence listing was provided as a file of approximately 43kb created on February 2, 2021, with the filename SCRI269WOSEQLIST. The information contained herein is incorporated herein by reference in its entirety.

[0003] The embodiments provided herein include methods and compositions comprising an anti-dinitrophenol chimeric antigen receptor (CAR). Some embodiments include nucleic acids encoding this CAR, polypeptides encoded by this nucleic acid, cells comprising this polypeptide, and methods utilizing these cells. Some embodiments further include the use of dinitrophenol (DNP) and its derivatives. [Background technology]

[0004] Immunotherapy utilizing adoptive cell transfer of T cells possessing chimeric antigen receptors (CARs) is an effective treatment method for cancer. The structure of a CAR includes an antigen-binding domain, a spacer domain, a transmembrane domain, and one or more costimulatory activation domains. CAR T cells may be generated from T cells obtained from the patient or a donor. In some cases, CARs exert their function by binding to specific antigens on the cell surface, causing lysis of cells presenting that antigen. Various studies have focused on designing the ligand-binding domain of CARs to target desired cell surface antigens and reduce toxicity, but further therapies utilizing CAR T cells are still needed. [Overview of the Initiative] [Means for solving the problem]

[0005] Some embodiments of the methods and compositions provided herein are nucleic acids encoding chimeric antigen receptors (CARs), wherein the CAR is A ligand-binding domain that specifically binds to the dinitrophenol (DNP) moiety; Spacer; Transmembrane domain; and Intracellular signal transduction domains It contains nucleic acids characterized by including the following:

[0006] In some embodiments, the ligand-binding domain includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12.

[0007] In some embodiments, the ligand-binding domain includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 9, or 10.

[0008] In some embodiments, the spacer is selected from the group consisting of short spacers consisting of (preferably 2 or more and) 12 or fewer consecutive amino acid residues, medium-length spacers consisting of (preferably 2 or more and) 119 or fewer consecutive amino acid residues, and long spacers consisting of more than 119 consecutive amino acid residues.

[0009] In some embodiments, the spacer is selected from the group consisting of a short spacer containing an IgG4 hinge domain, a medium-length spacer containing an IgG4 hinge-CH3 domain, and a long spacer containing an IgG4 hinge-CH2-CH3 domain.

[0010] In some embodiments, the spacer is a long spacer consisting of at least 229 consecutive amino acid residues.

[0011] In some embodiments, the transmembrane domain includes the transmembrane domain of CD28.

[0012] In some embodiments, the intracellular signaling domain includes a portion of CD3ζ and / or a portion of 4-1BB.

[0013] Some embodiments further include a selection gene, a cell surface selection marker, or a polynucleotide encoding a cleavable linker. In some embodiments, the selection gene comprises a double mutant of dihydrofolate reductase (DHFRdm). In some embodiments, the cell surface selection marker is selected from the group consisting of cleaved EGFR (EGFRt), cleaved Her2 (Her2tG), and cleaved CD19 (CD19t). In some embodiments, the cleavable linker comprises a ribosome skip sequence selected from the group consisting of P2A, T2A, E2A, and F2A.

[0014] Some embodiments of the methods and compositions provided herein include a vector comprising one of the nucleic acids provided herein. In some embodiments, the vector includes a lentiviral vector.

[0015] Some embodiments of the methods and compositions provided herein are methods for preparing cell populations for infusion, A step of introducing one of the nucleic acids encoding anti-DNP CARs provided herein into a cell; and culturing the cells under conditions suitable for obtaining a sufficient number of cell populations for infusion comprising a method comprising.

[0016] Some embodiments of the methods and compositions provided herein include a CAR encoded by any one of the nucleic acids provided herein.

[0017] Some embodiments of the methods and compositions provided herein include cells comprising any one of the CARs provided herein.

[0018] In some embodiments, the cells are cells derived from CD4+ T cells, CD8+ T cells, progenitor T cells or hematopoietic stem cells. In some embodiments, the CD8+ T cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some embodiments, the central memory CD8+ T cells are CD45RO positive and CD62L positive. In some embodiments, the CD4+ T cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells and bulk CD4+ T cells. In some embodiments, the naive CD4+ T cells are CD45RA positive, CD62L positive and CD45RO negative.

[0019] In some embodiments, the cells are ex vivo cells. In some embodiments, the cells are in vivo cells.

[0020] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0021] Some embodiments of the methods and compositions provided herein include a composition comprising one of the CARs provided herein and a DNP moiety attached to a target cell, wherein the CAR specifically binds to the DNP moiety.

[0022] In some embodiments, the DNP portion is attached to the target cell via an antibody or antigen-binding fragment that binds to the target cell.

[0023] In some embodiments, the DNP portion is attached to the target cells via the folic acid portion.

[0024] In some embodiments, the DNP portion is attached to the surface of the target cell via a lipid incorporated into the surface of the target cell. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group comprises a group selected from choline, phosphatidylcholine, phosphocholine, sphingomyelin, phosphoethanolamine, sugar residues, phosphatidylserine, phosphatidylinositol, piperidine, and trimethylarsenoethyl phosphate. In some embodiments, the hydrophobic group comprises a fatty acid chain or a terpenoid portion. In some embodiments, the hydrophobic group comprises an ether linkage, the ether linkage located between the polar head group and the fatty acid chain. In some embodiments, the fatty acid chain is C 10-20 It contains an alkyl chain. In some embodiments, the lipid is an ether phospholipid (PLE).

[0025] In some embodiments, the target cells are cancer cells. In some embodiments, the cancer cells are selected from the group consisting of breast cancer cells, brain tumor cells, colorectal cancer cells, kidney cancer cells, pancreatic cancer cells, and ovarian cancer cells.

[0026] In some embodiments, the target cells are ex vivo cells. In some embodiments, the target cells are in vivo cells.

[0027] In some embodiments, the target cells are mammalian cells. In some embodiments, the target cells are human cells.

[0028] Some embodiments of the methods and compositions provided herein are methods for treating or alleviating cancer in a subject, This includes a method comprising administering to one of the anti-DNP CAR T cells provided herein in combination with a composition containing a DNP moiety.

[0029] In some embodiments, the composition is administered after the cells are administered. In some embodiments, the cells are administered after the composition is administered. In some embodiments, the cells and the composition are administered simultaneously.

[0030] In some embodiments, the composition is configured to target the cancer.

[0031] In some embodiments, the DNP portion is linked to an antibody or its antigen-binding fragment that specifically binds to the cancer.

[0032] In some embodiments, the DNP portion is linked to folic acid.

[0033] In some embodiments, the DNP portion is linked to a lipid. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group comprises a group selected from choline, phosphatidylcholine, phosphocholine, sphingomyelin, phosphoethanolamine, sugar residues, phosphatidylserine, phosphatidylinositol, piperidine, and trimethylarsenoethyl phosphate. In some embodiments, the hydrophobic group comprises a fatty acid chain or a terpenoid portion. In some embodiments, the hydrophobic group comprises an ether linkage, the ether linkage located between the polar head group and the fatty acid chain. In some embodiments, the fatty acid chain is C 10-20 It contains an alkyl chain. In some embodiments, the lipid is an ether phospholipid (PLE).

[0034] In some embodiments, the cancer includes target cells selected from the group consisting of breast cancer cells, brain tumor cells, colorectal cancer cells, kidney cancer cells, pancreatic cancer cells, and ovarian cancer cells.

[0035] In some embodiments, the cells are autologous cells obtained from the subject.

[0036] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0037] Some embodiments of the methods and compositions provided herein involve the use of any one of the anti-DNP CAR T cells provided herein in combination with a composition comprising a DNP moiety for treating cancer in a subject.

[0038] Some embodiments of the methods and compositions provided herein involve the use of any one of the anti-DNP CAR T cells provided herein in combination with a composition containing a DNP moiety in the manufacture of a pharmaceutical product for treating cancer in a subject.

[0039] Some embodiments of the methods and compositions provided herein include one of the anti-DNP CAR T cells provided herein for use in pharmaceuticals. [Brief explanation of the drawing]

[0040] [Figure 1] An embodiment of a DNP-ether phospholipid (DNP-PLE) comprising (i) a DNP portion; (ii) a polyethylene glycol (PEG) portion; (iii) a polar head; and (iv) a hydrophobic tail is shown.

[0041] [Figure 2A] Flow cytometry data for MDA-MB-231 control cells and MDA-MB-231 control cells incubated with anti-DNP-Alexa Fluor 488 antibody are shown.

[0042] [Figure 2B] Flow cytometry data for MDA-MB-231 control cells, MDA-MB-231 control cells incubated with anti-DNP-Alexa Fluor 488 antibody, and MDA-MB-231 cells labeled with 5 μM DNP-PLE and stained with anti-DNP-Alexa Fluor 488 antibody are shown.

[0043] [Figure 2C] Flow cytometry data for MDA-MB-231 control cells, MDA-MB-231 control cells incubated with anti-DNP-Alexa Fluor 488 antibody, and MDA-MB-231 cells labeled with 500 nM DNP-PLE and stained with anti-DNP-Alexa Fluor 488 antibody are shown.

[0044] [Figure 2D]Flow cytometry data are shown for MDA-MB-231 control cells, MDA-MB-231 control cells incubated with anti-DNP-Alexa Fluor 488 antibody, and MDA-MB-231 cells labeled with 50 nM DNP-PLE and stained with anti-DNP-Alexa Fluor 488 antibody.

[0045] [Figure 2E] Figures 2A to 2D show histogram plots of the flow cytometry data.

[0046] [Figure 3A] Confocal images of MDA-MB-231 control cells incubated with anti-DNP-Alexa Fluor 488 antibody are shown.

[0047] [Figure 3B] This image shows a confocal image of MDA-MB-231 cells incubated with 5 μM DNP-PLE.

[0048] [Figure 3C] The image shows confocal images of MDA-MB-231 cells incubated with 5 μM DNP-PLE and stained with anti-DNP-Alexa Fluor 488 antibody.

[0049] [Figure 3D] The image shows confocal images of MDA-MB-231 cells incubated with 1 μM DNP-PLE and stained with anti-DNP-Alexa Fluor 488 antibody.

[0050] [Figure 4] A schematic diagram of a second-generation CAR cassette with a long spacer for expressing anti-DNP CARs is shown.

[0051] [Figure 5A] Confocal images of MDA-MB-231 control cells co-cultured with anti-DNP CAR H9 cells are shown.

[0052] [Figure 5B] The image shows a confocal image of MDA-MB-231 cells stained with 5 μM DNP-PLE and co-cultured with anti-DNP CAR H9 cells. [Modes for carrying out the invention]

[0053] The embodiments provided herein include methods and compositions comprising an anti-dinitrophenol chimeric antigen receptor (CAR). Some embodiments include nucleic acids encoding this CAR, polypeptides encoded by this nucleic acid, cells comprising this polypeptide, and methods utilizing these cells. Some embodiments further include the use of dinitrophenol (DNP) and its derivatives.

[0054] Adoptive transfer of T cells modified by transgenes has been successful under selected conditions (e.g., malignancies of the CD19 B cell lineage), but generalizing T cell adoptive transfer therapy to other types of cancer is difficult because there is no single target antigen that is found in all forms of cancer but not in normal healthy cells. CAR T cell therapy can be used to treat various types of cancer that afflict many people, but its development is hindered by the daunting challenge of identifying and meticulously investigating tens of thousands of antigens (e.g., antigens targeted by CARs) presented on cancer cells. In some embodiments described herein, this challenge is solved by using DNP-ether phospholipid (DNP-PLE). This DNP-PLE associates with tumor cells and interacts with T cells possessing DNP-specific CARs by presenting a specific target molecule to those CARs, thereby inducing the lysis of DNP-presenting tumor cells. This approach eliminates the need to identify and validate tens of thousands of unique antigen-specific CARs.

[0055] Some embodiments provided herein include CARs having specificity or selected affinity or avidity to a DNP moiety. In some embodiments, the DNP moiety is linked to a molecule that can associate with or bind to the surface of tumor cells. In some embodiments, the molecule is a PLE. In some embodiments, the molecule may include an antibody or its antigen-binding fragment that specifically binds to cells. In some embodiments, a method can be carried out to make the reactivity of antitumor T cells target-directed using the DNP-PLE or other DNP molecule and T cells having the CAR. In some embodiments, the DNP-PLE is a synthetic molecule having a structure designed to be incorporated into the cell membrane of tumor cells, wherein the DNP moiety of the synthetic molecule is positioned adjacent to the outer layer of the cell membrane of tumor cells and is presented in the extracellular space in an orientation or proximity that enables desired interaction with T cells having a CAR having desired affinity, specificity or avidity to the DNP moiety. Similarly, in some embodiments, the CARs described herein have a unique structure designed such that the anti-DNP ligand-binding domain of the CAR is presented on the cell membrane of T cells in an orientation or proximity that enables a desired interaction with DNP-PLE-carrying tumor cells, or in an orientation or proximity that provides a desired avidity to DNP-PLE-carrying tumor cells. Thus, in some embodiments, the DNP-PLE molecules and CARs described herein possess therapeutically important features, and it is envisioned that the DNP-PLE molecule be used as a pharmaceutical product, either in combination with or without an anti-DNP-specific CAR. In another embodiment, one or more of the CARs and DNP-PLE molecules described herein are useful for the treatment or mitigation of human diseases or conditions such as cancer.

[0056] Another embodiment relates to a CAR that targets and interacts with a DNP moiety linked to a PLE molecule, which may be constitutively expressed and controlled in cells (preferably T cells); a nucleic acid encoding the CAR; cells (preferably T cells) having the nucleic acid or CAR; a method for producing the same; and a method for treating diseases such as human cancer using these compositions. Some of the embodiments of the methods and compositions provided herein include embodiments disclosed in WO2018 / 148224, WO2019 / 156795, WO2019 / 144095, US2019 / 0224237 and PCT / US2019 / 044981 (these documents are expressly incorporated herein by reference in their entirety).

[0057] Definition of Terms In this specification, "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of monomers made from natural nucleotide monomers (such as DNA or RNA), or analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications to the sugar moiety and / or the pyrimidine base moiety or purine base moiety. Modifications to the sugar moiety include, for example, the substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Furthermore, the entire sugar moiety may be substituted with a structure that is stereochemically similar or electronically similar, such as aza sugars or carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Similar bonds to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoranilothioate bonds, phosphoranilidate bonds, and phosphoramidate bonds. "Nucleic acid molecule" also includes "peptide nucleic acid," which includes native nucleic acid bases or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded. In this specification, "encodes" refers to the property that a particular nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA functions as a template for synthesizing another macromolecule, such as a given amino acid sequence.Therefore, when mRNA corresponding to a particular gene is transcribed and translated to produce a protein in a cell or other biological system, that gene encodes this protein. A “nucleic acid sequence encoding a polypeptide” includes any degenerate nucleotide sequence encoding the same amino acid sequence. “Specific” or “specificity” may refer to the properties of a ligand to its binding partner, or the properties of a binding partner to a ligand, such as complementary shape, charge, or specificity involved in hydrophobic binding. Specificity involved in binding includes stereospecificity, region selectivity, and / or chemoselectivity. In some embodiments, a method is provided for producing nucleic acids encoding chimeric antigen receptors specific to a DNP portion linked to or associated with a PLE molecule, which can be associated with or ligated to tumor cells.

[0058] A “vector” or “construct” is a nucleic acid used to introduce heterologous nucleic acids into cells and can contain various regulatory elements, thereby enabling the expression of heterologous nucleic acids in cells. Examples of vectors include, but are not limited to, plasmids, minicircles, yeast, or viral genomes. In some embodiments, the vector is a plasmid, minicircle, viral vector, DNA, or mRNA. In some embodiments, the vector is a lentiviral vector or retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0059] In this specification, “chimeric receptor” refers to a synthetically designed receptor that includes a ligand-binding domain of an antibody sequence or other protein sequence that binds to a target molecule, and this ligand-binding domain is linked to one or more intracellular signaling domains (e.g., costimulatory domains) of a T cell receptor or other receptor via a spacer domain. Chimeric receptors may also be called artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, or chimeric antigen receptors (CARs). By utilizing chimeric receptors, the specificity of a monoclonal antibody or its binding fragment or other ligand-binding domain can be transferred to T cells, and the coding sequence required for the expression of the chimeric receptor is transferred to the T cells using a viral vector such as a retroviral vector or lentiviral vector. A CAR is a recombinant T cell receptor designed to confer directivity to T cells towards target cells that express or present a specific cell surface antigen targeted by the CAR. In a method called “adoptive cell transfer,” T cells are first obtained from a subject, and then the T cells are genetically engineered to express a receptor specific to the desired antigen. These T cells are then reintroduced into the patient. Reintroduced T cells recognize the antigen-presenting molecule presented to the cell and bind to it as a target. Such CARs are recombinant receptors that can transfer selected specificity to cells expressing immune receptors. Some researchers understand "chimeric antigen receptors," or "CARs," to include an antibody or antibody fragment, a spacer, a signaling domain, and a transmembrane domain. The CARs described herein have various components, or domains (e.g., epitope-binding domains (e.g., antibody fragments, scFv or parts thereof), spacers, transmembrane domains, and / or signaling domains), and because remarkable effects have been obtained as a result, each component of the CAR can often be clearly distinguished as an independent element through the disclosures herein.

[0060] A "single-chain variable region fragment (scFv)" is a fusion protein that may contain the heavy-chain variable region (VH) and light-chain variable region (VL) of an immunoglobulin, linked by a short linker peptide consisting of 10 to 25 amino acids. The short linker peptide may contain 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, or any two of these numbers as upper and lower limits. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, scFv retains the specificity of the original immunoglobulin. scFv may be antigen-specific. In this specification, “antigen” or “Ag” refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immune cells, or both. Antigens can be prepared, synthesized, and manufactured by recombinant techniques, or obtained from biological samples. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids (e.g., blood, plasma, or ascites). In some embodiments described herein, a composition is provided comprising cells (preferably T cells) prepared by any of the methods according to the embodiments described herein. In some embodiments, the cells (preferably T cells) comprise a chimeric antigen receptor comprising an scFv specific to a DNP portion linked to or associated with a PLE molecule, which can associate with or ligate to tumor cells.

[0061] The "antigen-specific binding domain" may include a protein or protein domain that specifically binds to an epitope on a protein with low or high binding affinity (fM to mM binding strength). In some embodiments, the fusion protein includes a protein or a portion thereof that modulates the immune response. In some embodiments, the protein includes the antigen-specific binding domain.

[0062] Several types of “spacers” are described herein. With respect to CARs, spacers refer to polypeptide spacers, the length of which the spacer is configured or selected to enhance the binding to or interaction with the chimeric antigen receptor, or to reduce or minimize the adverse side effects associated with CAR T cell therapy. In some embodiments, short spacer domains of CARs have 2 to 12 or 2 to about 12 amino acids and include all or part of the hinge region sequence of IgG4, or all or part of a variant thereof. In some embodiments, medium-length spacer domains of CARs have 2 to 119 or 2 to about 119 amino acids and include all or part of the sequence consisting of the hinge region sequence and the CH3 region of IgG4, or all or part of a variant thereof. In some embodiments, the long spacer domain of the CAR has 2 to 229 or 2 to approximately 229 amino acids and includes all or part of the sequence consisting of the hinge region sequence of IgG4 and the CH2 and CH3 regions, or all or part of a variant thereof. In some embodiments, the length of the spacer, its sequence, or both is selected based on desired avidity or interaction with a DNP portion linked to or associated with a PLE molecule, which can associate with or link to tumor cells.

[0063] A "transmembrane domain" is a hydrophobic protein region located in the cell membrane bilayer that plays a role in tethering proteins embedded in biological membranes. The topology of the transmembrane domain may, but is not limited to, a transmembrane α-helix. In some embodiments of the method for producing recombinant T cells having a chimeric antigen receptor, the vector includes a sequence encoding a transmembrane domain. In some embodiments of the method, the transmembrane domain includes a transmembrane sequence or fragment of CD28, the transmembrane sequence or fragment of CD28 having a length within the range defined by 10 amino acid lengths, 11 amino acid lengths, 12 amino acid lengths, 13 amino acid lengths, 14 amino acid lengths, 15 amino acid lengths, 16 amino acid lengths, 17 amino acid lengths, 18 amino acid lengths, 19 amino acid lengths, 20 amino acid lengths, 21 amino acid lengths, 22 amino acid lengths, 23 amino acid lengths, 24 amino acid lengths, 25 amino acid lengths, 26 amino acid lengths, 27 amino acid lengths, or 28 amino acid lengths, or any two of these lengths. In some embodiments of the method, the transmembrane sequence or fragment of CD28 is 28 amino acid lengths long. In some embodiments, the chimeric receptor of the present invention includes a transmembrane domain. This transmembrane domain plays a role in tethering the chimeric receptor to the cell membrane.

[0064] The terms “costimulatory domain” or “intracellular signaling domain” have the general and ordinary meanings as used herein and include, but are not limited to, signaling portions that provide T cells with signals that mediate T cell responses, such as activation, proliferation, differentiation, and cytokine secretion, in addition to primary signals provided by, for example, the CD3ζ chain of the TCR / CD3 complex. Examples of costimulatory domains include, but are not limited to, whole molecules such as CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or whole ligands that specifically bind to CD83, or parts thereof. In some embodiments, the costimulatory domain is an intracellular signaling domain that mediates a cellular response, including one or more of activation, proliferation, differentiation, and cytokine secretion, by interacting with other intracellular mediators.

[0065] In this specification, “marker sequence” encodes a cell containing the protein of interest or a protein used to select or track the protein of interest. In embodiments described herein, the fusion protein provided herein may include a marker sequence that can be selected in experiments such as flow cytometry. In some embodiments, the marker is Her2tG, CD19t, or EGFRt.

[0066] In this specification, a “ribosome skip sequence” refers to a sequence that has the function of causing ribosomes to “skip” the ribosome skip sequence during translation, thereby preventing the formation of peptide bonds and causing translation to begin from the region immediately following the ribosome skip sequence. For example, some viruses have ribosome skip sequences, allowing for the sequential translation of multiple proteins from a single nucleic acid, and the translated proteins are obtained as separate proteins without being linked by peptide bonds. In this specification, the ribosome skip sequence is used as a “linker” sequence. In some embodiments of the nucleic acid provided herein, the nucleic acid of the present invention includes a ribosome skip sequence between the sequence encoding a chimeric antigen receptor and the sequence encoding a marker protein, so that the chimeric antigen receptor and the marker protein are co-expressed without being linked by peptide bonds. In some embodiments, the ribosome skip sequence is a P2A sequence, a T2A sequence, an E2A sequence, or an F2A sequence. In some embodiments, the ribosome skip sequence is a T2A sequence. In some embodiments, a ribosome skipping sequence is present between two chimeric antigen receptors, and a second ribosome skipping sequence is present between one of the chimeric antigen receptors and a marker.

[0067] In this specification, 2,4-dinitrophenol (2,4-DNP or simply DNP) is an organic compound represented by the formula HOC6H3(NO2)2 and has the general and ordinary meaning as used herein. DNP is used, in particular, as a preservative, a non-selective bioaccumulative pesticide, a herbicide, and the like. DNP is also an intermediate chemical in the manufacture of sulfur dyes, wood preservatives, and picric acid. In some embodiments described herein, DNP is a target moiety on a lipid that is recognized by a chimeric antigen receptor and undergoes binding to the chimeric antigen receptor. In some embodiments, the hapten is DNP or a derivative thereof. In some embodiments, the lipid is a phospholipid (e.g., ether phospholipid (PLE)).

[0068] As used herein, “lipids” are a type of organic compound comprising a carbon chain, fatty acid, or fatty acid derivative, which are typically insoluble in water but can be miscible or mixed with hydrophobic solvents or organic solvents. Examples of lipids include, but are not limited to, fats, waxes, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, sphingolipids, cerebrosides, ceramides, or phospholipids. This specification describes amphiphilic lipids that may have a polar head group and a hydrophobic moiety or hydrophobic moiety. As used herein, a “hydrophobic group,” or hydrophobic moiety, is a molecule or part of a molecule that repels water and tends to be non-polar. Examples of hydrophobic moieties include alkanes, oils, or fats. Examples of lipids include, but are not limited to, glycerolipids, glycerophospholipids, sphingolipids, sterollipids, prenolipids, saccharolipids, or polyketides. Embodiments described herein provide complexes containing lipids. In some embodiments, the lipids include a polar head group and a hydrophobic moiety. In some embodiments, the hydrophobic portion is a hydrophobic carbon chain tail. In some embodiments, the hydrophobic carbon chain tail is a saturated carbon chain tail or an unsaturated carbon chain tail. In some embodiments, the hydrophobic carbon chain tail contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms, or a number of carbon atoms within the range defined by any of these values. In some embodiments, the hydrophobic portion is a steroid or cholesterol. In some embodiments, the lipid includes glycerolipids, glycerophospholipids, sphingolipids, sterollipids, prenolipids, saccharolipids, or polyketides. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the lipid includes a branched alkyl tail.

[0069] In some embodiments, the lipid is a sphingolipid. The sphingolipid may contain a back chain consisting of a sphingoid base, such as a series of aliphatic amino alcohols including sphingosine. A sphingolipid in which the R group consists only of hydrogen atoms is a ceramide. Other common R groups include phosphocholine (when the R group is phosphocholine, sphingomyelin is obtained), or various sugar monomers or sugar dimers (when the R group is a sugar monomer, a cerebroside is obtained; when the R group is a sugar dimer, a globoside is obtained). Cerebrosides and globosides are known as sphingoglycolipids. In some embodiments, the lipid is a sphingoglycolipid.

[0070] According to this specification, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the hydrophobic group comprises a fatty acid, such as a fatty acid chain. The fatty acid may be a saturated fatty acid or an unsaturated fatty acid. In some embodiments, the hydrophobic group comprises an alkyl group, an alkenyl group, or an alkynyl group. In some embodiments, the hydrophobic group comprises a terpenoid lipid, such as a steroid or cholesterol. In some embodiments, the hydrophobic group comprises an ether linkage, the ether linkage located between the polar head group and the fatty acid chain. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the polar head group comprises choline, phosphatidylcholine, sphingomyelin, a phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidylserine, or phosphatidylinositol. In some embodiments, the sugar is glycerol or a sugar alcohol.

[0071] In some embodiments, the lipid is a single-chain alkylphospholipid.

[0072] In some embodiments, the lipid comprises a synthetic alkylphospholipid structure such as edelhosine, perifosine, or erucylphosphocholine. In some embodiments, the lipid is lysophosphatidylcholine, edelhosine, erucylphosphocholine, D-21805, or perfisone. Such lipids have been reported, for example, by van der Lui et al. ("A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells" Mol Cancer Ther 2007; 6(8), 2007 (this document is expressly incorporated herein by reference in its entirety)). In some embodiments of the lipids described herein, the choline in the polar head group can be substituted with a piperidine moiety. In some embodiments, the lipid is an anticancer alkylphospholipid. Anticancer phospholipids were reported by van der Lui et al. ("A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells" Mol Cancer Ther 2007; 6(8), 2007 (this document is explicitly incorporated herein by reference in its entirety)).

[0073] In some embodiments, the lipids provided herein are structurally related synthetic antitumor agents characterized by their interaction with cell membranes. These synthetic lipids are alkylphospholipids, as reported, for example, by van Blitterswijk et al. ("Anticancer mechanisms and clinical application of alkylphopholipids," Biochimica et Biophysica Acta 1831 (2013) 663-674 (this document is incorporated herein by reference in its entirety)). Examples of such synthetic alkylphospholipids include, but are not limited to, edelfosine, miltefosine, perifosine, erucylphosphocholine, or erufosine. In some embodiments, the lipid is edelfosine, miltefosine, perifosine, erucylphosphocholine, or erufosine. In some embodiments, the lipid is a stable analog of lysophosphatidylcholine. In some embodiments, the lipid is a thioether-linked variant of edelfosine, or 1-hexadecylthio-2-methoxymethyl-rac-glycero-3-phosphocholine. In some embodiments, the lipid is LysoPC, edelfosine, ylmofosine, miltefosine, perifosine, erucylphosphocholine, or erufosine.

[0074] The “polar head group” as described herein is a hydrophilic group of a lipid (such as a phospholipid). The “phospholipid” as described herein is a special type of lipid that has amphiphilic properties and can form a lipid bilayer. A phospholipid molecule comprises at least one hydrophobic fatty acid “tail” and a hydrophilic “head” or “polar head group”. In embodiments of this specification, a phospholipid or ether phospholipid comprises a polar head group. In some embodiments, the polar head group comprises a phosphocholine, piperidine moiety, or trimethylarsenoethyl phosphate moiety. In some embodiments, the lipid comprises a target moiety, which is preferably a DNP, and the CAR is linked to the lipid or configured to be linked to the lipid via interaction with the target moiety. In some embodiments, the lipid comprises a polar head group (e.g., a polar head group comprising an aromatic ring) and an alkyl carbon chain. Embodiments of the present invention provide a complex comprising one or more of the above lipids. In some embodiments, the lipid comprises a polar head group. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the ether phospholipid comprises a target moiety, which is preferably a DNP, and the CAR is linked to or configured to be linked to the ether phospholipid via interaction and / or binding to the target moiety. In some embodiments, the ether phospholipid comprises a polar head group and an alkyl carbon chain. In some embodiments, the polar head group comprises choline, phosphatidylcholine, sphingomyelin, a phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidylserine, or phosphatidylinositol. In some embodiments, the polar head group comprises phosphocholine, a piperidine moiety, or a trimethylarsenoethyl phosphate moiety. In some embodiments, the lipid is an ether phospholipid (PLE). In some embodiments, the sugar is glycerol or a sugar alcohol. In some embodiments, the polar head group comprises a sugar group. In some embodiments, the lipid comprises a head group comprising mannose.In some embodiments, the polar head group comprises sphingosine. In some embodiments, the polar head group comprises glucose. In some embodiments, the polar head group comprises a disaccharide, trisaccharide, or tetrasaccharide. In some embodiments, the lipid is a glucosylcerebroside. In some embodiments, the lipid is a lactosylceramide. In some embodiments, the lipid is a glycolipid. In some embodiments, the glycolipid comprises sugar units such as n-glucose, n-galactose, and N-acetyl-n-galactosamine. In some embodiments, the lipid comprises a hydrocarbon ring such as a sterol.

[0075] In some embodiments, the polar head group of the lipid includes glycerol or a sugar alcohol. In some embodiments, the polar head group of the lipid includes a phosphate group. In some embodiments, the polar head group of the lipid includes choline. In some embodiments, the lipid is phosphatidylethanolamine. In some embodiments, the lipid is phosphatidylinositol. In some embodiments, the lipid includes a backbone consisting of a sphingoid base. In some embodiments, the lipid includes sterol lipids such as cholesterol or its derivatives. In some embodiments, the lipid includes saccharolipids. In some embodiments, the polar head group includes choline, a phosphate, or glycerol.

[0076] In some embodiments, the lipid is a glycolipid. In some embodiments, the lipid contains sugars. In some embodiments, the lipid is derived from sphingosine. In some embodiments, the lipid is a glyceroglycolipid or a sphingoglycolipid.

[0077] In some embodiments, the lipid is an ether lipid having a hydrophobic branched chain.

[0078] As used herein, "terpenoids" are molecules derived from a five-carbon isoprene unit. Steroids and sterols can be produced from terpenoid precursors. For example, steroids and cholesterol can be biosynthesized from terpenoid precursors.

[0079] As used herein, “ether phospholipids (PLEs)” are lipids in which one or more carbon atoms of a polar head group are linked to an alkyl chain via an ether bond, rather than a more common ester bond. In some embodiments, the polar head group is glycerol.

[0080] Several types of “spacers” are described herein. With respect to lipids, the lipids may contain spacers, for example, spacers linked to the polar head groups of the lipids, which can be oriented in a desired direction or reduce steric hindrance to obtain a desired degree of freedom by separating the target portion (preferably DNP) from the lipid and from cells linked to or associated with the lipid. The spacers of the lipids may include PEG spacers, hapten spacers, small peptides, or alkane chains. In some embodiments, the hapten spacer contains two haptens (hapten (2×) spacers). In some embodiments, the lipids contain hydrophobic groups such as alkane chains. In some embodiments, the alkane chains may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms, or a number of carbon atoms within a range where any two of these numbers are the upper and lower limits. In some embodiments, the PEG spacer contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen PEG molecules, or a number of PEG molecules within a range of any two of these numbers as upper and lower limits. The length and type of the spacer are preferably selected so that the target region (preferably DNP) is presented in an orientation or proximity that yields the desired affinity or avidity to the anti-DNP CAR presented on the cell (e.g., a T cell).

[0081] In this specification, "T cells" or "T lymphocytes" may be obtained from any mammalian species, for example, from monkeys, dogs, or humans, but are preferably obtained from primates. In some embodiments, the T cells are of the same species as the recipient (same species but derived from a different donor). In some embodiments, the T cells are autologous T cells (the donor and recipient are the same). In some embodiments, the T cells are syngeneic (the donor and recipient are different, but are identical twins).

[0082] In some embodiments, the T cells are "memory" T cells that have previously experienced the antigen (T M It is preferable that the cells are progenitor T cells. In some embodiments, the progenitor T cells are hematopoietic stem cells. In some embodiments, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

[0083] In this specification, “cytotoxic T lymphocytes (CTLs)” refers to T lymphocytes that express CD8 on their cell surface (e.g., CD8+ T cells). In some embodiments, such cells are “memory” T cells (T) that have previously experienced an antigen. M Preferably, the cells are cytotoxic T lymphocytes. In some embodiments, cells for secreting fusion proteins are provided. In some embodiments, the cells are cytotoxic T lymphocytes. In this specification, "central memory" T cells (or "T") are used. CMA central memory T cell (T) is a cytotoxic T lymphocyte (CTL) that has experienced an antigen and, compared to naive cells, expresses CD62L, CCR-7, and / or CD45RO on its surface, but does not express CD45RA or has reduced CD45RA expression. In some embodiments, cells are provided for secreting a fusion protein. In some embodiments, the cells are central memory T cells (T). CM )

[0084] In some embodiments, central memory cells may be positive for CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95 expression, but have reduced CD54RA expression, compared to naive cells. In this specification, "effector memory" T cells (or "T") are used. EM A T cell is an antigen-experienced T cell that, compared to a central memory cell, does not express CD62L on its surface or has reduced CD62L expression, and compared to a naive cell, does not express CD45RA or has reduced CD45RA expression. In some embodiments, cells for secreting a fusion protein are provided. In some embodiments, the cells are effector memory T cells. In some embodiments, the effector memory cells are negative for CD62L and / or CCR7 expression and may or may not be positive for CD28 and / or CD45RA expression compared to a naive cell or a central memory cell.

[0085] As used herein, a "naive" T cell is a T lymphocyte that has not experienced an antigen, and refers to a T lymphocyte that expresses CD62L and / or CD45RA and does not express CD45RO as compared to central memory cells or effector memory cells. In some embodiments, a cell for secreting a fusion protein is provided. In some embodiments, the cell is a naïve T cell. In some embodiments, naïve CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naïve T cells, and examples of phenotypic markers of naïve T cells include CD62L, CCR7, CD28, CD127, and / or CD45RA.

[0086] As used herein, a "T cell" or "T lymphocyte" may be obtained from any mammalian species, for example, may be obtained from a monkey, a dog, a human, etc., and is preferably obtained from a primate. In some embodiments, the T cell is allogeneic (the same species but derived from a different donor) to the recipient subject. In some embodiments, the T cell is an autologous T cell (the donor and the recipient are the same). In some embodiments, the T cell is syngeneic (the donor and the recipient are different but are monozygotic twins).

[0087] As used herein, a "progenitor T cell" refers to a lymphoid progenitor cell that migrates to the thymus and can become a progenitor T cell, and the progenitor T cell does not express a T cell receptor. All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells (lymphoid progenitor cells) derived from hematopoietic stem cells colonize the thymus and expand by cell division to create a large population of immature thymocytes. Very early thymocytes do not express either CD4 or CD8, and thus are classified as double-negative (CD4 - CD8 - ) cells. As their development progresses, these become double-positive thymocytes (CD4 + CD8 + ), and ultimately single-positive (CD4 + CD8 - or CD4- CD8 + They mature into thymocytes and are then released from the thymus into peripheral tissues.

[0088] In this specification, “CD8 T cell” or “killer T cell” refers to a T lymphocyte capable of killing cancer cells, virus-infected cells, or damaged cells. CD8 T cells recognize proteins or specific antigens produced by cancer cells or viruses that can stimulate an immune response. When the T cell receptor of a CD8 T cell recognizes an antigen, the CD8 T cell can bind to the presented antigen and destroy the cell.

[0089] In this specification, "central memory T cell (T)" is used. CM "Effector memory" T cells (or "T cells") refer to CTLs that have experienced an antigen and, compared to naive cells, express CD62L or CCR-7 and CD45RO on their surface, but do not express CD45RA or have reduced CD45RA expression. In some embodiments, central memory cells are positive for CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95 expression and have reduced CD54RA expression compared to naive cells. In this specification, "effector memory" T cells (or "T cells") are used. EM Effector memory cells are T cells that have experienced an antigen and, compared to central memory cells, do not express CD62L on their surface or have reduced CD62L expression, and compared to naive cells, do not express CD45RA or have reduced CD45RA expression. In some embodiments, effector memory cells may be negative for CD62L and / or CCR7 expression and positive or negative for CD28 and / or CD45RA expression compared to naive cells or central memory cells. In this specification, "effector T cells (T)" refers to T cells that have experienced an antigen and, compared to central memory cells, do not express CD62L or have reduced CD62L expression on their surface, and compared to naive cells or central memory cells. EA "Cell" refers to an antigen-experienced cytotoxic T lymphocyte that, compared to a central memory T cell or naive T cell, does not express CD62L, CCR7, and / or CD28, or has reduced expression of CD62L, CCR7, and / or CD28, and is positive for granzyme B and / or perforin. In some embodiments, cells for secreting fusion proteins are provided. In some embodiments, the cells are effector T cells. In some embodiments, the cells, compared to a central memory T cell or naive T cell, do not express CD62L, CCR7, and / or CD28, or have reduced expression of CD62L, CCR7, and / or CD28, and are positive for granzyme B and / or perforin.

[0090] As used herein, “combination therapy” refers to treatment using two or more drugs or treatments. Combination therapy also refers to, for example, the use of multiple treatments to treat a single disease, often involving the combined use of multiple pharmaceutical formulations. Furthermore, combination therapy includes the administration of two or more active ingredients by prescribing and administering separate drugs. In some embodiments, combination therapy is provided, including the administration of recombinant immune cells to modify the tumor microenvironment. In some embodiments, the combination therapy includes the administration of recombinant immune cells to modulate the suppression of the immune response in the tumor microenvironment. In some embodiments, the combination therapy includes the administration of recombinant immune cells to a subject (e.g., human) requiring such administration to minimize the proliferation of tumor cells and immunosuppressive cells. In some embodiments, the combination therapy includes the administration of recombinant immune cells to a subject (e.g., human) requiring such administration to improve the effectiveness of anti-cancer therapy, anti-infective therapy, antibacterial therapy, antiviral therapy, or anti-tumor therapy. In some embodiments, the combination therapy further includes the administration of an inhibitor. In some embodiments, the inhibitor is not an enzyme inhibitor. In some embodiments, the inhibitor is an enzyme inhibitor. In some embodiments, the combination therapy comprises administering a therapeutic dose of an inhibitor or antibody or a conjugated fragment thereof. In some embodiments, the antibody or conjugated fragment thereof may be humanized. In some embodiments, the combination therapy further comprises administering CAR-expressing T cells to a subject (e.g., a human) that requires them.

[0091] In this specification, “subject” or “patient” means any organism that may be used or administered with the embodiments described herein, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Examples of subjects or patients include animals. In some embodiments, the subject is a mouse, rat, rabbit, non-human primate, or human. In some embodiments, the subject is a cattle, sheep, pig, horse, dog, cat, primate, or human.

[0092] As used herein, “cancer” refers to a group of diseases characterized by the proliferation of abnormal cells that may invade or spread to other parts of the body. Subjects who can be treated using the methods described herein include subjects identified or selected for having cancer, such as colorectal cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including malignant melanoma), bone cancer, leukemia, multiple myeloma, or brain tumors, but are not limited to these. Identification and / or selection of cancer patients can be made by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or tumor-associated molecules are known, such as malignant melanoma, breast cancer, brain tumors, squamous cell carcinoma, colorectal cancer, leukemia, myeloma, or prostate cancer. Such cancers include, but are not limited to, B-cell lymphoma, breast cancer, brain tumors, prostate cancer, and / or leukemia. In some embodiments, one or more tumorigenic polypeptides are associated with kidney cancer, uterine cancer, colorectal cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer (including malignant melanoma), bone cancer, brain tumor, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, or leukemia.

[0093] In some embodiments, methods are provided for treating, alleviating, or suppressing one or more of the aforementioned cancers in a subject. In some embodiments, the cancers are breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colorectal cancer, skin cancer (including malignant melanoma), bone cancer, or brain tumor. In some embodiments, a subject receiving any of the therapies described herein, for example, a subject receiving T cells having CARs with selective avidity to DNP and DNP-PLE as described herein, is further selected to carry out another cancer therapy, which may include cancer drugs, radiotherapy, chemotherapy, or cancer therapy agents. In some embodiments, the cancer therapy drugs offered include abiraterone, alemtuzumab, anastrozole, aprepitant, arsenic trioxide, atezolizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, combinations of chemotherapy drugs, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil, and flu. Examples include Bestrant, gemcitabine, imatinib, imiquimod, ipilimumab, ixabepirone, lapatinib, lenalidomide, letrozole, leuprolide, mesna, methotrexate, nivolumab, oxaliplatin, paclitaxel, palonosetron, pembrolizumab, pemetrexed, prednisone, radium-223, rituximab, Sipuleucel-T, sorafenib, sunitinib, talc intrapleural suspension, tamoxifen, temozolomide, temsirolimus, thalidomide, trastuzumab, vinorelbine, or zoledronic acid.

[0094] As used herein, the “tumor microenvironment” refers to the cellular environment in which a tumor resides. The tumor microenvironment may include, but is not limited to, surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, or the extracellular matrix (ECM).

[0095] Specific nucleic acids that encode CARs Some embodiments of the methods and compositions provided herein include one or more nucleic acids encoding a CAR specific to a DNP moiety associated with or ligated to a PLE molecule. In some embodiments, the DNP moiety is associated with or ligated to an antibody or folic acid. In some embodiments, the DNP moiety is presented on the extracellular cell surface in an orientation or proximity that yields a desired affinity or avidity to an anti-DNP CAR (an anti-DNP CAR on a T cell or a component of the anti-DNP CAR, such as a ligand-binding domain). In some embodiments, the CAR includes a selected ligand-binding domain that binds to the DNP moiety, a selected spacer of a desired length, a transmembrane domain, and an intracellular signaling domain. The CAR is preferably presented on the surface of a T cell in an orientation that enhances the affinity or avidity to a DNP-PLE, which may be associated with or attached to tumor cells, to a desired degree, or in an orientation that yields a desired affinity or avidity to this DNP-PLE.

[0096] In some embodiments, one or more nucleic acids encode the ligand-binding domain of one or more CARs described herein, the ligand-binding domain comprising an scFv domain containing a VH sequence and a VL sequence derived from an antibody. In some embodiments, these VH and VL sequences are linked via a linker. Table 1 shows examples of a series of CAR embodiments, and Table 2 shows the sequences of components of a particular CAR. Specifically, Table 2 shows examples of embodiments of the VH sequence, VL sequence, and linker encoded by one or more nucleic acids described herein. In some embodiments, one or more nucleic acids encoding the ligand-binding domain of one or more CARs described herein encode a ligand-binding domain comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 12. In some embodiments, the one or more nucleic acids encode a ligand-binding domain comprising the amino acid sequence shown in any of SEQ ID NOs: 1 to 12. In some embodiments, the one or more nucleic acids encode a ligand-binding domain comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NOs: 1, 2, 9, or 10. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0097] In some embodiments, one or more nucleic acids described herein include a short spacer having 2 to 12 consecutive amino acid residues. In some embodiments, one or more nucleic acids described herein encode a medium-length spacer having 2 to 119 consecutive amino acid residues. In some embodiments, one or more nucleic acids described herein encode a long spacer having more than 119 consecutive amino acid residues. In some embodiments, this long spacer has 2 to 229 consecutive amino acid residues. In some embodiments, the nucleic acids described herein encode a spacer selected from the group consisting of a short spacer containing an IgG4 hinge domain, a medium-length spacer containing an IgG4 hinge-CH3 domain, and a long spacer containing an IgG4 hinge-CH2-CH3 domain. In some embodiments, the nucleic acids described herein encode a long spacer.

[0098] In some embodiments, the transmembrane domain includes the transmembrane domain of CD28.

[0099] In some embodiments, the nucleic acids described herein encode an intracellular signaling domain comprising a portion of CD3ζ and / or a portion of 4-1BB.

[0100] Some embodiments further include a selection gene, a cell surface selection marker, or a polynucleotide encoding a cleavable linker. In some embodiments, the selection gene comprises a double mutant of dihydrofolate reductase (DHFRdm). In some embodiments, the cell surface selection marker is selected from the group consisting of cleaved EGFR (EGFRt), cleaved Her2 (Her2tG), and cleaved CD19 (CD19t). In some embodiments, the cleavable linker comprises a ribosome skip sequence selected from the group consisting of P2A, T2A, E2A, and F2A.

[0101] Some embodiments of the methods and compositions provided herein include a vector comprising one of the nucleic acids provided herein. In some embodiments, the vector includes a lentiviral vector.

[0102] Specific Car Some embodiments of the methods and compositions provided herein include a CAR that specifically binds to a DNP moiety. In some embodiments, the DNP moiety is associated with a PLE molecule. In some embodiments, the DNP moiety is associated with an antibody or folic acid. In some embodiments, the CAR is encoded by one or more nucleic acids provided herein.

[0103] In some embodiments, the ligand-binding domain of the CAR used in the present invention as described herein includes an scFv domain containing a VH sequence and a VL sequence derived from an antibody. In some embodiments, these VH and VL sequences are linked via a linker. Examples of VH sequences, VL sequences, and linkers used in one or more CARs as described herein are shown in Table 2. In some embodiments, the ligand-binding domain of the CAR described herein includes an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 12. In some embodiments, the ligand-binding domain of the CAR described herein includes an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NOs: 1 or 2. In some embodiments, the ligand-binding domain of the CAR described herein includes an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NOs: 1, 2, 9, or 10.

[0104] In some embodiments, the CAR described herein includes a short spacer having 2 to 12 consecutive amino acid residues. In some embodiments, the CAR described herein includes a medium-length spacer having 2 to 119 consecutive amino acid residues. In some embodiments, the CAR described herein includes a long spacer having more than 119 consecutive amino acid residues. In some embodiments, the CAR described herein includes a long spacer having 2 to 229 consecutive amino acid residues. In some embodiments, the CAR described herein includes a spacer selected from the group consisting of a short spacer containing an IgG4 hinge domain, a medium-length spacer containing an IgG4 hinge-CH3 domain, and a long spacer containing an IgG4 hinge-CH2-CH3 domain. In some embodiments, the CAR described herein includes a long spacer.

[0105] In some embodiments, the CAR described herein includes a transmembrane domain comprising the transmembrane domain of CD28. In some embodiments, the CAR described herein includes an intracellular signaling domain comprising a portion of CD3ζ and / or a portion of 4-1BB.

[0106] In some embodiments, the DNP portion is bound to an antibody or its antigen-binding fragment presented on a CAR, which may be presented by a cell (e.g., a T cell).

[0107] In some embodiments, the DNP portion is linked to a lipid. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group comprises a group selected from choline, phosphatidylcholine, phosphocholine, sphingomyelin, phosphoethanolamine, sugar residues, phosphatidylserine, phosphatidylinositol, piperidine, and trimethylarsenoethyl phosphate. In some embodiments, the hydrophobic group comprises a fatty acid chain or a terpenoid portion. In some embodiments, the hydrophobic group comprises an ether linkage, the ether linkage located between the polar head group and the fatty acid chain. In some embodiments, the fatty acid chain is C 10-20 It contains an alkyl chain. In some embodiments, the lipid is PLE. One embodiment of the DNP moiety linked to the lipid is shown in Figure 1.

[0108] Certain cells including CAR Some embodiments of the methods and compositions provided herein include cells containing any one of the CARs provided herein and / or cells containing any one of the nucleic acids encoding the CARs provided herein.

[0109] In some embodiments, the cells are derived from CD4+ T cells, CD8+ T cells, progenitor T cells, or hematopoietic stem cells. In some embodiments, the CD8+ T cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the central memory CD8+ T cells are CD45RO-positive and CD62L-positive. In some embodiments, the CD4+ T cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the naive CD4+ T cells are CD45RA-positive, CD62L-positive, and CD45RO-negative.

[0110] In some embodiments, the cells are ex vivo cells. In some embodiments, the cells are in vivo cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0111] Some embodiments of the methods and compositions provided herein involve preparing a cell population containing one or more of the CARs described herein. In some embodiments, the cell population containing the CARs described herein is incorporated into an infusion administered to a subject (e.g., a cancer patient) that requires this cell population. Some embodiments involve introducing one or more nucleic acids encoding the CARs or their components provided herein. Some embodiments further involve culturing the cells under conditions suitable for obtaining a sufficient number of cells for the infusion.

[0112] Specific treatments Some embodiments of the methods and compositions provided herein include methods for treating, suppressing, or mitigating cancer in a subject. Some such methods include the step of administering one of the anti-DNP CAR T cells provided herein to a subject, and may include the step of selecting a subject to receive such treatment based on a diagnostic evaluation, a clinical evaluation, or both. In some embodiments, the anti-DNP CAR T cells are administered to the subject in combination with a composition containing a DNP moiety, such as an antitumor antigen antibody containing a DNP moiety or an antigen-binding fragment thereof, or DNP-PLE.

[0113] In some embodiments, after administering the cells, an antitumor antigen antibody containing a DNP moiety, or its antigen-binding fragment, or a DNP moiety such as DNP-PLE, is administered. In some embodiments, after administering an antitumor antigen antibody containing a DNP moiety, or its antigen-binding fragment, or a DNP moiety such as DNP-PLE, the cells are administered. In some embodiments, an antitumor antigen antibody containing a DNP moiety, or its antigen-binding fragment, or a DNP moiety such as DNP-PLE, and the cells are administered simultaneously.

[0114] In some embodiments, compositions containing a DNP moiety (such as DNP-PLE) are configured to target cancer or are suitable for targeting cancer. In another embodiment, in some embodiments, the DNP moiety is linked to an antibody or its antigen-binding fragment that specifically binds to cancer.

[0115] The DNP portion is preferably linked to a lipid. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group comprises a group selected from choline, phosphatidylcholine, phosphocholine, sphingomyelin, phosphoethanolamine, sugar residues, phosphatidylserine, phosphatidylinositol, piperidine, and trimethylarsenoethyl phosphate. In some embodiments, the hydrophobic group comprises a fatty acid chain or a terpenoid portion. In some embodiments, the hydrophobic group comprises an ether linkage, the ether linkage located between the polar head group and the fatty acid chain. In some embodiments, the fatty acid chain is C 10-20 It contains an alkyl chain. In some embodiments, the lipid is PLE.

[0116] In some embodiments, the cancer includes target cells selected from the group consisting of breast cancer cells, brain tumor cells, colorectal cancer cells, kidney cancer cells, pancreatic cancer cells, and ovarian cancer cells.

[0117] In some embodiments, the anti-DNP CAR T cells are autologous cells obtained from the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0118] Specific compositions, systems, and kits Some embodiments of the methods and compositions provided herein include a composition comprising one of the anti-DNP CARs provided herein and a dinitrophenol (DNP) moiety attached to a target cell, wherein the CAR specifically binds to the DNP moiety.

[0119] In some embodiments, the DNP portion is attached to the target cell via an antibody or antigen-binding fragment that binds to the target cell.

[0120] In some embodiments, the DNP portion is attached to the surface of the target cell via a lipid incorporated into the surface of the target cell. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group comprises a group selected from choline, phosphatidylcholine, phosphocholine, sphingomyelin, phosphoethanolamine, sugar residues, phosphatidylserine, phosphatidylinositol, piperidine, and trimethylarsenoethyl phosphate. In some embodiments, the hydrophobic group comprises a fatty acid chain or a terpenoid portion. In some embodiments, the hydrophobic group comprises an ether linkage, the ether linkage located between the polar head group and the fatty acid chain. In some embodiments, the fatty acid chain is C 10-20 It contains an alkyl chain. In some embodiments, the lipid is PLE.

[0121] In some embodiments, the target cells are cancer cells. In some embodiments, the cancer cells are selected from the group consisting of breast cancer cells, brain tumor cells, colorectal cancer cells, kidney cancer cells, pancreatic cancer cells, and ovarian cancer cells. In some embodiments, the target cells are ex vivo cells. In some embodiments, the target cells are in vivo cells. In some embodiments, the target cells are mammalian cells. In some embodiments, the target cells are human cells.

[0122] Some embodiments of the methods and compositions provided herein include a system and / or kit comprising one nucleic acid encoding an anti-DNP CAR and a composition comprising a DNP moiety. In some embodiments, the DNP moiety is linked to an antibody or its antigen-binding fragment. In some embodiments, the DNP moiety is linked to a lipid. [Examples]

[0123] Example 1 - Anti-DNP antibody that binds to cells labeled with DNP-PLE Adenocarcinoma-derived MDA-MB-231 cells were incubated overnight in complete medium containing 50 nM, 500 nM, or 5 μM DNP-PLE. The integration of DNP-PLE into the cell membrane was analyzed by flow cytometry after contact of MDA-MB-231 cells with anti-DNP-Alexa Fluor 488 antibody. In control cells, no shift was observed between untreated and MDA-MB-231 cells stained with anti-DNP-Alexa Fluor 488 antibody (Figure 2A). In cells treated with 5 μM DNP-PLE, a clear shift from untreated control cells to treated cells was observed (Figure 2B). In cells treated with 50 nM DNP-PLE, the shift from untreated control cells to treated cells was smaller (Figure 2D). In cells treated with 500 nM DNP-PLE, the shift from untreated control cells to treated cells was intermediate between these levels (Figure 2C). Histograms of the data shown in Figures 2A to 2D are shown in Figure 2E.

[0124] In further studies, MDA-MB-231 cells were incubated overnight with 1 μM or 5 μM DNP-PLE, then washed and imaged using confocal microscopy to identify where the DNP-PLE was incorporated into the cells. The nuclei were stained with DAPI (i), the cell surface with wheat germ agglutinin (WGA) (ii), and DNP was stained with anti-DNP-Alexa Fluor 488 antibody (iii). Figure 3A shows a confocal image of MDA-MB-231 control cells incubated with anti-DNP-Alexa Fluor 488 antibody. Figure 3B shows a confocal image of MDA-MB-231 cells incubated with 5 μM DNP-PLE. Figure 3C shows a confocal image of MDA-MB-231 cells incubated with 5 μM DNP-PLE and stained with anti-DNP-Alexa Fluor 488 antibody. Figure 3D shows a confocal image of MDA-MB-231 cells incubated with 1 μM DNP-PLE and stained with anti-DNP-Alexa Fluor 488 antibody. Staining with the anti-DNP antibody was identified as localizing to the cell surface (iii), confirming that DNP-PLE was incorporated into the cell surface (Figures 3C and 3D). Furthermore, this study confirmed that the DNP portion is accessible by antibody binding.

[0125] Example 2 - Preparation of anti-DNP CAR Figure 4 shows the components of a polynucleotide encoding an anti-DNP CAR as an example. This anti-DNP CAR includes a leader sequence, a ligand-binding domain containing anti-DNP scFv, a spacer, a transmembrane domain of CD28, an intracellular costimulatory domain containing a 41BB domain and a CD3ζ domain, a P2A sequence, a selection marker containing DHFRdm, a T2A sequence, and a cell surface selection marker containing EGFRt.

[0126] A series of polynucleotides encoding various anti-DNP CARs are prepared. Some of these polynucleotides encode various CARs with different spacers, such as long spacers, medium-length spacers, and short spacers. Some of these polynucleotides also encode various CARs with different anti-DNP ligand-binding domains, for example, CARs with various scFvs derived from various anti-DNP antibodies (see, e.g., Brunger, AT, et al., (1991) Journal of Molecular Biology, 5:239-56 (this document is incorporated herein by reference in its entirety)). Some of the ligand-binding domains contain VH and VL sequences. Some of the ligand-binding domains also contain linker-linked VH and VL sequences. Table 1 shows the series of CARs to be prepared, and Table 2 shows the sequences of the components of these CARs. For example, the CAR containing a ligand-binding domain consisting of VH-linker-VL of Ab-1(1BAF) with a long spacer has a sequence from the NH terminus to the COOH terminus, Sequence ID 29 , GM-CSF signal sequence][ Sequence ID 1 VH of anti-DNP scFv(Ab-1;1BAF) Sequence ID 19 Linker Sequence ID 2 VL of anti-DNP scFv(Ab-1;1BAF)][ Sequence ID: 20 Long spacer: IgG4 hinge-CH2(L235D)-CH3][ Sequence ID 23 ,CD28tm][ Sequence ID 24 , 4-1BB][ Sequence ID 25 , CD3ζ][P2A nucleic acid][ Sequence ID 28 ,DHFRdm] and [ Sequence ID 28 It contains a polynucleotide encoding an amino acid sequence consisting of EGFRt linked to the signal sequence of the GM-CSF receptor.

[0127] Example 3 - Production of anti-DNP CAR T cells Polynucleotides encoding anti-DNP CARs with long spacers were transduced into H9 cells (CD4+CD3+ cutaneous T lymphocytes). Transduced cells were selected with methotrexate. Transduction efficiency was measured by quantitatively evaluating the presence or absence of EGFRt cell surface markers on transduced cells using flow cytometry. Flow cytometry plots showed an anti-DNP CAR H9 population with 92% positivity.

[0128] Example 4 - Anti-DNP CAR that binds to cells labeled with DNP-PLE MDA-MB-231 cells were incubated overnight with 5 μM DNP-PLE, washed, and co-cultured with anti-DNP CAR T cells. Cells were imaged using confocal microscopy to confirm the interaction between anti-DNP CAR T cells and DNP-labeled cells. The nuclei were stained with DAPI (i), the cell surface with wheat germ agglutinin (WGA) (ii), and DNP was stained with anti-DNP-Alexa Fluor 488 antibody (iii) and (iv). In addition, H9 CAR T cells and MDA-MB-231 cells were distinguished using anti-CD3 antibody (red). Below each color image, the layers that make up the entire confocal image, namely the nucleus (i), cell surface (ii), DNP-PLE (iii), and (iv) anti-DNP CAR H9 cells, are shown in grayscale.

[0129] Figure 5A shows unlabeled MDA-MB-231 control cells co-cultured with anti-DNP CAR H9 cells. In this figure, no binding was observed between MDA-MB-231 cells and H9 cells. The upper left image of Figure 5A shows a confocal image created by superimposing all images (i) to (iv). Figure 5B shows DNP-PLE labeled MDA-MB-231 cells co-cultured with anti-DNP CAR H9 cells. In this figure, interaction was observed between MDA-MB-231 cells and H9 cells. The upper left image of Figure 5B shows a confocal image created by superimposing all images (i) to (iv). In Figure 5B, synapse formation between cells was shown, confirming that DNP exposed on the surface of target cells was recognized by anti-DNP CAR. This study confirmed that anti-DNP CAR can bind to DNP-labeled cells.

[0130] Example 5 - In vitro activity of anti-DNP CAR Chromium-releasing assays and cytokine production assays are used to measure the activity of anti-DNP CARs in vitro. See, for example, Gonzalez, S., Naranjo, A., Serrano, LM, Chang, W.-C., Wright, CL, & Jensen, MC (2004). Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma. The Journal of Gene Medicine, 6(6), 704-711 (this document is explicitly incorporated herein by citation).

[0131] To perform a chromium release assay, target cells 51 Incubate overnight with Cr. For target cells to be labeled with DNP-PLE, add DNP-PLE to the culture medium. 51 Incubate overnight with Cr. The next day, wash the target cells and seed them in a 96-well plate at a concentration of 5000 cells / well. As effector cells, wash CD8+ anti-DNP CAR T cells and mock T cells (usually from 8-16 days of rapid expansion culture) and seed them in triplets with the target cells at various E:T ratios (30:1, 10:1, 3:1, 1:1) and co-culture at 37°C for 4 hours. Also, as a control... 51 To evaluate Cr release, each target cell line is seeded in a culture medium without any additives. Furthermore, 51 To evaluate the maximum release of Cr, each target cell line is seeded and lysed with 2% SDS. The control group is operated in a 6-cell chain. After co-culturing, the supernatant is collected, seeded on a LUMA plate, and dried overnight. The next day, the sample is measured using a Top Count scintillation counter. The specific lysis rate is calculated using the following formula.

number

[0132] The chromium release assay measures the relative lytic activity levels of various anti-DNP CAR T cells against DNP-labeled cells. Unlabeled K562 control cells do not induce lysis when in contact with anti-DNP CAR T cells. OKT3 cells, which activate T cells via the TCR, are used as a positive control. Anti-DNP CAR T cells induce specific lysis of DNP-labeled cells.

[0133] Perform a cytokine release assay. For target cells to be labeled with DNP-PLE, add DNP-PLE to the culture medium and incubate overnight. The next day, harvest all target cells, wash them, and set them to 5 × 10⁴ 4 Seed in a 96-well plate at a concentration of cells / well. Wash CD8+ anti-DNP CAR T cells and mock T cells (usually from 8-16 days of rapid expansion culture) as effector cells and seed them together with the target cells (1 × 10⁶). 5 Co-culture cells (individual cells / well) at 37°C for 24 hours. After 24 hours, collect the supernatant and measure the concentrations of IFN-γ, TNF-α, and IL-2 in the supernatant using the Bio-Plex® 200 system (Bio-Rad). Measure the amount of cytokines released by anti-DNP CAR T cells. In DNP-labeled cells, contact with anti-DNP CAR T cells induces the release of IFN-γ, IL-2, and TNF-α.

[0134] Example 6 - In vivo targeting with DNP-PLE and integration of DNP-PLE This study investigates in vivo targeting and uptake of DNP-PLE in tumor sites. Glioblastoma cells (U87 cells) are established in mice via intracranial injection, followed by intravenous injection of DNP-PLE into the mice. Mice are sacrificed and their brains are extracted at various time points after DNP-PLE injection. Specifically, mice with orthotopic xenografts of glioblastoma are intravenously administered DNP-PLE, and their brains are evaluated for 14 days. Brain tissue sections are prepared after 48 hours. Nuclei are stained with DAPI. Further staining is performed using a fluorescently labeled anti-DNP antibody to examine whether DNP-PLE integrated into the cell membrane is available for binding. Glioblastoma cells hold an excess amount of DNP-PLE compared to the contralateral hemisphere without the same tumor. Fluorescence imaging shows tumors to be significantly brighter than normal healthy tissue. These results confirm that DNP-PLE is selectively taken up by the tumor cell membrane, and that the DNP portion is available for binding.

[0135] Similar studies were conducted by injecting adenocarcinoma cells (MDA-MB-231) or osteosarcoma cells (143B) into the flanks of mice to establish tumors. In each mouse group, after tumor establishment by subcutaneous injection, DNP-PLE was intravenously injected into the mice. Mice were sacrificed and tumors were excised at various time points after DNP-PLE injection. The tumors were collected and rapidly imaged using a fluorescently labeled anti-DNP antibody to confirm the presence or absence of DNP-PLE. It was shown that DNP-PLE was specifically taken up in all three types of cancer and that DNP-PLE was retained for several days.

[0136] Example 7 - In vivo activity of anti-DNP CAR The activity of anti-DNP CAR in vivo will be measured using a xenograft model. Neuroblastoma (Be2) or glioma (U87, U251T, or T98) will be established by intracranial injection into mice. Mice will be intracranially injected with DNP-PLE and anti-DNP CAR T cells. The control group will be intracranially injected with anti-DNP CAR T cells only. Mice that received intracranial injection of anti-DNP CAR T cells in combination with intracranial injection of DNP-PLE will have increased survival rates, reduced tumor burden over time, and / or decreased tumor volume compared to the control group.

[0137] A similar study was conducted by injecting adenocarcinoma cells (MDA-MB-231) into the flanks of mice to establish tumors. In each mouse group other than the control group, after establishing tumors by subcutaneous injection, DNP-PLE was intravenously injected into the mice. Next, anti-DNP CAR T cells were intravenously administered to the mice. Mice that received intravenous injection of DNP-PLE showed increased survival rates, reduced tumor burden over time, and / or decreased tumor volume compared to the control group.

[0138] As used herein, the term “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is open-ended and comprehensive, not excluding any further elements or processes not described herein.

[0139] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention may be modified, as may the manufacturing methods and apparatus. Such modifications will be readily apparent to those skilled in the art, taking into account the practices of the present invention disclosed herein or the present disclosure. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but encompasses any possible modifications and other embodiments within the true scope and spirit of the invention.

[0140] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and academic literature, are incorporated herein by reference in their entirety and constitute part of this Specified. If any cited document, patent, or patent application conflicts with any disclosure herein, the provisions of this Specified shall prevail and / or take precedence over such conflict.

Claims

1. A system including (i) and (ii) below: (i) Effector cells comprising a chimeric antigen receptor (CAR) or a nucleic acid encoding a CAR, wherein the CAR A ligand-binding domain that specifically binds to the dinitrophenol (DNP) moiety; Spacer; Transmembrane domain; and Intracellular signal transduction domains Includes, Effector cells in which the ligand-binding domain comprises a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 2, and the spacer comprises an IgG4 hinge-CH2-CH3 domain containing the amino acid sequence shown in SEQ ID NO: 20; (ii) A composition comprising a DNP moiety covalently linked to a phospholipid, wherein the phospholipid is incorporated into the cell membrane of a cancer cell.

2. The system according to claim 1, wherein the ligand-binding domain comprises the amino acid sequence shown in SEQ ID NO: 9 or 10.

3. The system according to claim 1 or 2, wherein the transmembrane domain comprises the transmembrane domain of CD28, and the intracellular signaling domain comprises a portion of CD3ζ and / or a portion of 4-1BB.

4. The system according to any one of claims 1 to 3, wherein the transmembrane domain comprises a transmembrane domain of CD28 consisting of the amino acid sequence shown in SEQ ID NO: 23, and the intracellular signaling domain comprises a portion of CD3ζ consisting of the amino acid sequence shown in SEQ ID NO: 25 and / or a portion of 4-1BB consisting of the amino acid sequence shown in SEQ ID NO:

24.

5. The system according to any one of claims 1 to 4, wherein the transmembrane domain comprises a transmembrane domain of CD28 consisting of the amino acid sequence shown in SEQ ID NO: 23, and the intracellular signaling domain comprises a part of CD3ζ consisting of the amino acid sequence shown in SEQ ID NO: 25 and a part of 4-1BB consisting of the amino acid sequence shown in SEQ ID NO:

24.

6. The system according to any one of claims 1 to 5, wherein the nucleic acid further comprises a polynucleotide encoding at least one selected from (i) to (iii) below: (i) Double mutant of dihydrofolate reductase (DHFRdm); (ii) A cell surface selective marker selected from the group consisting of cleaved EGFR (EGFRt), cleaved Her2 (Her2tG), and cleaved CD19 (CD19t); (iii) A cleavable linker comprising a ribosome skip sequence selected from the group consisting of P2A, T2A, E2A, and F2A.

7. The system according to any one of claims 1 to 6, wherein the effector cells are (i) CD4+ T cells or CD8+ T cells, (ii) cells derived from CD4+ T cells or CD8+ T cells, or (iii) progenitor T cells or hematopoietic stem cells.

8. The system according to claim 7, wherein the CD8+ T cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells.

9. The system according to claim 8, wherein the central memory CD8+ T cells are CD45RO-positive and CD62L-positive.

10. The system according to claim 7, wherein the CD4+ T cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

11. The system according to claim 10, wherein the naive CD4+ T cells are CD45RA-positive, CD62L-positive, and CD45RO-negative.

12. The system according to any one of claims 1 to 11, wherein the phospholipid is an ether phospholipid (PLE).

13. Effector cells for the treatment or palliative care of the target cancer, The effector cells are used to be administered to a target in combination with a composition containing a dinitrophenol (DNP) moiety covalently linked to a phospholipid, characterized in that the phospholipid is incorporated into the cell membrane of the cancer cells. The effector cells contain a chimeric antigen receptor (CAR), and the CAR is A ligand-binding domain that specifically binds to the aforementioned DNP portion; Spacer; Transmembrane domain; and Intracellular signal transduction domains Includes, Effector cells wherein the ligand-binding domain comprises a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 2, and the spacer comprises an IgG4 hinge-CH2-CH3 domain containing the amino acid sequence shown in SEQ ID NO:

20.

14. The effector cell according to claim 13, wherein the phospholipid is ether phospholipid (PLE).

15. The effector cell according to claim 13 or 14, wherein the ligand-binding domain comprises the amino acid sequence shown in SEQ ID NO: 9 or 10.

16. The effector cell according to any one of claims 13 to 15, wherein the transmembrane domain comprises a transmembrane domain of CD28 consisting of the amino acid sequence shown in SEQ ID NO: 23, and the intracellular signaling domain comprises a portion of CD3ζ consisting of the amino acid sequence shown in SEQ ID NO: 25 and / or a portion of 4-1BB consisting of the amino acid sequence shown in SEQ ID NO:

24.

17. The effector cell according to any one of claims 13 to 16, wherein the transmembrane domain comprises a transmembrane domain of CD28 consisting of the amino acid sequence shown in SEQ ID NO: 23, and the intracellular signaling domain comprises a part of CD3ζ consisting of the amino acid sequence shown in SEQ ID NO: 25 and a part of 4-1BB consisting of the amino acid sequence shown in SEQ ID NO:

24.

18. The effector cell according to any one of claims 13 to 17, wherein (i) a CD4+ T cell or a CD8+ T cell, (ii) a cell derived from a CD4+ T cell or a CD8+ T cell, or (iii) a progenitor T cell or a hematopoietic stem cell.

19. The effector cell according to claim 18, wherein the CD8+ T cell is a CD8+ cytotoxic T lymphocyte selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells.

20. The effector cell according to claim 19, wherein the central memory CD8+ T cell is CD45RO positive and CD62L positive.

21. The effector cell according to claim 18, wherein the CD4+ T cell is a CD4+ helper T lymphocyte selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

22. The effector cells according to claim 21, wherein the naive CD4+ T cells are CD45RA-positive, CD62L-positive, and CD45RO-negative.

23. Effector cells according to any one of claims 13 to 22, which are autologous cells obtained from the subject mentioned above.

24. The effector cell according to any one of claims 13 to 23, characterized in that the effector cell is administered to the subject after the composition has been administered to the subject.

25. The effector cell according to any one of claims 13 to 23, characterized in that the composition is administered to the subject after the effector cell is administered to the subject.

26. The effector cells according to any one of claims 13 to 25, wherein the cancer is selected from the group consisting of breast cancer cells, brain tumor cells, colorectal cancer cells, kidney cancer cells, pancreatic cancer cells, and ovarian cancer cells.

27. The use of effector cells in the manufacture of therapeutic or palliative drugs for the target cancer, The aforementioned pharmaceutical is used to administer to the subject a combination of the effector cells and a dinitrophenol (DNP) moiety covalently linked to a phospholipid, characterized in that the phospholipid is incorporated into the cell membrane of the cancer cells. The effector cells contain a chimeric antigen receptor (CAR), and the CAR is A ligand-binding domain that specifically binds to the aforementioned DNP portion; Spacer; Transmembrane domain; and Intracellular signal transduction domains Includes, The ligand-binding domain comprises a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 2, and the spacer comprises an IgG4 hinge-CH2-CH3 domain containing the amino acid sequence shown in SEQ ID NO: 20.

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