Methods for stimulating chimeric antigen receptor T cells with hapten-labeled cells and compositions therefor

Abstract

Some embodiments of the methods and compositions provided herein relate to the use of hapten-labeled cells to stimulate chimeric antigen receptor (CAR) T cells. In some embodiments, the CAR T cells may comprise a CAR that specifically binds to a hapten. Some embodiments relate to the stimulation of CAR T cells with hapten-labeled cells in vivo or in vitro.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 969,917, filed February 4, 2020, entitled "Methods for Stimulating Chimeric Antigen Receptor T Cells with Hapten-Labeled Cells and Compositions Therefor," which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application has been filed with an electronic Sequence Listing, which has been provided as an approximately 43 kb file created on February 1, 2021, under the file name SCRI272WOSEQLIST. The information set forth in this electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Some embodiments of the methods and compositions provided herein relate to the use of hapten-labeled cells to stimulate chimeric antigen receptor (CAR) T cells. In some embodiments, the CAR T cells comprise a CAR that specifically binds to a hapten. Furthermore, some embodiments relate to the stimulation of CAR T cells by hapten-labeled cells in vivo or in vitro. [Background technology]

[0004] Immunotherapy using adoptive cell transfer (ACT) of T cells bearing chimeric antigen receptors (CARs) has previously been reported to be useful for cancer treatment. The structure of a CAR includes an antigen-binding domain, a linker and spacer sequence, a costimulatory activation domain, and a transmembrane region. CAR-expressing cells can be derived from the patient in need of treatment or from donor cells (related or unrelated). CARs act by binding to specific proteins or antigens on cells or tumor cells. Once infused into a patient, the engineered CAR T cells further proliferate in the patient's body, recognize cancer or tumor cells bearing specific proteins or antigens on their surface, and kill these cells.

[0005] It is desirable for CAR T cells to maintain their efficacy over a long period of time. As hematologic cancers reach the final stage of regression, the number of cancer cells decreases, resulting in a lower antigen load, causing the CAR T cell population to shrink and lose its efficacy. Furthermore, solid tumors exhibit a highly immunosuppressive environment within the tumor. Therefore, further stimulation of CAR T cells may be necessary to complete treatment by eliminating remaining cancer cells. Furthermore, stimulation and restimulation of CAR T cells can overcome the immunosuppressive tumor environment.

[0006] Stimulation and restimulation of CAR T cells have been reported previously. For example, stimulation of CAR T cells can be performed in vitro by adding anti-CD3 / CD28 beads before infusion into patients. The embodiments provided herein describe a new approach for stimulating CAR T cells in vivo and in vitro. Summary of the Invention [Means for solving the problem]

[0007] Some embodiments of the methods and compositions provided herein include a method of inducing the expansion of chimeric antigen receptor (CAR) T cells, comprising incubating CAR T cells with hapten-antigen-presenting cells (H-APCs) and allowing the CAR of the CAR T cells to specifically bind to the hapten bound to the H-APCs. In some embodiments, the CAR T cells and the H-APCs are derived from a single subject, such as a mammal (preferably a human).

[0008] Some embodiments of the methods and compositions provided herein include a method of treating, inhibiting, or alleviating cancer in a subject, comprising: administering to a subject an effective amount of CAR T cells having a chimeric antigen receptor (CAR) that specifically binds to a tumor-specific antigen of the cancer; and Incubating the CAR T cells with hapten-antigen-presenting cells (H-APCs) and inducing expansion of the CAR T cells by specifically binding the CAR of the CAR T cells to the hapten bound to the H-APCs. The method includes: In some embodiments, the CAR T cells and the H-APCs are derived from the subject, such as a human.

[0009] In some embodiments, the CAR T cells comprise a bispecific CAR.

[0010] In some embodiments, the CAR T cells comprise two or more CARs.

[0011] In some embodiments, the CAR T cell comprises a first ligand-binding domain capable of specifically binding to a tumor-specific antigen and a second ligand-binding domain capable of specifically binding to the hapten.

[0012] In some embodiments, the CAR T cell comprises a monospecific CAR, hi some embodiments, the CAR comprises a single ligand-binding domain capable of specifically binding to a tumor-specific antigen and the hapten.

[0013] In some embodiments, the incubation is performed in vitro.

[0014] In some embodiments, the incubation occurs in vivo.

[0015] In some embodiments, the CAR specifically binds to a tumor-specific antigen, which in some embodiments is selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B-cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epithelial cell adhesion molecule (EpCAM), epidermal growth factor variant III, receptor tyrosine kinase-like orphan receptor 1 (ROR1), CD123, prostate stem cell antigen (PSCA), CD5, Lewis Y antigen, B7H3, CD20, CD43, HSP90, and IL13.

[0016] In some embodiments, the hapten is selected from the haptens listed in Table 1, or the ligand-binding domain comprises a binding fragment of an antibody selected from an antibody to a hapten listed in Table 1 and an antibody listed in Table 2, or a sequence selected from Table 3, or the CAR comprises one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the hapten is selected from fluorescein and dinitrophenol, and derivatives thereof.

[0017] In some embodiments, the hapten is covalently bound to the extracellular surface of the H-APC, hi some embodiments, the hapten is bound to the H-APC via an ether phospholipid (PLE).

[0018] In some embodiments, the CAR T cells are derived from CD4+ cells or CD8+ cells.

[0019] In some embodiments, the CD8+ 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 CD8+ cells are CD8+ cytotoxic T lymphocytes, and the CD8+ cytotoxic T lymphocytes are central memory T cells, and the central memory T cells are CD45RO+, CD62L+, and CD8+.

[0020] In some embodiments, the CD4+ 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 CD4+ helper lymphocytes are naive CD4+ T cells, which are CD45RA+, CD62L+, CD4+, and CD45RO-.

[0021] In some embodiments, the CAR T cells are derived from progenitor T cells. In some embodiments, the CAR T cells are derived from hematopoietic stem cells.

[0022] In some embodiments, the H-APC is derived from a cell selected from the group consisting of a T cell and a B cell.

[0023] In some embodiments, the subject is a mammal, such as a livestock animal or domestic animal, hi some embodiments, the subject is a human.

[0024] Some embodiments of the methods and compositions provided herein include a composition comprising one or more nucleic acids encoding a first chimeric antigen receptor (CAR) and a second chimeric antigen receptor (CAR), the one or more nucleic acids comprise a first sequence encoding a first CAR and a second sequence encoding a second CAR; the first CAR comprises a first ligand-binding domain specific for a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain; the second CAR comprises a second ligand-binding domain specific for a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain; The composition includes:

[0025] In some embodiments, the first ligand-binding domain specifically binds to an antigen selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B-cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epithelial cell adhesion molecule (EpCAM), epidermal growth factor variant III, receptor tyrosine kinase-like orphan receptor 1 (ROR1), CD123, prostate stem cell antigen (PSCA), CD5, Lewis Y antigen, B7H3, CD20, CD43, HSP90, and IL13.

[0026] In some embodiments, the hapten is selected from the haptens listed in Table 1, or the ligand-binding domain comprises a binding fragment of an antibody selected from an antibody to a hapten listed in Table 1 and an antibody listed in Table 2, or a sequence selected from Table 3, or the CAR comprises one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the hapten is selected from fluorescein and dinitrophenol, and derivatives thereof.

[0027] In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain comprises an antibody or binding fragment thereof or an scFv. In some embodiments, the second ligand-binding domain comprises a binding fragment of an antibody selected from an antibody against a hapten listed in Table 1 and an antibody listed in Table 2, or a sequence selected from Table 3, or the CAR comprises one or more sequences listed in Table 4.

[0028] In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer is 1 to 24 amino acids in length, 25 to 50 amino acids in length, 51 to 75 amino acids in length, 76 to 100 amino acids in length, 101 to 125 amino acids in length, 126 to 150 amino acids in length, 151 to 175 amino acids in length, 176 to 200 amino acids in length, 201 to 225 amino acids in length, 226 to 250 amino acids in length, or 251 to 275 amino acids in length.

[0029] In some embodiments, the nucleic acid further comprises a leader sequence.

[0030] In some embodiments, the first intracellular signaling domain and / or the second intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ, or both.

[0031] In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB.

[0032] Some embodiments further comprise a sequence encoding a marker sequence, hi some embodiments, the marker is EGFRt, CD19t, or Her2tG.

[0033] In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises the transmembrane domain of CD28.

[0034] In some embodiments, the one or more nucleic acids further comprise a sequence encoding a cleavable linker.

[0035] In some embodiments, the linker is a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is P2A, T2A, E2A, or F2A.

[0036] Some embodiments of the methods and compositions provided herein include vectors that include compositions according to certain embodiments provided herein.

[0037] Some embodiments of the methods and compositions provided herein include a composition comprising one or more nucleic acids encoding a first chimeric antigen receptor (CAR) and a second chimeric antigen receptor (CAR), a first nucleic acid comprising a first sequence encoding a first CAR and a second nucleic acid comprising a second sequence encoding a second CAR; the first CAR comprises a first ligand-binding domain specific for a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain; the second CAR comprises a second ligand-binding domain specific for a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain; The composition includes:

[0038] In some embodiments, the first ligand-binding domain specifically binds to an antigen selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B-cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epithelial cell adhesion molecule (EpCAM), epidermal growth factor variant III, receptor tyrosine kinase-like orphan receptor 1 (ROR1), CD123, prostate stem cell antigen (PSCA), CD5, Lewis Y antigen, B7H3, CD20, CD43, HSP90, and IL13.

[0039] In some embodiments, the hapten is selected from the haptens listed in Table 1, or the ligand-binding domain comprises a binding fragment of an antibody selected from an antibody to a hapten listed in Table 1 and an antibody listed in Table 2, or a sequence selected from Table 3, or the CAR comprises one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the hapten is selected from fluorescein and dinitrophenol, and derivatives thereof.

[0040] In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain comprises an antibody or binding fragment thereof or an scFv. In some embodiments, the second ligand-binding domain comprises a binding fragment of an antibody selected from an antibody against a hapten listed in Table 1 and an antibody listed in Table 2, or a sequence selected from Table 3, or the CAR comprises one or more sequences listed in Table 4.

[0041] In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer is 1 to 24 amino acids in length, 25 to 50 amino acids in length, 51 to 75 amino acids in length, 76 to 100 amino acids in length, 101 to 125 amino acids in length, 126 to 150 amino acids in length, 151 to 175 amino acids in length, 176 to 200 amino acids in length, 201 to 225 amino acids in length, 226 to 250 amino acids in length, or 251 to 275 amino acids in length.

[0042] In some embodiments, the one or more nucleic acids further comprise a leader sequence.

[0043] In some embodiments, the first intracellular signaling domain and / or the second intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ, or both.

[0044] In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB.

[0045] Some embodiments further comprise a sequence encoding a marker sequence, hi some embodiments, the marker is EGFRt, CD19t, or Her2tG.

[0046] In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises the transmembrane domain of CD28.

[0047] In some embodiments, the nucleic acid further comprises a sequence encoding a cleavable linker.

[0048] In some embodiments, the linker is a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is P2A, T2A, E2A, or F2A.

[0049] Some embodiments of the methods and compositions provided herein include or utilize multiple (e.g., two) vectors that contain one or more nucleic acids according to any of the embodiments provided herein.

[0050] Some embodiments of the methods and compositions provided herein include compositions comprising one or more nucleic acids encoding a bispecific chimeric antigen receptor (CAR), wherein the one or more nucleic acids comprise a sequence encoding a first ligand-binding domain specific for a tumor antigen, a glycine-serine linker, a second ligand-binding domain specific for a hapten, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain.

[0051] In some embodiments, the first ligand-binding domain specifically binds to an antigen selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B-cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epithelial cell adhesion molecule (EpCAM), epidermal growth factor variant III, receptor tyrosine kinase-like orphan receptor 1 (ROR1), CD123, prostate stem cell antigen (PSCA), CD5, Lewis Y antigen, B7H3, CD20, CD43, HSP90, and IL13.

[0052] In some embodiments, the hapten is selected from the haptens listed in Table 1, or the ligand-binding domain comprises a binding fragment of an antibody selected from an antibody to a hapten listed in Table 1 and an antibody listed in Table 2, or a sequence selected from Table 3, or the CAR comprises one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the hapten is selected from fluorescein and dinitrophenol, and derivatives thereof.

[0053] In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain comprises an antibody or binding fragment thereof or an scFv. In some embodiments, the second ligand-binding domain comprises a binding fragment of an antibody selected from an antibody against a hapten listed in Table 1 and an antibody listed in Table 2, or a sequence selected from Table 3, or the CAR comprises one or more sequences listed in Table 4.

[0054] In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer is 1 to 24 amino acids in length, 25 to 50 amino acids in length, 51 to 75 amino acids in length, 76 to 100 amino acids in length, 101 to 125 amino acids in length, 126 to 150 amino acids in length, 151 to 175 amino acids in length, 176 to 200 amino acids in length, 201 to 225 amino acids in length, 226 to 250 amino acids in length, or 251 to 275 amino acids in length.

[0055] In some embodiments, the one or more nucleic acids further comprise a leader sequence.

[0056] In some embodiments, the intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or CD83, or the cytoplasmic domain of CD3ζ, or both. In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB.

[0057] Some embodiments further comprise a sequence encoding a marker sequence, hi some embodiments, the marker is EGFRt, CD19t, or Her2tG.

[0058] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28.

[0059] Some embodiments of the methods and compositions provided herein include a vector for expressing a bispecific CAR, which includes one or more nucleic acids according to any of the embodiments provided herein.

[0060] Some embodiments of the methods and compositions provided herein include a bispecific chimeric antigen receptor encoded by one or more nucleic acids according to any of the embodiments provided herein or a vector according to any of the embodiments provided herein.

[0061] Some embodiments of the methods and compositions provided herein include cells comprising one or more nucleic acids according to any of the embodiments provided herein, one or more vectors according to any of the embodiments herein, or a bispecific chimeric antigen receptor according to any of the embodiments herein.

[0062] 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 CD8+ cytotoxic T lymphocytes are central memory T cells, and the central memory T cells are CD45RO+, CD62L+, and CD8+.

[0063] 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. In some embodiments, the cells are naive CD4+ T cells, which are CD45RA+, CD62L+, CD4+, and CD45RO-.

[0064] In some embodiments, the cells are progenitor T cells. In some embodiments, the cells are hematopoietic stem cells.

[0065] Some embodiments of the methods and compositions provided herein include a method of generating a cell that expresses a first chimeric antigen receptor specific for a hapten and a second chimeric antigen receptor specific for a tumor antigen, the method comprising: introducing one or more nucleic acids according to any of the embodiments provided herein, or one or more vectors according to any of the embodiments provided herein, into a cell under conditions in which the first chimeric antigen receptor and the second chimeric antigen receptor are expressed. The method includes:

[0066] 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 CD8+ cytotoxic T lymphocytes are central memory T cells, and the central memory T cells are CD45RO+, CD62L+, and CD8+.

[0067] 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. In some embodiments, the CD4+ helper lymphocytes are naive CD4+ T cells, which are CD45RA+, CD62L+, CD4+, and CD45RO-.

[0068] In some embodiments, the cells are progenitor T cells. In some embodiments, the cells are hematopoietic stem cells.

[0069] Some embodiments of the methods and compositions provided herein include a method of generating a cell that expresses a bispecific chimeric antigen receptor specific for a hapten and a tumor antigen, the method comprising: introducing one or more nucleic acids according to certain embodiments provided herein, or one or more vectors according to certain embodiments provided herein, into a cell under conditions in which the first chimeric antigen receptor and the second chimeric antigen receptor are expressed. The method includes:

[0070] 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 CD8+ cytotoxic T lymphocytes are central memory T cells, and the central memory T cells are CD45RO+, CD62L+, and CD8+.

[0071] 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. In some embodiments, the CD4+ helper lymphocytes are naive CD4+ T cells, which are CD45RA+, CD62L+, CD4+, and CD45RO-.

[0072] In some embodiments, the cells are progenitor T cells. In some embodiments, the cells are hematopoietic stem cells.

[0073] Some embodiments of the methods and compositions provided herein include a method of stimulating or restimulating chimeric antigen receptor (CAR)-bearing T cells in a subject (preferably a human) with a disease, such as cancer, comprising: providing or administering to said subject a cell according to any of the specific embodiments provided herein; monitoring said subject for suppression of said disease; and providing hapten antigen-presenting cells (H-APCs) to said subject. Including, The subject may be selected or identified for CAR T cell therapy using CAR T cells having a receptor specific for an antigen associated with the disease (such as a tumor antigen). The method includes: Such selection or identification can be made by clinical or diagnostic evaluation, or both.

[0074] In some embodiments, the H-APCs are cells generated by labeling healthy cells of the subject with a hapten ex vivo.

[0075] In some embodiments, the hapten is selected from the haptens listed in Table 1.

[0076] In some embodiments, the monitoring step and the providing or administering step are performed repeatedly.

[0077] In some embodiments, the subject has cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the subject, such as a human, is a subject selected or identified for cancer therapy, e.g., by clinical or diagnostic evaluation, or both. In some embodiments, the subject, such as a human, is administered a combination therapy, such as chemotherapy or radiation therapy.

[0078] Some embodiments of the methods and compositions provided herein include a method of stimulating or restimulating T cells bearing a chimeric antigen receptor (CAR) ex vivo, comprising: providing a cell according to certain embodiments provided herein; providing hapten antigen-presenting cells (H-APCs) or haptens; mixing the cells with the H-APC cells to obtain activated cells; and isolating the activated cells The method includes: In some embodiments, the hapten is selected from the haptens listed in Table 1. In some embodiments, the H-APCs comprise a hapten selected from the haptens listed in Table 1. In some embodiments, the isolation of activated cells comprises affinity isolation by conjugating the hapten to affinity beads. In some embodiments, the isolation of activated cells comprises affinity isolation by conjugating EGFRt, CD19t, or Her2tG to affinity beads. [Brief explanation of the drawings]

[0079] [Figure 1A] Schematic diagrams of three types of chimeric antigen receptors (CARs). Panel (1) shows a second-generation CAR with an antigen recognition moiety (i) displayed at a desired distance from the cell surface via a spacer domain (ii). This spacer is linked to a transmembrane domain (iii), which is linked to two signaling domains (iv and v). Panel (2) shows a CAR with an extended, long spacer. This CAR has a different antigen recognition moiety (vi) and a long spacer domain (vii) from the CAR in panel (i). Panel (3) shows a bispecific CAR containing two linked antigen recognition domains. This CAR can be activated by recognition of either epitope.

[0080] [Figure 1B] FIG. 1 is a schematic diagram of a CAR T cell containing two CARs (dual CAR T cell).

[0081] [Figure 1C] Schematic diagram of a CAR T cell containing a bispecific CAR. Bispecific CAR T cells express a single CAR capable of recognizing two epitopes.

[0082] [Figure 2]

[0023] Figure 1C is a schematic diagram illustrating an example of a therapeutic embodiment. Hapten antigen-presenting cells (H-APCs) are generated by carrying a hapten on the surface of healthy cells. The generated H-APCs are then infused into patients. Dual CAR T cells and bispecific CAR T cells (Figure 1C) can be activated via tumor cell recognition or H-APCs. One CAR (i) shown in the diagram is designed to target epitope (ii) on tumor cells, while the other CAR (iii) is engineered to recognize hapten (iv) on the hapten-APCs. Hapten-APCs are generated by carrying a hapten on the surface of healthy cells. The hapten-APCs are then reinfused into the patient, where they can be recognized and lysed by CAR T cells, activating the CAR T cells. If the hapten-APCs are not lysed by CAR T cells, the hapten is metabolized and reverts to normal healthy cells. It should be noted that in some embodiments, a single anti-hapten CAR T cell is used, for example, when labeling tumor cells with the same hapten used to label the hapten-APCs.

[0083] [Figure 3A] The structure of fluorescein-linked ether phospholipid (FL-PLE) as a hapten is shown, which contains (i) a fluorescein moiety, (ii) a polyethylene glycol (PEG) moiety that functions as a spacer to extend the hapten from the cell surface, (iii) a polar head group, and (iv) a hydrophobic tail that integrates into the cell membrane.

[0084] [Figure 3B] The structure of N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (FL-DHPE) is shown.

[0085] [Figure 3C]The structure of N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (BODIPY-DHPE) containing BODIPY as the hapten is shown.

[0086] [Figure 4A] The results of flow cytometry performed after incubating CD19+ Raji cells with FL-DHPE or anti-CD19-FITC antibody are shown.

[0087] [Figure 4B] The results of flow cytometry performed after incubating K562 cells with 0.5 μM or 5 μM FL-PLE are shown.

[0088] [Figure 4C] The results of flow cytometry performed after incubating Be2 cells, U87 cells, or Daoy cells with 5 μM FL-PLE are shown.

[0089] [Figure 5A] 1 shows one embodiment of confocal images of U87 cells stained with DAPI after incubation with 5 μM FL-PLE.

[0090] [Figure 5B] 1 shows one embodiment of confocal images of U87 cells stained with DAPI and Alexa Fluor 647 fluorochrome-labeled anti-fluorescein antibody after incubation with 5 μM FL-PLE.

[0091] [Figure 6A] The results show that signal retention was measured over time after incubating Be2 cells or U87 cells with 5 μM FL-DHPE.

[0092] [Figure 6B]The results show that Be2 cells or U87 cells were incubated with 5 μM FL-PLE, and then signal retention was measured over time.

[0093] [Figure 7A] Figure 1 is a series of graphs showing the results of a cytotoxicity assay. A chromium release assay was used to test the lytic ability of two anti-FL CAR T cells (4M5.3 and FITC-E2) against hapten-labeled cells. To generate hapten-labeled cells, CD19+ K562 cells were incubated with 5 μM FL-DHPE or stained with anti-CD19-FITC antibody. OKT3 cells were used as a positive control to intrinsically activate T cells via the TCR.

[0094] [Figure 7B]

[0023] Figure 1 is a series of graphs showing the results of a cytokine release assay measuring cytokine production. Two anti-FL CAR T cells (4M5.3 and FITC-E2) were tested against hapten-labeled cells generated by incubating CD19+ K562 cells with 5 μM FL-DHPE or anti-CD19-FITC antibody.

[0095] [Figure 8A] The results of flow cytometry performed after incubating K562 cells with 0.5 μM or 5 μM FL-PLE are shown.

[0096] [Figure 8B]

[0023] Figure 1 is a series of graphs showing the results of a cytotoxicity assay performed by incubating K562 cells with 0.5 μM or 5 μM FL-PLE to generate hapten-labeled cells, and then incubating the hapten-labeled cells with anti-FL CAR T cells. K562 parental cells were used as a negative control, and K562 + OKT3 cells were used as a positive control.

[0097] [Figure 8C]This is a series of graphs showing the results of measuring cytokine production in a cytokine release assay performed by incubating K562 cells with 0.5 μM or 5 μM FL-PLE to create hapten-labeled cells, and then incubating these hapten-labeled cells with anti-FL CAR T cells.

[0098] [Figure 9A] Flow cytometry analysis shows that anti-FL CAR T cells express similar phenotypic markers when expanded by either the FREP or REP method.

[0099] [Figure 9B] 1 shows the results of flow cytometry analysis performed after incubating K562 cells with 5 μM FL-PLE in the presence or absence of fetal bovine serum (FBS).

[0100] [Figure 9C] 9B is a series of graphs showing the results of a cytotoxicity assay of anti-FL CAR T cells expanded by the FREP or REP methods. The assay was performed using cells recovered from the experiment in FIG. 9B.

[0101] [Figure 9D] Figure 9B is a series of graphs showing cytokine stimulation by anti-FL CAR T cells expanded by FREP or REP. Assays were performed using cells recovered from the experiment in Figure 9B.

[0102] [Figure 10A]Figure 1 shows the structure of an ether phospholipid (DNP-PLE) linked to 2,4-dinitrophenol (shown in (i)) as a hapten target for CAR T cells. (ii) represents polyethylene glycol (PEG), which is a spacer that provides an ideal distance between the target and the cell surface. In the PLEs shown in (iii) and (iv), (iii) represents the polar head group, and (iv) is a hydrophobic tail that is incorporated into or tethered to the cell membrane.

[0103] [Figure 10B] The NMR graph showing the precise structure of DNP-PLE is shown.

[0104] [Figure 11] 11A-11E show data on the generation of cells with extracellularly exposed haptens (specifically DNP using DNP-PLE) tethered to the cell surface.

[0105] [Figure 11A] Flow cytometry data of parental MDA-MB-231 cells and MDA-MB-231 cells stained with Alexa Fluor 488-labeled anti-DNP antibody alone are shown.

[0106] [Figure 11B] Flow cytometry data of parental MDA-MB-231 cells and MDA-MB-231 cells incubated with 5 μM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody are shown.

[0107] [Figure 11C] Flow cytometry data of MDA-MB-231 parental cells and MDA-MB-231 cells incubated with 500 nM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody are shown.

[0108] [Figure 11D]Flow cytometry data of MDA-MB-231 parental cells and MDA-MB-231 cells incubated with 50 nM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody are shown.

[0109] [Figure 11E] 11A-11D show histogram plots of the flow cytometry data shown in FIGS. 11A-11D.

[0110] [Figure 12] 12A-12D show confocal microscopy data on the cellular incorporation of DNP-PLE.

[0111] [Figure 12A] Confocal images of MDA-MB-231 parental cells not carrying DNP-PLE stained with Alexa Fluor 488-labeled anti-DNP antibody are shown.

[0112] [Figure 12B] Confocal images of MDA-MB-231 parental cells incubated with 5 μM DNP-PLE but not stained with Alexa Fluor 488-labeled anti-DNP antibody are shown.

[0113] [Figure 12C] Confocal images of MDA-MB-231 parental cells incubated with 5 μM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody are shown.

[0114] [Figure 12D] Confocal images of MDA-MB-231 parental cells incubated with 1 μM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody are shown.

[0115] [Figure 13]13A to 13D show data confirming the extracellular accessibility of the hapten carried on the cells and the fact that PLE was carried on the cell membrane.

[0116] [Figure 13A] A schematic diagram of a second-generation CAR cassette with a long spacer for expressing an anti-DNP CAR is shown.

[0117] [Figure 13B] Flow cytometry data showing the H9 parental cell population, the H9 parental cell population stained with Erbitux antibody, and the anti-DNP CAR-expressing H9 cell population stained with Erbitux antibody are shown.

[0118] [Figure 13C] Confocal images of MDA-MB-231 co-cultured with anti-DNP CAR H9 cells are shown.

[0119] [Figure 13D] Confocal images of 5 μM DNP-PLE-loaded MDA-MB-231 cells co-cultured with anti-DNP CAR H9 cells are shown.

[0120] [Figure 14] Graphs showing data on cytokine production by CD19 CAR T cells co-cultured with various target cells and non-autologous T-APCs are shown.

[0121] [Figure 15] 15A-15C show data regarding the activation of autologous T-APCs in vitro.

[0122] [Figure 15A] This shows the results of flow cytometry detection of the expression of CD19t and cleaved EGFR (EGFRt) on the cell surface of CD4+ / CD8+ mixed antigen-presenting cells (T-APCs) prepared from clinical specimens by transduction with cleaved CD19 (CD19t).

[0123] [Figure 15B] EGFRt expression in transduced CD4+CD19 CAR T cells and transduced CD8+CD19 CAR T cells was detected by flow cytometry.

[0124] [Figure 15C] Graphs showing cytokine production by transduced CD4+ CD19 CAR T cells and transduced CD8+ CD19 CAR T cells.

[0125] [Figure 16] 16A-16C show data on the activation of autologous hapten-APCs in vitro.

[0126] [Figure 16A] Flow cytometric analysis of fluorescence of K562 leukemia cells incubated overnight in the presence or absence of 5 μM FL-PLE is shown.

[0127] [Figure 16B] Flow cytometric fluorescence analysis of primary CD8+ T cells incubated overnight in the presence or absence of 5 μM FL-PLE is shown.

[0128] [Figure 16C] Graphs showing cytokine production by activated anti-FL CAR T cells.

[0129] [Figure 17] Figures 17A-17D show data regarding the persistence of CAR T cells in the peripheral blood of two pediatric patients and the use of T-APCs to stimulate CAR T cells.

[0130] [Figure 17A] Graphs showing the status of CAR T cell, T-APC and CD19+ B cell populations in the peripheral blood of patients after treatment.

[0131] [Figure 17B] Graph showing the status of CAR T cell, T-APC and CD19+ B cell populations in peripheral blood after treatment of the second patient.

[0132] [Figure 17C] In the second patient shown in Figure 17B, CAR T cells were detected by flow cytometry on day 1 (C1.T2.D1).

[0133] [Figure 17D] In the second patient shown in Figure 17B, CAR T cells were detected by flow cytometry on day 14 (C1.T3.D14).

[0134] [Figure 18A] 1 shows the results of flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) that were depleted of T cells by isolating T cells using CD8+ and CD4+ magnetic beads sequentially.

[0135] [Figure 18B] The PBMCs shown in Figure 18A were depleted of T cells by separating them using CD8+ and CD4+ magnetic beads sequentially, and then labeled with 5 μM FL-PLE and analyzed by flow cytometry.

[0136] [Figure 18C] After T cell depletion by sequentially separating T cells using CD8+ and CD4+ magnetic beads, PBMCs shown in Figure 18B were labeled with 5 μM FL-PLE, frozen, thawed, and analyzed by flow cytometry.

[0137] [Figure 18D] A histogram of the data shown in Figure 18A is shown, demonstrating the incorporation of FL-PLE into PBMCs.

[0138] [Figure 18E] A histogram of the data shown in Figure 18B is shown, demonstrating the incorporation of FL-PLE into PBMCs.

[0139] [Figure 18F] A histogram of the data shown in Figure 18C is shown, demonstrating the incorporation of FL-PLE into PBMCs.

[0140] [Figure 18G] FIG. 18B shows a side scatter plot of the data shown in FIG. 18A demonstrating the incorporation of FL-PLE into PBMCs.

[0141] [Figure 18H] FIG. 18B shows a side scatter plot of the data shown in FIG. 18B, demonstrating the incorporation of FL-PLE into PBMCs.

[0142] [Figure 18I] FIG. 18C shows a side scatter plot of the data shown in FIG. 18C, demonstrating the incorporation of FL-PLE into PBMCs.

[0143] [Figure 19A] 1 shows a graph showing the time course of cell numbers expanded using irradiated TM-LCL and PBMC by the standard rapid expansion method (REP method).

[0144] [Figure 19B] This graph shows the time course of cell numbers proliferated using the fluorescein-based REP method (FREP method) with TM-LCLs loaded with 5 μM FL-PLE after irradiation at a target cell to effector cell ratio of 7:1.

[0145] [Figure 19C]This graph shows the time course of the number of cells expanded by the FREP method using autologous PBMCs (T cell-depleted) loaded with 5 μM FL-PLE after irradiation, with a target cell to effector cell ratio of 7:1.

[0146] [Figure 19D] This graph shows the time course of the number of cells expanded by the FREP method using autologous PBMCs (T cell-depleted) that were irradiated and then loaded with 5 μM FL-PLE, with a target cell to effector cell ratio of 14:1.

[0147] [Figure 19E] This graph shows the time course of the number of cells expanded by the FREP method at a target cell to effector cell ratio of 7:1 using autologous PBMCs (T cell-depleted) that were irradiated, loaded with 5 μM FL-PLE, and then frozen and thawed.

[0148] [Figure 20A] Graphs showing the time course of flux in mice administered anti-FL CAR T cells are shown, representing the average results for each group: (A) a group administered anti-FL CAR T cells alone (circles); (B) a group administered anti-FL CAR T cells and 20×10 irradiated TM-LCLs (squares); (C) a group administered anti-FL CAR T cells and 5×10 TM-LCLs that had been irradiated and loaded with 5 μM FL-PLE (triangles); and (D) a group administered anti-FL CAR T cells and 20×10 TM-LCLs that had been irradiated and loaded with 5 μM FL-PLE (inverted triangles).

[0149] [Figure 20B] A graph showing the time course of flux in the group of mice administered anti-FL CAR T cells alone (A) is shown.

[0150] [Figure 20C]A graph showing the time course of flux in the group of mice treated with anti-FL CAR T cells and 20x106 irradiated TM-LCLs (B).

[0151] [Figure 20D] A graph showing the time course of flux in the group of mice (C) that received anti-FL CAR T cells and 5x106 TM-LCLs that were irradiated and then loaded with 5 μM FL-PLE is shown.

[0152] [Figure 20E] A graph showing the time course of flux in the group of mice that received anti-FL CAR T cells and 20x106 TM-LCLs that were irradiated and then loaded with 5 μM FL-PLE (D). DETAILED DESCRIPTION OF THE INVENTION

[0153] Definition of Terms As used herein, "about" may indicate that a particular value includes the degree of error inherent in the method employed to determine the value or experimental variation.

[0154] As used herein, "nucleic acid" or "nucleic acid molecule" refers to a polynucleotide, including, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may be composed of natural nucleotide monomers (e.g., DNA or RNA), or monomers consisting of analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications in the sugar moiety or the pyrimidine or purine base moiety. Modifications in the sugar moiety include, for example, replacement 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 replaced with a sterically or electronically similar structure, such as an azasugar or carbocyclic sugar analog. 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 linkages analogous thereto. Linkages analogous to phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoramidate linkages. "Nucleic acid molecule" also encompasses "peptide nucleic acids," which contain natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. As used herein, "encode" refers to the property that a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, serves as a template for the synthesis of another macromolecule, such as a predetermined amino acid sequence.Thus, 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 that protein. A "nucleic acid sequence encoding a polypeptide" includes all degenerate nucleotide sequences that encode the same amino acid sequence. "Specific" or "specificity" may refer to the properties of a ligand relative to a binding partner, or the properties of a binding partner relative to a ligand, including, for example, specificity involving complementary shape, charge, and hydrophobic binding. Specificity involved in binding includes stereospecificity, regioselectivity, or chemoselectivity. In some embodiments, a method for producing a nucleic acid encoding a chimeric antigen receptor is provided, which allows for obtaining a nucleic acid encoding a chimeric antigen receptor specific to a hapten or tumor antigen.

[0155] A "vector" or "construct" is a nucleic acid used to introduce heterologous nucleic acid into a cell and can contain various regulatory elements, thereby allowing the expression of the heterologous nucleic acid in the cell. 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.

[0156] The terms "chimeric antigen receptor," "CAR," or "chimeric T cell receptor" have their common, ordinary meanings in the context of this specification, including, but not limited to, a synthetically designed receptor in which a ligand-binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with the disease or disorder is linked via a spacer domain to one or more intracellular signaling domains (e.g., costimulatory domains) of a T cell receptor or other receptor. Chimeric receptors can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, or chimeric antigen receptors (CARs). Such CARs are engineered receptors that can be grafted with any specificity onto 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. However, the CARs described herein have been engineered with various components or domains, such as epitope-binding regions (e.g., antibody fragments, scFvs, or portions thereof), spacers, transmembrane domains, or signaling domains, that have been modified to produce surprising effects, and therefore the components of the CAR can often be distinguished as separate elements throughout this disclosure. In some embodiments, the spacer of the chimeric antigen receptor is selected (e.g., so that the spacer is of a particular amino acid length) to confer desired orientation, avidity, or binding properties to the CAR. Next, multiple CARs with spacers of various lengths, displayed, e.g., on cells, are screened for their ability to bind to or interact with the targeting moiety to which the CAR is directed. Exemplary targeting moieties include, but are not limited to, biotin, digoxigenin, dinitrophenol, green fluorescent protein (GFP), yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, far-red fluorescent protein, and fluorescein (e.g., fluorescein isothiocyanate (FITC)).The targeting moiety to which the CAR binds or interacts can be presented on a substrate such as a membrane, bead, or support (e.g., a well), a binder such as a lipid (e.g., a PLE) or a hapten, or a cell such as a hapten-presenting cell (e.g., a cancer cell bound to a hapten containing the target). The CAR may also be specific for a hapten on another cell, or for an antigen presented on a cancer cell or a pathogen (e.g., a virus or bacterium). One approach involves contacting a substrate or binder containing the desired targeting moiety with a plurality of cells containing a CAR or TCR specific for the targeting moiety, and measuring the amount of binding of the CAR- or TCR-containing cells to the targeting moiety presented on the substrate or binder. Assessment of such binding may include staining cells bound to the targeting moiety, or assessing fluorescence or loss of fluorescence. Modifications to the CAR structure, such as varying the length of the spacer, can also be assessed using the same method. In some approaches, the methods of the present invention involve providing cells containing a hapten and contacting T cells bearing a second CAR or TCR specific for a target moiety or antigen on a target cell, such as a cancer cell, tumor cell, or target virus, with the hapten-bearing cells to stimulate the T cells.

[0157] "Specific" or "specificity" may refer to the properties of a ligand relative to a binding partner, or the properties of a binding partner relative to a ligand, including, for example, specificities involving complementary shape, charge, and hydrophobic binding. Specificities involving binding include stereospecificity, regioselectivity, and / or chemoselectivity. In some embodiments, methods are provided for producing nucleic acids encoding chimeric antigen receptors, which can yield nucleic acids encoding chimeric antigen receptors specific to tumor antigens or haptens.

[0158] As used herein, "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunocompetent cells, or both. It is readily understood that antigens can be produced, synthesized, and manufactured by recombinant technology, or obtained from biological samples. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or body fluids (e.g., blood, plasma, or ascites). As used herein, "anti-tumor effect" refers to a biological effect indicated by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastatic lesions, an extension of life expectancy, or a reduction in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be indicated by a reduction in recurrence or an extension of the time to recurrence. In some embodiments provided herein, the CAR-bearing T cells have an anti-tumor effect.

[0159] A "bispecific chimeric antigen receptor" refers to a CAR comprising two domains: a first domain specific for a first ligand and a second domain specific for a second ligand. In some embodiments, the first ligand is a hapten. In some embodiments, the second ligand is a tumor-specific ligand. In some embodiments, the bispecific CAR comprises two scFv domains, the first scFv domain specific for a tumor-specific ligand and the second scFv domain specific for a hapten.

[0160] As used herein, a "ligand" refers to a substance that specifically binds to another substance to form a complex. Examples of ligands include epitopes on antigens, molecules that bind to receptors, substrates, inhibitors, hormones, or activators. As used herein, a "ligand-binding domain" refers to a substance or portion thereof that binds to a ligand. Examples of ligand-binding domains include the antigen-binding portion of an antibody, the extracellular domain of a receptor, or the active site of an enzyme. The "percent amino acid sequence identity" identified for the chimeric receptor polypeptides of the present invention is defined as the percentage of amino acid residues in the ligand-binding domain, spacer, transmembrane domain, or lymphocyte activation domain in a candidate sequence that match the amino acid residues in that domain in a reference sequence. This amino acid sequence identity is calculated after aligning the candidate and reference sequences and inserting gaps, if necessary, to calculate the maximum percent sequence identity; conservative substitutions are not considered as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed by a variety of methods within the skill of the art, including publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to maximize alignment across the entire length of the sequences being compared. For example, to calculate percent amino acid sequence identity using the WU-BLAST-2 computer program [Altschul et al., Methods in Enzymology, 266:460-480 (1996)], several search parameters are used, most of which are set to default values. Parameters that are not set to default values ​​(i.e., adjustable parameters) are set to overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62.The percent amino acid sequence identity is determined by dividing (a) the number of identical amino acid residues that match between the amino acid sequence of each or all of the polypeptides in the chimeric receptor reference sequence. In some embodiments, a nucleic acid encoding a CAR or a nucleic acid encoding a CAR polypeptide may have a percent sequence identity to a sequence shown in Table 3 or Table 4.

[0161] In some embodiments, cells can be engineered to express two CARs or bispecific CARs using vectors such as viral vectors, such as gammaretroviral vectors or lentiviral vectors, or CRISPR / CAS9 systems. Such techniques for genetically engineering T cells to express CARs or bispecific CARs are known to those skilled in the art. In some embodiments, the vector is a transposon, an integrase vector system, or an mRNA vector.

[0162] The terms "costimulatory domain" or "intracellular signaling domain," as used herein, have their common, ordinary meaning, and include, but are not limited to, a signaling moiety that provides a signal to a T cell that mediates a T cell response, such as (but not limited to) activation, proliferation, differentiation, or cytokine secretion, in addition to the primary signal provided by, for example, the CD3ζ chain of the TCR / CD3 complex. Examples of costimulatory domains include, but are not limited to, entire 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 the entire ligand that specifically binds to CD83, or portions thereof. In some embodiments, the costimulatory domain is an intracellular signaling domain that mediates a cellular response, including activation, proliferation, differentiation, and / or cytokine secretion, by interacting with other intracellular mediators.

[0163] In some embodiments described herein, the CAR of the present invention is specific for a hapten. In some embodiments described herein, a second CAR is present on a T cell specific for an antigen on a cell or tumor cell. In some embodiments described herein, the CAR of the present invention comprises a costimulatory domain. In some embodiments, the costimulatory domain is CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or a ligand that specifically binds to CD83; or a portion thereof.

[0164] A "transmembrane domain" is a hydrophobic protein region present in the cell membrane bilayer and serves to anchor proteins embedded in biological membranes. The topology of the transmembrane domain may be, but is not limited to, a transmembrane alpha helix. In some embodiments of the method for generating genetically engineered T cells having a chimeric antigen receptor, the vector comprises a sequence encoding the transmembrane domain. In some embodiments of the method, the transmembrane domain comprises a CD28 transmembrane sequence or a fragment thereof, the CD28 transmembrane sequence or a fragment thereof being 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids in length, or within a range defined by any two of these lengths. In some embodiments of the method, the CD28 transmembrane sequence or a fragment thereof is 28 amino acids in length. In some embodiments, a chimeric receptor of the present invention comprises a transmembrane domain, which serves to anchor the chimeric receptor to a membrane.

[0165] "T cell receptor" or "TCR" has its common and ordinary meaning in the context of this specification, including, but not limited to, a molecule present on the surface of a T lymphocyte or T cell, which is responsible for recognizing antigen fragments bound to major histocompatibility complex molecules.

[0166] As used herein, the term "hapten" has its general, ordinary meaning in the context of this specification, including, but not limited to, a binding moiety consisting of a small molecule. In some embodiments, a hapten alone may not induce an immune response or may not induce a significant immune response, but an immune response may be induced by a hapten bound to a carrier. In some embodiments, the hapten may be tethered to a carrier (e.g., a cell).

[0167] In some embodiments, the hapten may be any type of Alexa Fluor fluorescent dye. In some embodiments, a "hapten" may be any small molecule that can induce an immune response only when bound to a larger carrier, such as a protein, which may not be capable of inducing an immune response by itself. In some embodiments, a hapten may be any small molecule that, when combined with a larger carrier, such as a protein, can induce the production of antibodies that specifically bind to the hapten (free or bound to the carrier). In some embodiments, a hapten may be a peptide, other large chemical entity, or an aptamer. In some embodiments, a hapten may be any hapten provided in a database of haptens accessible from the World Wide Web.

[0168] Non-limiting examples of haptens useful in the embodiments provided herein are listed in Table 1. Table 1: Examples of haptens useful in the embodiments described herein Alexa Fluor 405;Alexa Fluor 430;Alexa Fluor 500;Alexa Fluor 514;Alexa Fluor 532;Alexa Fluor 546;Alexa Fluor 555;Alexa Fluor 568;Alexa Fluor 594;Alexa Fluor 610;Alexa Fluor 633;Alexa Fluor 635 647;Alexa Fluor 660;Alexa Fluor 680;Alexa Fluor 700;Alexa Fluor 750;Alexa Fluor 790;Cascade Blue;Alexa Fluor 488; BODIPY; Dansyl chloride; Oregon Green; Lucifer Yellow; Rhodamine; Tetramethylrhodamine; Nitrotyrosine; Digoxigenin; 2,4-Dichlorophenoxyacetic acid; Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine); Nicotine (3-(1-methyl-2-pyrrolidyl)pyridine; Black leaf; Morphine (morphine; morphine sulfate); 2,4-Dinitrochlorobenzene (1-chloro-2,4-dinitrobenzene; DNCB; dinitrochlorobenzene); 4-Chloro-6-(ethylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid); Structurally related s-triazines (modified: H / Cl / C6, R 1 = NH2-, R 2 = -Cl, R 3 = -NH-(CH2)5-COOH; iPr / Cl / nBu, R 1 = (CH3)2-CH-NH-, R 2 = -Cl, R 3= -NH-(CH2)3-(CH3)); Ametryn (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine); Deethylatrazine (DEA) (structurally related s-triazines); Deisopropylatrazine (DIA) (structurally related s-triazines); Deethyldeisopropylatrazine (DEDIA) (structurally related s-triazines); Deethyldeisopropylatrazine (DEDIA) (structurally related s-triazines); Hydroxyatrazine (HA) (structurally related s-triazines); Deisopropylhydroxyatrazine Desmetryn (structurally related s-triazines); 2-hydroxyatrazine (atrazine derivative); 2-hydroxypropazine (structurally related s-triazines); 2-hydroxysimazine; N-(4-amino-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (modified: R 1 = NH2, R 2 = NH(CH2)3COOH, R 3= OH); Sulkofuron; 5-chloro-2-{4-chloro-2-[3-(3,4-dichlorophenyl)ureido]phenoxy}benzenesulfonic acid; Flucofuron (1,3-bis(4-chloro-α,α,α-trifluoro-m-tolyl)urea); Agatharesinol; Sequilin C; Sugiresinol; Hydroxysugiresinol; Hinokiresinol; Coniferyl alcohol; Cinnamyl alcohol; p-Coumaric acid; Cinnamic acid; p-Coumaric acid; Cinnamic acid; Hinokinin; Guaiacylglycerol-β-guaiacyl ether; Morphine-3-glucuronide (M3 G); Codeine; Norcodeine; 6-Monoacetylmorphine; (+)-Methamphetamine; Ceftazidime; Phenobarbital; p-Hydroxyphenobarbital; p-Aminophenobarbital; Cyclobarbital; 3'-Ketocyclobarbital; 3'-Hydroxycyclobarbital; Secobarbital; Barbital; Methalbital; Barbituric Acid; Thiopental; Thiobarbituric Acid; Primidone; Glutethimide; Pentobarbital; Heroin; Diacetylmorphine; Levallorphan; L-11-Allyl-1,2,3,9,10 ,10a-Hexahydro-4H-10,4a-iminoethanophenanthrene-6-ol;Pethidine (Demerol;Drantin;Meperidine;Ethyl 1-methyl-4-phenylpiperidine-4-carboxylate;Isonipecaine);Methamphetamine;d-Desoxyephedrine;Methedrine;Tolpropamine;Pratalgin;Pragman;Benzoylecgonine;3-Carboxymethylmorphine;Cocaine;5-Benzimidazolecarboxylic acid;ABA (4-acetylbenzoic acid);Dexamethasone;Flumethasone;6α,9α-Difluoro- 11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione; 9α-fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione; 9-α-fluoroprednisolone; desoximetasone; triamcinolone; 9α-fluoro-11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20-dione; fluocortolone; 6α-fluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione; cortisol;11β,17,21-Trihydroxypregn-4-ene-3,20-dione;Prednisone;17,21-Dihydroxypregn-4-ene-3,11,20-trione;Methylprednisolone;11β,17,21-Trihydroxy-6α-methylpregna-1,4-diene-3,20-dione;Triamcinolone hexacetonide;21-(3,3-Dimethyl-1-oxobutoxy)-9α-fluoro-11-hydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione;Carbofuran;Me 2,3-Dihydro-2,2-dimethyl-7-benzofuranyl thiocarbamate;BFNP(3-[[(2,3-Dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]amino]propanoic acid);Carbofuran derivatives;2,3-Dihydro-2,2-dimethyl-7-benzofuranol;Bendiocarb;Carbaryl;Methiocarb;Propoxur;Aldicarb;Methomyl;Benalaxyl;Methyl N-(phenylacetyl)-N-(2,6-xylyl)-DL-alaninate;Bn-Ba(4-[2-(N-phenylacetyl-N-2,6-xylyl) (N-phenylacetyl-N-2,6-xylyl-DL-alanine);Bn-COOH(4-[2-(N-phenylacetyl-N-2,6-xylyl-DL-alanine);Benalaxyl derivatives;Furalaxyl;Metalaxyl;Acetochlor;Dimetachlor;Metolachlor;2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide;Diethatylethyl;Benzoylpropethyl;Benzoylpropethyl;2,4,5-Trichlorophenoxyacetic acid;2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide;Diethatylethyl Triethyl benzoylpropethyl; Propachlor; Propachlor; 2,4,5-trichlorophenoxyacetic acid; 2,4,5-T; Weedone; 2,4-Dichlorophenoxybutyric acid (2,4-DB); 2,4-DB; Butanoic acid; 4-(2,4-dichlorophenoxy)-; Butoxone; Embutone; MCPA; 2-Methyl-4-chlorophenoxyacetic acid; Metaxon; Dichlorprop (2,4-DP); 1-[(2-chloro)phenylsulfonyl]monoamidosuccinic acid; Chlorsulfuron; Chlorbromuron;Amidosulfuron;Chlorotoluron;Isoproturon;Diuron;Linuron;O-Methyl-O-(4-nitrophenyl)-N-(4-carboxybutyl)-phosphoramidothioate;Parathion methyl;O,O-Dimethyl O-4-nitrophenyl phosphorothioate;Metaphos;Wolfatox;Dimethyl parathion;Metacide;Parathion ethyl;Diethyl p-nitrophenylthiophosphate;O,O-Diethyl O-(p-nitrophenyl) phosphorothioate;Fenitrothion;O,O-Dimethyl O-4-nitro-m-tolyl phosphorothioate;Fenthion, O,O-Dimethyl O-4-methylthio-m-tolyl phosphorothioate;Bromophos, O-4-bromo-2,5-dichlorophenyl O,O-dimethyl phosphorothioate;Chlorpyrifos-methyl, O,O-Dimethyl O-3,5,6-trichloro-2-pyridyl Phosphorothioate; Parathion methyl oxide, Paraoxon; Phosphate; O,O-Diethyl O-(4-nitrophenyl) ester, Diazinon, O,O-Diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate; Azinphos methyl; Pirimiphosmethyl;O-2-Diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate;Methidathion;S-2,3-Dihydro-5-methoxy-2-oxo-1,3,4-thiadiazol-3-ylmethyl O,O-dimethyl phosphorodithioate;Dimethylchlorothiophosphate;4-Nitrophenol;p-Nitrophenol;Phenol derivatives (modifications on the benzene ring:R 1 = OH, R 2 = NO2, R 3 = H, R 4 = CH2COOH, R 5 = H, R 6= H); 2-nitrophenol; o-nitrophenol; 3-nitrophenol; m-nitrophenol; 2,4-dinitrophenol; 3,4-dinitrophenol; 2,5-dinitrophenol; 2,4-dinitro-6-methylphenol; 2,3,6-trinitrophenol; 2-chlorophenol; 4-chloro-3-methylphenol, fenitroxone; 3-methyl-4-nitrophenol; nonylphenol; HOM (3-[2-hydroxy-5-nitrobenzylthio]propionic acid); phenol, Delor 103; polychlorinated biphenyls; Delor 104; polychlorinated biphenyls; Delor 105, polychlorinated biphenyls, Delor 106;4,4'-Dichlorobiphenyl, PCBs;2,4,4'-Trichlorobiphenyl;PCBs, 2,4'-;PCBs;2,2'-Dichlorobiphenyl, PCBs;2,4,5-Trichlorobiphenyl, PCBs;3,3',4,4'-Tetrachlorobiphenyl, PCBs;PCBs;2,2',4,4',5,5'-Hexachlorobiphenyl;2-(5-Carboxypentanoylamino)-4,4'-dichlorobiphenyl;Biphenyl derivatives;4-Chlorophenoxyacetic acid;2-Chlorophenoxyacetic acid;DDT, 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane;DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene;p-Chlorophenol;4-Chlorophenol;m-Chlorophenol;3,4-Dichlorophenol 3,5-Dichlorophenol;2,3,4-Trichlorophenol;2,3,5-Trichlorophenol;3-Methylindole;3-Methylindole derivatives;4-(3-Methylindol-5-yloxy)butanoic acid;4-(3-Methylindol-5-yloxy)butanoic acid;3-Methylindole derivatives;6-[n-3-Methylindol-5-yloxycarbonyl)amino]hexanoic acid;6-[n-3-Methylindol-5-yloxycarbonyl)amino]hexanoic acid;3-Methylindole derivatives;2-[4-(3-Methylindol-6-yl)but-1-ylsulo]acetic acid;2-[4-(3-Methylindol-6-yl)but-1-ylsulo]acetic acid;3-Methylindole derivatives;4-(3-Methylindol-6-yl-4-oxo)butanoic acid4-(3-Methylindol-6-yl-4-oxo)butanoic acid;3-Methylindole derivative;6-(3-Methylindol-7-yloxy)hexanoic acid;6-(3-Methylindol-7-yloxy)hexanoic acid;Indole;Indole-3-carboxylic acid;Indole-3-acetic acid, an indole derivative;Indole-3-acetic acid;Indole-3-propionic acid, an indole derivative;Indole-3-propionic acid;Indole derivative Indole-3-carbinol, indole-3-carbinol; tryptophan; tryptamine; 5-methoxyindole-3-carboxaldehyde, 5-methoxytryptamine; 5-methoxyindole; 6-methoxyindole; 7-methoxyindole, EB1089 (seocalcitol); EB1089 (seocalcitol) derivatives; (22E,24E)-des-A,B-24-homo-26,27-dimethyl-8-[(E)-N-(2-carboxyethyl)- Carbamoylmethylidene]-cholesta-22,24-dien-25-ol; 1α-25-dihydroxyvitamin D3; 25(OH)D3, 25-hydroxyvitamin D3, 24R,25(OH)2D3; 24R,25-dihydroxyvitamin D3; vitamin D2, ergocalciferol; vitamin D3; cholecalciferol; EB1446; EB1436; EB1445; EB1470; deethylhydroxyatrazine (DEHA) (structurally related s-to triazines); Irgarol 1051; fluorescein isothiocyanate; FITC, metanephrine, normetanephrine; propazine; terbuthylazine; terbuthylazine; 6-chloro-N-(1,1-dimethylethyl)-N'-ethyl-1,3,5-triazine-2,4-diamine; (structurally related s-triazines); ametryn (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine (modification: iPr / SCH3 / Et, R); 1 = (CH3)2-CH-NH-, R 2 = -SCH3, R 3 = -NH-CH2-CH3; Irgarol; Cyanazine (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3= NHCCN(CH3)2); OH-Terbuthylazine; Terbuthylazine-2OH; Hydroxytriazine (EQ-0027); Deisopropylatrazine (structurally related s-triazines); Desethylterbuthylazine (structurally related s-triazines); Desethyl-deisopropylatrazine (structurally related s-triazines); Atratone; Terbutryn (structurally related s-triazines); Atrazine derivatives (modified: R 1 = -NHCH(CH3)2, R 2 = -S(CH2)2COOH, R 3 = -NHC2H5); cyanuric chloride; trifluralin; (structurally related s-triazines) tBu / C4 / SCH3 (modification: R 1 = -NH-C-(CH3)3, R 2 = -NH(CH2)3COOH, R 3 = -SCH3); sulfamethazine; (structurally related s-triazines) 6-[[[4-chloro-6-(methylamino)]-1,3,5-triazin-2-yl]amino]hexanoic acid (modification: Me / Cl / C6, R 1 = -NHCH3, R 2 = -Cl, R 3 = -NH(CH2)5COOH); (structurally related s-triazines) procyadins (modification: R 1 = -Cl, R 2 = -NHcyclopropyl, R 3 = -NHCCN(CH3)2); (structurally related s-triazines); Prometon (modification: R 1 = -OCH3, R 2 = -NHCH(CH3)2, R 3 = -NHCH(CH3)2); (Structurally related s-triazines) atrazine mercapturic acid (AM) (modification: R 1 = -SCH2CH(NHAc)COOH, R 2 = -NHCH2CH3, R 3 = -NHCH(CH3)2); (structurally related s-triazines), desethyl atrazine mercapturic acid (desethyl AM) (modified: R 1 = -NAcCys, R2 = -NH2, R 3 = -NHCH(CH3)2); (structurally related s-triazines); deisopropyl atrazine mercapturic acid (deisopropyl AM) (modification: R 1 = -NAcCys, R 2 = -NHCH2CH3, R 3 = -NH2); (structurally related s-triazines); didealkylated atrazine mercapturic acid (didealkylated AM) (modification: R 1 = -NAcCys, R 2 = -NH2, R 3 = -NH2); (structurally related s-triazines); simazine mercapturate (modification: R 1 = -NAcCys, R 2 = -NHCH2CH3, R 3 = -NHCH2CH3); (structurally related s-triazines) (modification: R 1 = -S(CH2)2COOH, R 2 = -NHCH2CH3, R 3 = -NHCH2CH3); (structurally related s-triazines) (modification: R 1 = -Cl, R 2 = -NHCH(CH3)2, R 3 = -NH(CH2)2COOH); (structurally related s-triazines) (modification: R 1 = -Cl, R 2 = -NHCH2CH3, R 3 = -NH(CH2)2COOH); (structurally related s-triazines); atrazine mercapturic acid methyl ester (AM methyl ester) (modification: R 1 = -NAcCysME, R 2 = -NHCH2CH3, R 3 = -NHCH(CH3)2); N-acetylcysteine; S-benzylmercapturic acid; (structurally related s-triazines); Simetryne (modification: R 1 = -SCH3, R 2 = -NHCH2CH3, R 3= -NHCH2CH3); Metribuzin; 4-Amino-6-tert-butyl-4,5-dihydro-3-methylthio-1,2,4-triazin-5-one; Sulfonamides; N 4 -acetylsulfamethazine (modified: N 4-Acetylsulfamethazine);Sulfa drugs;Sulfathiazole;Sulfathiazole;Sulfamerazine;Sulfamerazine;Sulfaquinoxaline;Sulfaquinoxaline;Sulfachlorpyridazine;Sulfachlorpyridazine;Sulfapyridine;Sulfadimethoxine;Sulfadimethoxine;Sulfamethoxazole;Sulfamethoxazole;Sulfisoxazole;Sulfamethizole;Sulfamethizole;Sulfanilamide;Sulfanilamide;Sulfaguanidine;Sulfag Anidine;Sulfadiazine;Sulfadiazine;Sulfamethoxypyridiazine;Sulfamethoxypyridiazine;Pentachlorophenoxypropionic acid;Pentachlorophenol;PCP;2,3,5,6-Tetrachlorophenol;1,2,4,5-Tetrachlorobenzene;2,4,6-Trichlorophenol;2-Methoxy-3,5,6-trichloropyridine;1,3,5-Trichlorobenzene;1,3-Dichlorobenzene;2,4,5-Trichlorophenol;2,6-Dichlorophenol;3,5,6-Trichloro-2 -Pyridinoxyacetic acid;3,5,6-Trichloro-2-pyridinol;TCP;2,4-Dichlorophenol;2,5-Dichlorophenol;DNC;4,4'-Dinitrocarbanilide;(Structurally related s-triazines);Dichloroatrazine;(Structurally related s-triazines);Dichlorosimazine;1-((6-Chloropyridin-3-yl)methyl)imidazolidin-2-imine;Pyridine derivatives;6-Chloropyridin-3-carboxylic acid;Nicotinic acid;Pyridine derivatives;N-((6-Chloropyridin-3-yl)methyl )-N-Methylacetamide;(6-Chloropyridin-3-yl)-N-methylmethanamine;(6-Chloropyridin-3-yl)methanol;Imidacloprid;1-(6-Chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine;Acetamiprid;(E)-N1-[(6-Chloro-3-pyridyl)methyl]-N2-cyano-N1-methylacetamidine;Nitenpyram;Deltamethrin;1(R)-cis-α(S)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylic acid cyano(3-phenoxyphenyl)methyl ester;DON;Deoxynivalenol;DON derivatives; 15-AcDON (15-acetyldeoxynivalenol); DON derivatives; -AcDON (3-acetyldeoxynivalenol); DON derivatives; 3,15-DiacDON (3,15-diacetyldeoxynivalenol); DON derivatives; 3,7,15-TriacDON (3,7,15-triacetyldeoxynivalenol); NIV (nivalenol); Nivalenol; NIV derivatives; 4-AcNIV (fusarenon X); Flutolanil; α,α,α-trifluoro-3'-isopropoxy-o-toluanilide; Mepronil; Mebenil; Benodaniol; 24,25(OH)2D3; (24R)-24,25-dihydroxyvitamin D3; 24S,25(OH)2D3; 24S,25-dihydroxyvitamin D3; 25R,26(OH)2D3; 25R,26-dihydroxyvitamin D3; 25S,26(OH)2D3; 25S,26-dihydroxyvitamin D3; 1,24,25(OH)3D3; 1,24,25-trihydroxyvitamin D3; 1,25-lactone; (23S,25R)-1,25(OH)2D3-26,23-lactone; 24,25(OH)2-7-DHC; 24,25(O H) 2-7-Dehydrocholesterol; 25(OH)D3-3S; 25(OH)D3 3-Sulfate; 24,25(OH)2D3-Hemiglutarate Derivatives; 11α-Hemiglutaryloxy-(24R)-24,25-Dihydroxyvitamin D3; 24,25(OH)2D3-Hemiglutarate Derivatives; (24R)-24,25-Dihydroxyvitamin D3-3-Hemiglutarate; 24R,25(OH)2D2; 24S,25(OH)2D2; 25(OH)D2; 1,24(OH)2D3; 2,3,6-Trichlorophenol; Tetrachlorohydroquinone; Phenol Pentachloroaniline; Pentachlorobenzene; 2,3-Dinitrotoluene; 4-Dinitrotoluene; 2,4,5-Trichloronitrobenzene; 3-(3-Hydroxy-2,4,6-trichlorophenyl)propanoic acid; 2,3,4,6-Tetrachlorophenol; 2,4,6-Trichloroanisole; 2,4,6-TCA; Pentabromophenol; PBP; 2,4,6-Tribromophenol; 2,4,6-TBP; 2-Bromo-4-chlorophenol; 2-B-4-CP, 2,4-Dibromophenol; 2,4-DBP; 2,6-Dibromophenol;2,6-DBP; 4-bromophenol; 4-BP; furosemide; ampicillin; amoxicillin; 6-aminopenicillanic acid (6-APA); azlocillin; bacampicillin; carbenicillin; epicillin; cloxacillin; dicloxacillin; metampicillin; methicillin; moxalactam; oxacillin; penicillin G; benzylpenicillin; penicillin V; phenoxymethylpenicillin; phenethicillin; piperacillin; ticarcillin; ampicillin hydrolysate; Penicillin G hydrolysate; 3-phenoxybenzoic acid (3-PBAc); chlorpyrifos; chlorpyrifos derivatives; HClo1; derivatives synthesized directly by replacing the chlorine at position 6 of chlorpyrifos with a 3-mercaptopropanoic acid spacer arm; chlorpyrifos derivatives; HTCP (modified: the TCP metabolite HTCP was prepared by hydrolysis of the thiophosphate ester of HClo1); zeatin riboside (trans isomer); zeatin (trans isomer); N; 6 -(2-Isopentenyl)-adenosine;IPA;N 6-(2-Isopentenyl)-adenine; 2-iP; benzyladenine; kinetin; monuron; monolinuron; fenuron; nebron; propanil; propham; chloropropham; 4-chloroaniline; methylurea derivatives; 1-(3-carboxypropyl)-3-(4-chlorophenyl)-1-methylurea; methylurea derivatives; 1-(5-carboxypentyl)-3-(4-chlorophenyl)-1-methylurea; metobromuron; sennoside B; SB; sennoside B with erythro configuration between C-10 and C-10'; sennoside A ( Modified forms: Sennoside A with threo configuration between C-10 and C-10; Rhein; Emodin; Aloe-emodin; Barbaloin; 1,4-Dihydroxyanthraquinone; Rhaponticin; Gallic acid; Vanillic acid; Caffeic acid; Homogentisic acid; Esculin; Cinnamtannin B1; Baicalin; Naringin hydrate; Wogonin; Wogonin 7-O-β-glucuronide; Curcumin; δ1-Tetrahydrocannabinolic acid; δ1-Tetrahydrocannabinol; (±)-cis-4-Aminopermethrin; 3-(4-Aminophenoxy)benzyl (±)-cis-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate;Permethrin;trans-permethrin;cis-permethrin;Cypermethrin;Fenothrin;Resmethrin;Cyfluthrin;Trans-permethrinic acid esfenvalerate;Fulvalinate;Fenpropathrin;cis-permethrinic acid;4-Phenoxybenzoyl alcohol;Diuron derivatives;1-(3-carboxypropyl)-3-(3,4-dichlorophenyl)-1-methylurea;Siduron;Terbuthiuron;Burban;Trifluralin; 2,6-Dinitro-N-propyl-N-(2-carboxyethyl)-4-(trifluoromethyl)benzenamine;TR-13;2-Ethyl-7-nitro-1-propyl-5-(trifluoromethyl)-1H-benzimidazole;Benefin;2,6-Dinitro-N-butyl-N-ethyl-4-(trifluoromethyl)benzenamine;TR-2;2,6-Dinitro-N-propyl-4-(trifluoromethyl)benzenamine;Ethalfluralin;2,6-Dinitro-N-ethyl-N-(2-methyl-2-propenyl)-4-(trifluoromethyl)benzenamine;TR-40;N-(2,6-Dinitro-4-(trifluoromethyl)phenyl)-N-propylpropanamide;TR-15;2-Ethyl-4-nitro-6-(trifluoromethyl)-1H-benzimidazole;TR-3;2,6-Dinitro-4-(trifluoromethyl)benzenamine;TR-6;3-Nitro-5-(trifluoromethyl)-1,2-benzenediamine;TR-9;5-(Trifluoromethyl)-1,2,3-benzenetriamine;TR-21;4-(Dipropylamino)-3,5-dinitrobenzoic acid;TR-36M ;3-Methoxy-2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine;Oryzalin;3,5-Dinitro-4-(dipropylamino)benzenesulfonamide;Pendimethalin;2,6-Dinitro-N-(1-ethylpropyl)-3,4-dimethylbenzenamine;Pentagalloylglucose;Pyrene;Pyrene-1-carboxaldehyde;Phenanthrene;Benzopyrene;3,4-Benzopyrene;Anthracene;3,4-Benzopyrene;Acenaphthene;Fluorene;Chrysene;1,2-Benzophen Nanthrene;Benzo[g,h,i]perylene;Benzo[e]pyrene;Acenaphthylene;Fluoranthene;Benzo(j,k)fluorene;Indeno-1,2,3-cd-pyrene;1,10-(1,2-phenylene)pyrene;Benz[a]anthracene;1,2-Benzanthracene;Benzo(k)fluoranthene;Naphthalene;Benzo[a]fluoranthene;Dibenz[ah]anthracene;1,2,5,6-Dibenzanthracene;2,3-Diaminonaphthalene;2,6-Dinitroaniline;17-β-Estradiol (ED);Est N-(2,6-Dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid;Trifluralin derivatives;N-(2,6-Dinitro-4-trifluoromethylphenyl)-N-methyl-6-aminohexanoic acid;Trifluralin derivatives;N-(2,6-Dinitro-4-trifluoromethylphenyl)-N-propyl-6-aminohexanoic acid;Trifluralin derivatives;N-(2,6-Dinitro-4-trifluoromethylphenyl)-N-propyl-6-aminohexanoic acid;Trifluralin derivativesN-(2,6-Dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid methyl ester;Trifluralin derivatives;N-(2,6-Dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid tert-butyl ester;Benfluralin;Ethalfluralin;Trifluralin derivatives;2,6-Dinitro-4-trifluoromethylphenol;Isopropanol;Aniline;2-Hydroxybenzotrifluoride;N-Propyl-6-aminohexanoic acid;N-Methyl-6-aminohexanoic acid;MHPG derivatives;D-MHPG (D-3-methoxy-4-hydroxyphenylglycol);MHPG derivatives;L-MHPG (L-3-methoxy-4-hydroxyphenylglycol);MHP G derivatives; DL-MHPG (DL-3-methoxy-4-hydroxyphenylglycol); isomeric mixtures of D-MHPG and L-MHPG; MHPG derivatives; DL-MHPG-SO4 (DL-3-methoxy-4-hydroxyphenylglycol sulfate), modified forms may include isomeric mixtures of D-MHPG-SO4 and L-MHPG-SO4; serotonin; 5-HT; 5-hydroxydopamine (5-4HDA); 3,4-dihydroxyphenylglycol (DOPEG); dopamine; 4-(2-aminoethyl)pyrocatechol; 3-hydroxytyramine; 3,4-dihydroxyphenethylamine; L-3,4-dihydroxyphenylalanine; L-DOPA; vanillomandelic acid; DL-VMA; homovanillic acid; Norepinephrine; DL-NE; D-Epinephrine; DE; 3-Methoxytyramine; MTA; 3-Methoxytyrosine; MTyr; 3,4-Dihydroxymandelic acid; DL-DOMA; 3,4-Dihydroxyphenylacetic acid; DOPAC; L-Phenylalanine; Tyramine; p-Tyramine; 4-(2-Aminoethyl)phenol; D-Mandelic acid; Homocatechol; Octopamine; DL-Octopamine; Azinphosethyl; S-(3,4-Dihydro-4-oxobenzo[d]-[1,2,3]-triazin-3-ylmethyl) O,O-diethyl phosphorodithioate; Phosmet; O,O-Dimethyl S-Phthalimidomethylphosphorodithioate;Folpet;N-[(Trichloromethyl)thio]phthalimide;Tetramethrin;(1-Cyclohexene-1,2-dicarboximido)methyl-2,2-dimethyl-3-(2-methylpropenyl)-cyclopropanecarboxylate;N-(Bromomethyl)phthalimide;N-(Chloromethyl)benzazimide;6-(N-Phthalimidoylmethylthio)hexanoic acid (MFH);Bromacil;5-Bromo-3-sec-butyl-6-methyluracil;Bromacil derivatives;5-Bromo-6-(hydroxymethyl)-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinone;Bromacil derivatives;5-Bromo-3-(2-methylpropyl-6-methyl-2,4(1H,3H)-pyrimidine Dione;Metabolites of bromacil;Bromacil derivatives;3-Hydroxy-1-methylpropyl-6-methyl-2,4(1H,3H)-pyrimidinedione (modified; bromacil metabolites);Bromacil derivatives;6-Methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified; bromacil metabolites);Terbacil derivatives;[5-Chloro-3-(1,1-dimethylethyl)-6-(hydroxymethyl)-2,4(1H,3H)-pyrimidinedione;Terbacil;3-tert-butyl-5-chloro-6-methyluracil;Bromacil derivatives;Ethyl-5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-1-yl)hexanoate;N-1 alkylated bromacil derivatives;Bromacil derivatives include 5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-1-yl)hexanoic acid (modified: N-1 alkylated bromacil derivatives); bromacil derivatives include bromo-6-(bromomethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified: 6-methyl substituted bromacil derivatives); and bromacil derivatives include [5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]-2-carboxyl Propanoic acid (modified: 6-methyl-substituted bromacil derivative); 3-[5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]propanoic acid (modified: 6-methyl-substituted bromacil derivative); 5-bromo-1,6-dimethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione, a bromacil derivative; 5-bromo-1-butyl-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione, a bromacil derivative; Butachlor; N-butoxy Methyl-2-chloro-2',6'-diethylacetanilide; Amidochlor; N-[(acetylamino)methyl]-2-chloro-N-(2,6-diethylphenyl)acetamide; Nicarbazin; N,N'-bis(4-nitrophenyl) compounds with 4,6-dimethyl-2(1H)-pyrimidinone (modified: DNC+HDP); 2-Hydroxy-4,6-dimethylpyrimidine; HDP; Imazalil; [1-(β-Allyloxy-2,4-dichlorophenethyl)imidazole]; Imazalil derivatives; EIT-0073 (Imazalil original -OC Modified with -O(CH2)5-COOH group instead of H2CH=CH2 group; penconazole; (RS)-1-(2,4-dichloro-β-propylphenethyl)-1H-1,2,4-triazole; hexaconazole; (RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)hexan-2-ol; propiconazole; cis-trans-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole; diclobutazole;(2RS,3RS)-1-(2,4-Dichlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol; Triflumizole; (E)-4-Chloro-α,α,α-trifluoro-N-(1-imidazol-1-yl-2-propoxyethylidene)-o-toluidine; Imazalil derivatives; EIT-0183; Imazalil derivatives; EIT-0180; Imazalil derivatives; EIT-0111; Imazalil derivatives; EIT-0158; Imazalil derivatives; K-240; Chlorothalonil; Tetrachloroisophthalonitrile (Modification on the benzene ring: R; 1 = CN, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); chlorothalonil derivative 2,4,5,6-tetrachloro-3-cyanobenzamide (modification on the benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); chlorothalonil derivative 2,5,6-trichloro-4-hydroxyisophthalonitrile (modification on the benzene ring: R 1 = CN, R 2 = Cl, R 3 = CN, R 4 = OH, R 5 = Cl, R 6 = Cl); 3-carbamyl-2,4,5-trichlorobenzoic acid (modification on the benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = COOH, R 4 = H, R 5 = Cl, R 6 = Cl); pentachloronitrobenzene (modification on the benzene ring: R 1 = NO2, R 2 = Cl, R 3 = Cl, R 4 = Cl, R5 = Cl, R 6 = Cl); benzene hexachloride; hexachlorobenzene; BHC; lindane (modification on the benzene ring: R 1 = Cl, R 2 = Cl, R 3 = Cl, R 4 = Cl, R 5 = Cl, R 6 = Cl); 2,4,5,6-tetrachlorophenol (modification on the benzene ring: R 1 = OH, R 2 = Cl, R 3 = H, R 4 = Cl, R 5 = Cl, R 6 = Cl); carbaryl derivatives; ethyl carbamate (modification: R 1 = OCONHCH2CH3, R 3 = H); 1-naphthol; 1-naphthaleneacetamide; -(1-naphthyl)acetamide; carbaryl derivatives; 1-methyl carbonate (modified: R 1 = OCOOCH3, R 2 = H); carbaryl derivatives; 1-ethyl carbonate (modification: R 1 = OCOOCH2CH3, R 2 = H); carbaryl derivative 2-ethyl carbonate (modified: R 1 = H, R 2 = OCOOCH2CH3); carbaryl derivative; 1-ethylthiocarbonate (modification: R 1 = OCOSCH2CH3, R 2 = H); carbaryl derivatives; 2-ethylthiocarbonate (modification: R 1 = H, R 2 = OCOSCH2CH3); naptalam; N-1-naphthylphthalamic acid; carbaryl derivatives; 3-hydroxycarbaryl (modification: R 1 = OCONHCH3, R 2 = H, R 3 = OH, R 4 = H, R 5 = H); carbaryl derivative 4-hydroxycarbaryl (modification: R 1= OCONHCH3, R 2 = H, R 3 = H, R 4 = OH, R 5 = H); carbaryl derivative 5-hydroxycarbaryl (modification: R 1 = OCONHCH3, R 2 = H, R 3 = H, R 4 = H, R 5 = OH); carbaryl derivative; 1-(5-carboxypentyl)-3-(1-naphthyl)urea (modification: R 1 = NHCONH(CH2)5COOH, R 2 = H); (structurally related s-triazines adiprothrin; 4-azido-N-isopropyl-6-methylthio-1,3,5-triazin-2-ylamine (modification: R 1 = -SCH3, R 2 = -N3, R 3 = -CH(CH3)2); (structurally related s-triazines); 2-(ethylamino)-4-(methylthio)-6-aminotriazine (modification: R 1 = -SCH3, R 2 = -NH-C2H5, R 3 = -NH2); (Structurally related s-triazines, 2-amino-4-(methylthio)-6-(isopropylamino)triazine (modification: R 1 = -SCH3, R 2 = -NH2, R 3 = -NH-CH(CH3)2); (Structurally related s-triazines, 2-amino-4-methoxy-6-(isopropylamino)triazine (modification: R 1 = -OCH3, R 2 = -NH2, R 3= -NH-CH(CH3)2); TCP derivatives (3,5,6-trichloro-2-pyridinol derivatives); 3-(3,5-dichloro-6-hydroxy-2-pyridyl)thiopropanoic acid; p-nitrosuccinilidic acid (PNA-S); PNA-S; PNA-C; p-nitro-cis-1,2-cyclohexanedicarboxylic acid; Nitroaniline derivatives; 2-nitroaniline; o-nitroaniline; 3-nitroaniline, a nitroaniline derivative; m-nitroaniline; 4-nitroaniline, a nitroaniline derivative; p-nitroaniline; Aromatic alcohols; 4-nitrobenzyl alcohol; 4-nitrophenethyl alcohol, an aromatic alcohol; 2-nitrobenzyl alcohol, an aromatic alcohol; 3-nitrobenzyl alcohol; 1-benzyl-3-(4-nitrophenyl)urea, a urea derivative; 1-(3-chlorophenyl)- 3-(2-Methoxy-5-nitrophenyl)urea;Urea derivative 1-(3-chlorophenyl)-3-(4-methoxy-3-nitrophenyl)urea;Urea derivative 1-(4-chlorophenyl)-3-(4-nitrophenyl)urea;Urea derivative (2-fluorophenyl)-3-(2-methoxy-4-nitrophenyl)urea;1-(3-Methoxyphenyl)-3-(3-nitrophenyl)urea;Carbofuran derivative m-Carbofuranphenol;Hydroxycarbofuran;Ketocarbofuran;Carbosulfan;2,3-Dihydro-2,2-dimethylbenzofuran-7-yl (dibutylaminothio)methylcarbamate;Benfuracarb;N-[2,3-Dihydro-2,2-dimethylbenzofuran-7-yloxycarbonyl(methyl)aminothio]-N-isopropyl-β-anilinate;Furathiocarb;2,3-Dihydro-2,2-dimethyl-7-benzofuranyl 2,4-Dimethyl-5-oxo-6-oxa-3-thia-2,4-diazadecanoate; Carbofuran derivatives; 4-[[(2,3-Dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]butanoic acid (BFNB) (modification: n=3, X=CH2); Endrin; Nendrin; (1R,4S,4aS,5S,6S,7R,8R,8aR)- 1,2,3,4,10,10-Hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7-epoxy-1,4,5,8-dimethanonaphthalene;Heptachlor;1,4,5,6,7,8,8-Heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene;Chlordane;1,2,4,5,6,7,8,8-Octachloro-2,3,3a,4,7,7a-hexahydro-4,7-methanoindene;Endosulfan (modified: α- and β-isomer mixture);Endosulfan (modified: α-isomer);Endosulfan (modified beta isomer); endosulfan derivatives; endosulfan sulfate (modified: sulfate); endosulfan derivatives; endosulfan diol; diol metabolite of endosulfan; endosulfan derivatives; endosulfan ether (modified: ether metabolite of endosulfan); endosulfan derivatives; hydroxy ether; hydroxy ether metabolite of endosulfan; endosulfan derivatives; endosulfan lactone (modified: lactone metabolite of endosulfan); aldrin; dieldrin; fenvalerate isomers (modified : 1S,2R isomer, R:Ph; Fenvalerate isomers (modified: 1R,2S isomer, R:Ph); Fenvalerate isomers (modified: 1R,2R isomer, R:Ph); Fenvalerate isomers (modified: 1S,2R / S isomer, R:Ph); Fenvalerate isomers (modified: 1R,2R / S isomer, R:Ph); Fenvalerate isomers; Fenvalerate (modified: 1R / S,2R / S isomer, R:Ph); Thiabendazole; 2-(thiazol-4-yl)benzimidazole; Thiabendazole derivatives; 5-hydroxythiabendazole ( Modified: 5-OH-TBZ; Thiabendazole derivatives; 5-NH2-TBZ; Thiabendazole derivatives; Methyl benzimidazole carbamate; Albendazole; Mebendazole; Fenbendazole; Thiabendazole derivatives; 2-Succinamidothiabendazole; Thiabendazole derivatives; 2-Succinamidothiabendazole; Cambindazole; Fenvalerate haptens; (S)-4-Chloro-α-(1-methylethyl)benzeneacetic acid cyano[3-(4-aminophenoxy)phenyl]methyl (4-aminoesfenvalerate);Fenvalerate haptens; Benzyl 4-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]benzenepropanoate; Fenvalerate haptens; Benzyl 3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetate; Fenvalerate haptens; 3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetic acid; Fenvalerate haptens; Benzyl 6-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]hexanoate; Fenvalerate haptens; 6-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]hexanoic acid (fenvalerate haptens); 4-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]benzenepropanoic acid; (S)-fenvalerate acid; (structurally related s-triazines); atrazine mercapturate (modified: R; 1 = -SCH2CH(NHCOCH3)COOH, R 2 = -NHCH2CH3, R 3= -NHCH(CH3)2; Fenthion hapten; Methyl O-[3-methyl-4-(methylthio)phenyl] N-(3-carboxypropyl) phosphoramidothioate (modified form, called hapten B); Fenthion derivatives; Fenthion oxide; Fenthion derivatives; Fenthion oxide; Pirimiphos-ethyl; 4-(methylthio)-m-cresol; Chlorpyrifos derivatives; Chlorpyrifos-oxon; Fenchlorphos; O,O-Dimethyl O-2,4,5-trichlorophenyl phosphorothioate; Trichloronate; O-Ethyl O-2,4,5-trichlorophenyl ethylphosphonothioate; Diclofenthion; O-2,4-Dichlorophenyl O,O-Diethyl phosphorothioate; Parathion; O,O-Diethyl O-4-nitrophenyl phosphorothioate; Thiophos; Chlorpyrifos derivatives (modified form: synthesis of AR1 has been reported); Chlorpyrifos derivatives; O-Ethyl O-(3,5,6-trichloro-2-pyridyl) O-(3-carboxypropyl) phosphorothioate; (PO); Chlorpyrifos derivative, O-ethyl O-(3,5,6-trichloro-2-pyridyl) N-(5-carboxyethyl) phosphoramidothioate; (PN1) (modified: amide bond of thiophosphate reagent); Chlorpyrifos derivative; O-ethyl O-(3,5,6-trichloro-2-pyridyl) N-(2-carboxyethyl) phosphoramidothioate; (PN1) (modification: amide bond of appropriate thiophosphate reagent); triadimefon; (RS)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one; GR151004; (4-[[5-[3-[2-(dimethylamino)ethyl]]-5-benzofuranyl]-3-pyridinyl]acetyl]morpholine dihydrochloride; diflubenzuron; 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea; (structurally related s-triazines, SprAAT (modification: R 1 = SCH2CH2COOH, R 2 = NH2, R 3 = NH2); (structurally related s-triazines); SBeAAT (modification: R 1= S(C6H4)COOH, R 2 = NH2, R 3 = NH2); (structurally related s-triazines); SAAT (modification: R 1 = SH, R 2 = NH2, R 3 = NH2); (structurally related s-triazines); CDAT (modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH2); (Structurally related s-triazines, CDET (modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH(CH2CH3); (Structurally related s-triazines, CDIT (modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH(CH(CH3)2)); (structurally related s-triazines); CDDT (modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH[C(O)CH3]); (Structurally related s-triazines ammeline; OAAT (modification: R 1 = OH, R 2 = NH2, R 3 = NH2); (Structurally related s-triazines, ammelides; OOAT (modification: R 1 = OH, R 2 = OH, R 3 = NH2); (Structurally related s-triazines, cyanuric acid; OOOT (modification: R 1 = OH, R 2 = OH, R 3 = OH); (structurally related s-triazines); melamine; AAAT (modification: R 1 = NH2, R 2 = NH2, R 3 = NH2); N-isopropylammeline, a structurally related s-triazine; OIAT (modification: R 1 = OH, R2 = NH[CH(CH3)2], R 3 = NH2); N-ethylammeline, a structurally related s-triazine; OEAT (modification: R 1 = OH, R 2 = NHCH2CH3, R 3 = NH2); structurally related s-triazines; N-ethylammelide; OOET (modification: R 1 = OH, R 2 = OH, R 3 = NHCH2CH3); structurally related s-triazines, cyromazine, CyPAAT (modified: R 1 = NH(C3H5), R 2 = NH2, R 3 = NH2); diamino-s-triazine, a structurally related s-triazine; HAAT (modification: R 1 = H, R 2 = NH2, R 3= NH2); PCBs; 2,5,3',4'-tetrachlorobiphenyl (modification: IUPAC no.: 70); PCB 2,4,5,3',4'-pentachlorobiphenyl (modification: IUPAC no.: 118); PCB 2,2',5,5'-tetrachlorobiphenyl (modification: IUPAC no.: 52); PCBs; 6-[3,3',4'-trichlorobiphenyl-4-yl)oxy]hexanoic acid; metolazone; trade names: Mykrox, Zaroxolyn; furfuryl benzoate; DDT metabolites; DDA; paraquat; 1,1'-dimethyl-4,4'-bipyridinium ion; diethylcarbamazine; THP; 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium; o-DNCP; dinitrocarboxyphenol; PCBs; 3-chlorobiphenylol (modification: IUPAC no. 2); PCBs; 3,4'-dichlorobiphenyl (modified: IUPAC No. 13); PCBs; 3,5-dichlorobiphenyl (modified: IUPAC No. 14); PCBs; 3,4,5,3',4'-pentachlorobiphenyl (modified: IUPAC No. 126); 2,3,3',4'-tetrachlorobiphenyl (modified: IUPAC No. 56); 2',3,4,5-tetrachlorobiphenyl (modified: IUPAC No. 76); 3,3',5,5'-tetrachlorobiphenyl (modified: IUPAC No. 80); 2,4,5,2',5'-pentachlorobiphenyl (modified: IUPAC No. 101); 2,3,3',4,4'-pentachlorobiphenyl (modified: IUPAC No. 105); 2,3,6,3',4'-pentachlorobiphenyl (modified: IUPAC No. 110); 3,3',4,5,5'-pentachlorobiphenyl (modified: IUPAC No. 127); 3,4,5,3',4',5'-hexachlorobiphenyl (modified: IUPAC No. 169); 2,3,3',4,4',5-hexachlorobiphenyl (modified: IUPAC No.156); 3,4,3',4'-Tetrabromobiphenyl; 3,4,5,3',4',5'-Hexabromobiphenyl; 2,4,5,2',4',5'-Hexabromobiphenyl; Dibenzofurans and dioxins; 2,3,7,8-Tetrachlorobenzofuran; 2,3,7,8-Tetrachlorodibenzo-p-dioxin; 3,4',5-Trichloro-4-biphenylol; 3,3',5,5'-Tetrachloro-4,4'-biphenyldiol; 3,4,3',4'-Tetrachlorodiphenyl ether; 1,2-Dichlorobenzene; 1,4-Dichlorobenzene; 1,2,4-Trichlorobenzene; 3,4-Dichloroaniline; DDT metabolites; 4,4'-DDT; 4,4'-DDD-Retronecine; 3,4-Dichlorobiphenyl (modified: IUPAC No. 12); 3,4,3'-trichlorobiphenyl (modified: IUPAC No. 35); PCBs; 3,4,4'-trichlorobiphenyl (modified: IUPAC No. 37); 3,4,3',5-tetrachlorobiphenyl (modified: IUPAC No. 78); 3,4,3',5'-tetrachlorobiphenyl (modified: IUPAC No. 79); 3,4,4',5-tetrachlorobiphenyl (modified: IUPAC No. 81); DDT metabolites; p,p'-DDT (modified: p,p'-dichlorodiphenyltrichloroethane); o,p'-DDT (modified: o,p'-dichlorodiphenyltrichloroethane); p,p'-DDE (modified: p,p'-DDE); o,p'-DDE (modified: o,p'-); p,p'-DDD (modified: p,p'-DDD); o,p'-DDD (modified: o,p'-DDD); dicofol; 4,4-dichloro-α-(trichloromethyl)benzhydrol;. Ciprazine; 6-chloro-N-cyclopropyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine; Structurally related s-triazines; Dipropetrine; 6-(ethylthio)-N,N'-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine; Trietazine; 6-chloro-N,N,N'-triethyl-1,3,5-triazine-2,4-diamine; 6-hydroxyatrazine; Hexazinone; 3-cyclohexyl-6-dimethylamino-1-methyl-1,3,5-triazine-2,4( 1H,3H)-dione; TNT; 2,4,6-trinitrotoluene; Tetraconazole (M14360); 1-[2-(2,4-dichlorophenyl)-3-(1,1,2,2-tetrafluoroethoxy)propyl]-1H-1,2,4-triazole; DTP; 2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propanol; Imazalil; Fenarimol; (RS)-2,4'-Dichloro-α-(pyrimidin-5-yl)benzhydryl alcohol; Lupanine metabolites; (+)-Lupanine (modified: R = H); lupanine metabolites; (+)-13-hydroxylupanine (modification: R = OH); lupanine metabolites; hemisuccinate ester of (+)-13-hydroxylupanine (modification: R = OCO-(CH2)2COOH); lupanine metabolites; cis-hexahydrophthalate ester of (+)-13-hydroxylupanine (modification: R = OCO-H) 10 COOH); lupanine metabolites; α-isolupanine; lupanine metabolites; hydroxylupanine; sparteine; cysteine; multiflorin; epilupinine; (structurally related s-triazines); cyanazine acid (modified: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NHCCOOH(CH3)2); structurally related s-triazines (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)3COOH); structurally related s-triazines (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3= NHCH2COOH); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)4COOH); norflurazon; 4-chloro-5-(methylamino)-2-[3-(trifluoromethyl)phenyl]-3(2H)-pyridazinone; norflurazon derivatives; desmethylnorflurazon; metoflurane; chloro-5-(dimethylamino)-2-[(3-trifluoromethyl)phenyl]-3(2H)-pyridazinone; pyrazone; chloridazone; 5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone (active ingredient); dichlorophenyl-pyridazon; (a structurally related s-triazine, azidoatrazine (modified: R 1 = N3, R 2 = NHCH(CH3)2, R 3 = NHCH2CH3); alachlor, 2-chloro-2',6'-diethyl-N-methoxymethylacetanilide; trichothecolumn (modification: R 1 = H, R 2 = OH, R 3 = H, R 4 = O, R 5= H); DON derivatives; Acetyl-T-2; DON derivatives; T-2 tetrol tetraacetate; Chlorpyrifos derivatives; Monodechloro-CP; Bromophos derivatives; Bromophos methyl; Bromophos derivatives; Bromophos ethyl dicapton; 2-Chloro-4-nitrophenyl O,O-dimethyl phosphorothioate; Tetrachlorvinphos; (Z)-2-chloro-1-(2,4,5-trichlorophenyl)vinyldimethyl phosphate; Triclopyr; 3,5,6-trichloro-2-pyridyloxyacetic acid; Picloram; 4-Amino-3,5,6-trichloropyridine-2-carboxylic acid; Formononetin; Biochanin A; 5,7-dihydroxy-4'-methoxyisoflavone (modified: 4'-methyl ether of genistein); Equol; (7-hydrochloride hydroxy-3-(4'-hydroxyphenyl)-chroman; 2'-methoxyformononetin; daidzein; 7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one; genistein; quercetin; 3,3',4',5,7-pentahydroxyflavone; 3,5,7,3',4'-pentahydroxyflavone; matheucinol; coumestrol; (structurally related s-triazines); hydroxysimazine (modified: R 1 = OH, R 2 = NHCH2CH3, R 3 = NHCH2CH3);angustifoline;Alodan;1-methyl-4-phenyl-4-carbethoxypiperidine hydrochloride;zearalenone;RAL;F-2 toxin;fenpropimorph;(RS)-cis-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine;tridemorph;2,6-dimethyl-4-tridecylmorpholine;2,6-dimethylmorpholine;amorolfine;fenpropidin;(RS)-1-[3-(4-tert-butylphenyl)-2-methylpropyl]piperidine;(structurally related s-triazines)(modification:R 1 = Cl, R 2 = Cl, R 3 = NHCH2CH3); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = Cl, R3 = NHCH(CH3)2); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)5COOH); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = NHCH(CH3)2, R 3 = NHCH2COOH); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = NHCH(CH3)2, R 3 = NH(CH2)5COOH); structurally related s-triazines; cyanazine amide (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NHCCONH2(CH3)2); hydroxycyanadic acid (modification: R 1 = OH, R 2 = NHCH2CH3, R 3 = NHCCOOH(CH3)2); deethylsimazine (modification: R 1 = Cl, R 2 = NH2, R 3= NHCH2CH3);Albendazole sulfoxide;[5-(Propylthionyl)-1H-benzimidazol-2-yl]-, methyl ester;Albendazole sulfone;5(6)-Alkylbenzimidazoles;2-Amino-5-(propylthio)benzimidazole;5(6)-Alkylbenzimidazoles;2-Amino-5-(propylsulfonyl)benzimidazole;Oxibendazole;5-Propoxybenzimidazole-2-methylcarbamate;5(6)-Arylbenzimidazoles;Fenbendazole sulfone (modified: sulfone metabolite of fenbendazole);5(6)-Arylbenzimidazoles;4'-Hydroxy Droxifenbendazole; 5(6)-arylbenzimidazoles; Oxfendazole (modification: Oxfendazole is the sulfoxide metabolite of fenbendazole); 5(6)-arylbenzimidazoles; Flubendazole; Benzimidazole metabolites; 2-aminobenzimidazole; Benzimidazole metabolites; 5-aminobenzimidazole; Benzimidazole metabolites; 2-acetylbenzimidazole; Benzophenone; Diphenylmethanone; Phenyl ketone; Diphenyl ketone; Benzoylbenzene; Benzaldehyde; Benzaldehyde; 4-Bromo-2,5-dichlorophenol; Acephate; O,S-Dimethyl Acetyl phosphoramidothioate;Methamidophos;O,S-Dimethyl phosphoramidothioate;Dichlorvos;2,2-Dichlorovinyldimethyl phosphate;Phenthoate;S-α-Ethoxycarbonylbenzyl O,O-dimethyl phosphorodithioate;EPN;Ethyl p-nitrophenyl thionobenzenephosphonate;Bioresmethrin;5-benzyl-3-furylmethyl (1R,3R)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate (Modification: The ISO common name for the unseparated isomer mixture of this substance is resmethrin);Flufenoxuron;1-[4-(2-chloro-α,α,α-trifluoro-p-tolyloxy)-2-fluorophenyl]-3-(2,6-difluorobenzoyl)urea;Amitrole;1H-1,2,4-Triazol-3-ylamine;Molinate;S-Ethyl Azepane-1-carbothioate;Molinate derivatives (modified: S-2-carboxyethyl hexahydroazepine-1-carbothioate); Molinate derivatives (modified: S-5-carboxypentyl hexahydroazepine-1-carbothioate); Molinate derivatives (modified: molinate sulfone); Molinate derivatives (modified: S-(p-aminobenzyl) hexahydroazepine-1-carbothioate); Molinate derivatives (modified: S-2-(p-aminophenyl)ethyl hexahydroazepine-1-carbothioate); Hexamethyleneimine; Thiobencarb (Bolero); Butyrate (Sutan); EPTC (Eptam); Cycloate (Roneet); Pebulate (Tillam); Vernolate (Vernam); Aflatoxin M1; AFM1 (modified: AFM1); Aflatoxin B1; AFB1 (modified: AFB1); Aflatoxin G1; AFG1 (modified: AFG1); Aflatoxin M2; AFM2 (modified: AFM2); Aflatoxin B2; AFB2 (modified: AFB2); Aflatoxin G2; AFG2 (modified: AFG2); Aflatoxin B2α; AFB2α (modified: AFB2α); Aflatoxin G2α; AFG2α (modified: AFG2α); KB-6806; 6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl) (modified: R 1 = NH2, R 2 = CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivative) (modification: R 1 = NH2, R 2 = CH2CH(CH3)2, R 3 = CH3); Hapten name: KB-6806 (benzimidazole derivative) (modification: R 1 = NH2, R 2 = CH(CH2CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivative) (modification: R 1 = NHCOCH3, R 2 = CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivative) (modification: R 1 = H, R 2= CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivative) (modification: R 1 = NH2, R 2 = CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivative) (modification: R 1 = NH2, R 2 = CH(CH3)2, R 3 = =N(→O)CH3 (N-oxide); KB-6806 (benzimidazole) derivative (modification: R 1 = NH2, R 2 = CH(CH3)2, R 3 = H); KB-6806 (benzimidazole derivatives) (modification: R 1 = NH2, R 2 = CH2CH3, R 3= CH3);Aminopraoxon;Phosphoric acid;O,O-Diethyl O-(4-aminophenyl) ester;Methyl parathion;Phosphorothioic acid;O,O-Dimethyl O-(4-nitrophenyl) ester;Diethylphenyl phosphate;Phenylphosphonic acid;O,O-Diethyl ester;Diethyl phosphate;Ethylphosphonic acid;O,O-Diethyl ester;p-Nitrophenylphosphate;Phosphonic acid;O-(4-nitrophenyl) ester;Folate;Phosphorodithioic acid;O,O-Diethyl S-[(ethylthio)methyl] ester;Ethion;Bis(phosphorodithioic acid);S,S'-Methylene O,O,O',O'-tetraethyl ester;Carbophenothione;Phosphorodithioic acid;O,O-Diethyl S-[[(4-chlorophenyl)thio]methyl] ester;Disulfoton;Phosphorodithioic acid;O,O-Diethyl S-[(2-Ethylthio)ethyl] ester; TS; N-[4-(carboxymethyl)-2-thiazolyl)sulfanilamide; NS; N-(4-nitrophenyl)sulfanilamide; sulfamoxole; sulfacetamide; DNP-SL; spin-labeled dinitrophenyl (modification: the synthesis of DNP-SL was reported by Balakrishnan et al. (1982); the formula is given in Anglister et al. (1984)); β-ecdysone; benzimidazole derivatives; 5(6)-[carboxypentyl)thio]-2-(methoxycarbonyl)amino]-benzimidazole; 2-hydroxybiphenyl; H BP; atrazine caproate; lysophosphatidic acid (LPA); 1-acyl-2-hydroxy-sn-glycero-3-phosphate; berberine; palmatine; 9-acetylberberine; coridalin; coptisine; berberrubine; 8-oxoverbaine; papaverine; berberine derivatives; 9-O-carboxymethylberberine; phencyclidine; 1-(1-phenylcyclohexyl)piperidine; methoxychlor; endosulfan derivatives; 4-oxobutanoic acid, 4-(4,5,6,7,8,8-hexachloro-3a,4,7,7a-tetrahydro-4,7-methano-1H-indenyl-1-oxy); endosulfan derivatives;4-Oxybutanoic acid, 4-(1,3,4,5,6,7,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindanyl-2-oxy); Endosulfan derivatives (modified: hemisuccinate of endosulfandiol); Triazole derivatives; 5-(3-hydroxypropyl)-3-amino-2H-1,2,4-triazole; Triazole derivatives; 5-(3-hydroxypropyl)-3-(2-nitrophenylsulfenyl)amino-2H-1,2,4-triazole; Triazole derivatives; 3-amino-5-[(3-succinyloxy) 4-Methyl-1,2,4-triazole-3-thiol;Triazole derivatives;(1,2,4-Triazol-2-yl)acetic acid;1,2,4-Triazole;4-Nitrophenyl-4'-carboxymethylphenyl Phosphates;Triazole derivatives;4-Amino-1,2,4-triazole;Triazole derivatives;3-Acetamido-1H-1,2,4-triazole;Triazole derivatives;3-Amino-1,2,4-triazole-5-carboxylic acid hemihydrate;Triazole derivatives;2-(4-Chlorophenyl)-2-(1,2,4-triazol-1-yl)-methylhexanoic acid;Succinic acid;Imidazole;L-Histidine;L-Glutamic acid;Permethrin derivatives;3-Phenoxybenzyl 2,2-Dimethylcyclopropane-1,3-dicarboxylate;3-Phenoxybenzaldehyde;Flucythrinate;Chrysanthemic acid;2,4-Dinitrophenyl;DNP;Thiram haptens;4-[Carbodithioate(methyl)-amino]butanoic acid disodium salt;Thiram haptens;5,11-Dimethyl-6,10-dithioxo-7,9-dithia-5,11-diazadodecanoic acid;Thiram haptens;2-{[(dimethylamino)carbothioyl]sulfanyl}ethanoic acid;Thiram haptens;4-{[(dimethylamino)carbothioyl]sulfanyl}butanoic acid;Thiram haptens6-{[(Dimethylamino)carbothioyl]sulfanyl}hexanoic acid;Thiram haptens;11-{[(Dimethylamino)carbothioyl]sulfanyl}undecanoic acid;Thiram haptens;2-{[(Dimethylamino)carbothioyl]sulfanyl}ethanoic acid;Thiram;Tetramethylthiuram monosulfide;Tetraethylthiuram disulfide;Dimethyldithiocarbamic acid sodium salt;Dimethyldithiocarbamic acid zinc salt;Diethyldithiocarbamic acid sodium salt;N,N,N',N'-Tetramethylthiourea;Nabam;Zineb;Maneb;Ethylenethiourea;Chlorpyrifos hapten;O,O-Diethyl O-[3,5-dichloro-6-[(2-carboxyethyl)thio]-2-pyridyl] Phosphorothioates;2-Succinamidobenzimidazole;2-Benzimidazolecarbamic acid methyl ester;MBC;Benzimidazole;2-Benzimidazolylurea;Succinamide;Ethyl carbamate;Urea;N-Methylurea;N,N'-Dimethylurea;Brevetoxin PbTx-3;Organophosphorus haptens;O,O-Diethyl O-(5-carboxy-2-fluorophenyl) Phosphorothioate;Chlorpyrifos-ethyl;Anandamide hapten;N-arachidonyl-7-amino-6-hydroxyheptanoic acid;Anandamide;Arachidonic acid;Docosatetraenoylethanolamide;Dihomo-γ-linolenylethanolamide;2-Arachidonylglycerol;2-Arachidonylglycerol ether;Stearoylethanolamide;Heptadecanoylethanolamide;Prostaglandin E1;3-Hydroxy-2-(3-hydroxy-1-octenyl)-5-oxocyclopentaneheptanoic acid;Alprostadil;PGE1;Prostaglandin D2;PGD2;Prostaglandin A2;PGA2;Prostaglandin B2;PGB2;Prostaglandin F; 2α ;7-[3,5-dihydroxy-2-(3-hydroxy-1-octenyl)cyclopentyl]-5-heptenoic acid;Dinoprost;PGF 2α Prostaglandin F 1α ;PGF 1α ;6-keto-prostaglandin F 1α ;6-keto-PGF 1α;13,14-dihydro-15-keto-prostaglandin E2;13,14-dihydro-15-keto-PGE2;13,14-dihydro-15-keto-prostaglandin F 2α ;14-dihydro-15-keto-PGF 2α ;5α,7α-dihydroxy-11-ketotetranorpostane-1,16-dioic acid;15-keto-PGF 2α ;TXB2;prostaglandin E2;7-[3-hydroxy-2-(3-hydroxy-1-octenyl)-5-oxocyclopentyl]-5-heptenoic acid;dinoprostone;PGE2;hCG-α-(59-92)-peptide (34 residues); Paraquat derivatives; Paraquat hexanoate (PQ-h); Monoquat; Diquat; 9,10-Dihydro-8a,10a-diazoniaphenanthrene; MPTP; 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine; 1,2-Naphthoquinone; N-Acetyl-S-(1,2-dihydroxy-4-naphthyl)cysteine; N-Acetyl-S-(1,4-dihydroxy-2-naphthyl)cysteine; N-Acetyl-S-(1,2-dihydroxy-1-hydroxy-1-naphthyl)cysteine; 2-Chloro-2',6'-diethylacetoacetate 2-Chloro-2',6'-diethylacetanilide (CDA) hapten;2-[2-chloro-(2',6'-diethyl)acetanilide]ethanoic acid;2-Chloro-2',6'-diethylacetanilide (CDA) hapten;2-[2-chloro-(2',6'-diethyl)acetanilide]butanoic acid;2-Chloro-2',6'-diethylacetanilide (CDA) hapten;5-(4-chloroacetamido-3,5-diethyl)phenoxypentanoic acid;CDA;2-Chloro-2',6'-diethylacetanilide;HDA;2-Hydroxy-2',6'-diethylacetanilide;2,6-Diethyl aniline;Hydroxyalachlor;Alachlor ESA;Alachlorethanesulfonic acid;Isoproturon hapten;3-(4-Isopropylphenyl)-1-carboxypropyl-1-methylurea;Chlorotoluron;3-(3-chloro-p-tolyl)-1,1-dimethylurea;Metoxuron;3-(3-chloro-4-methoxyphenyl)-1,1-dimethylurea;Metamitron;4-Amino-4,5-dihydro-3-methyl-6-phenyl-1,2,4-triazin-5-one;Mecoprop;(RS)-2-(4-chloro-o-tolyloxy) Propionic acid;Propyzamide;3,5-Dichloro-N-(1,1-dimethylpropynyl)benzamide;Paraquat dichloride;MCPB;4-(4-Chloro-o-tolyloxy)butyric acid;Chlortoluron hapten;N-(3-chloro-4-methylphenyl)-N-methyl-N-carboxypropylurea;Metsulfuron;Methyl 2-[3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)ureidosulfonyl]benzoate;Captopril haptens;Captopril-4-(maleimidomethyl)-cyclohexanecarboxylic acid (MCC);Captopril haptens;Captopril disulfide modified;Mercaptoethanol-MCC;Mercaptoethanol-4-(maleimidomethyl)-cyclohexanecarboxylic acid, modified;Captopril haptens;Captopril without MCC;Acreaticide A;Acreaticide B;Solamardine;Solasonine;Solanine-S;Praprine;Solasodine;Casyanin;Tomatine;Lycopersicin;Tomatidine;3-O-β-D-glucopyranosyl-solasodine;O-α-L-Rhamnosyl-1(1→2)-3-O-β-D-glucopyranosyl -Solasodine;3-O-β-D-galactopyranosyl-solasodine;O-β-D-glucopyranosyl-1(1→3)-3-O-β-D-galactopyranosyl-solasodine;12-hydroxysolamardine;12-hydroxysolasonine;Isoanguivine;Solaberin I;Solaberin II;Xylosyl-β-solamardine;α-Solanine;α-Chaconine;Dioscin;Indole derivatives;β-Indoleacetic acid;2-Bromo-4,6-dinitroaniline;2-Chloro-4,6-dinitroaniline;Tetryl;2,4,6-Trinitrophenyl- n-Methylnitramine;Nitramine;Tetralite;Tetryl;2-Amino-4,6-dinitrotoluene;2,4-Dinitroaniline;3,5-Dinitroaniline;2-Amino-4,6-dinitrobenzoic acid;Disperse Blue 79;N-[5-[bis[2-(acetyloxy)ethyl]amino]-2-[(2-bromo-4,6-dinitrophenyl)azo]-4-ethoxyphenyl]acetamide;1,3-Dinitrobenzene;2,6-Dinitrotoluene;4-Amino-2,6-dinitrotoluene;1,3,5-Trinitrobenzene;Nicelle Golin; Ethylmorphine; 7,8-didehydro-4,5-epoxy-3-ethoxy-17-methylmorphinan-6-ol; Dihydromorphine; Dihydrocodeine; Dihydromorphinone; Hydromorphone; Dihydrocodeinone; Hydrocodone; Naltrexone; N-Cyclopropylmethyl-14-hydroxydihydromorphinone; Dextromethorphan; (±)-3-Methoxy-17-methylmorphinan; Homatropine; Endorphins and their modified derivatives (type: β-endorphin); Metenkephalin; DALEA;D-Ala(2)-D-Leu(5)-enkephalinamide; Vincristine; 22-oxovincaleukoblastine; Leulocristine; VCR; LCR; OCT; 22-oxacalcitriol; OCT-3-HG; 22-oxacalcitriol-3-hemiglutarate; 24(OH)OCT; 24(OH)-22-oxacalcitriol; 1,20(OH)2-hexanol-D3; Synephrine; Epinephrine; 4-[ (1R)-1-Hydroxy-2-(methylamino)ethyl]-1,2-benzenediol; Phenylephrine; Dopamine derivatives; 6-Hydroxydopamine; Tyramine derivatives; 3-Methoxytyramine; Phenethylamine; Benzenethanamine; PEA; m-Tyramine; o-Tyramine; Dimethoxyphenethylamine; Thymidine glycol monophosphate; 5,6-Dihydroxythymidine monophosphate; Thymidine monophosphate; Thymidine glycol Thymine glycol; 5,6-dihydrothymidine; Thymidine; Thymine; 5-methyluracil; 2,4-dihydroxy-5-methylpyrimidine; AMP; Adenosine monophosphate; CMP; Cytidine monophosphate; Carbamazepine; 5-carbamoyl-5H-dibenz[b,f]azepine; Neopterin isomers; D-erythroneopterin; Neopterin isomers; L-erythroneopterin; Neopterin isomers; D-threoneopterin; Bis Pterin isomers; L-erythrobiopterin; Biopterin isomers; D-erythrobiopterin; Biopterin isomers; L-threobiopterin; Biopterin isomers; D-threobiopterin; Pterin-6-carboxylic acid; C7H5NiO3; Pterin; Thromboxane B2; (5Z,9α,13E,15S)-9,11,15-trihydroxythromboxane-5,13-dien-1-oic acid; 15-keto prostaglandin F; 2α;Fumonisin B1;Macrofusin;FB1;Thyroliberin;TRH;Thyrotropin-releasing factor;Thyrotropin-releasing hormone;TRF;Protirelin;Lopremone;Thyroliberin-OH;TRH-OH;Diketopiperazine;Cyclo(HP);TRH analog;Methylated TRH;TRH analog;TRH extended peptide;TRH-Gly;TRH extended peptide;TRH-Gly-Lys-Arg;TRH extended peptide;TRH-Gly-Lys-Arg-Ala;TRH extended peptide;P7(modified:QHPGLRF);TRH extended peptide;P10(modified :SLRQHPGLRF); TRH extension peptide; Ps5 (modified: proTRH[178-199]); TRH extension peptide; TRH-Ps5 (modified: proTRH[172-199]); hypothalamic peptide; LHRH; cyanoginosin LA; cyanoginosin LB; cyanoginosin LR; cyanoginosin LY; cyanoginosin AY; cyanoginosin FR; cyanoginosin YR; Ne-acetyllysine-containing peptide; Gly-Lys(Ac)-e-aminocaproic acid (Aca)-Cys; benzoic acid; benzenecarboxylic acid; phenylformic acid; dolacyl acid; m -Hydroxybenzoic acid;3-Hydroxybenzoic acid;o-Methoxybenzoic acid;2-Methoxybenzoic acid;o-Toluic acid;2-Methylbenzoic acid;o-Chlorobenzoic acid;2-Chlorobenzoic acid;o-Aminobenzoic acid;2-Aminobenzoic acid;Thiosalicylic acid;2-Mercaptobenzoic acid;o-Sulfhydrylbenzoic acid;Salicylamide;2-Hydroxybenzamide;Saligenin;Saligenol;o-Hydroxybenzyl alcohol;Salicyl alcohol;2-Cyanophenol;2-Hydroxyphenylacetic acid;p-Hydroxybenzoic acid;p-Aminobenzoin Acid;4-Aminobenzoic acid;Vitamin Bx;Bacterial vitamin H1;p-Toluic acid;p-Methylaminobenzoic acid;p-Chlorosalicylic acid;4-Chloro-2-hydroxybenzoic acid;2,4-Dihydroxybenzoic acid;β-Resorcylic acid;2,4-Dihydroxybenzenecarboxylic acid;BRA;4-Aminosalicylic acid;4-Amino-2-hydroxybenzoic acid;p-Aminosalicylic acid;Gentisic acid;2,5-Dihydroxybenzoic acid;5-Hydroxysalicylic acid;Picolinic acid;o-Pyridinecarboxylic acid;2-Pyridinecarboxylic acid;Picolinic acid N-oxide;3-Hydroxypicolinic acid; 2-Hydroxynicotinic acid; 7-Methylguanine; N; 2 -Carboxymethyl-N 7 -Methylguanine;2-(7-methyl-6-oxo-6,7-dihydro-1H-purin-2-ylamino)acetic acid;7-methylxanthine;7-methyluric acid;7-methyladenine;Guanine;2-Amino-1,7-dihydro-6H-purin-6-one;2-Aminohypoxanthine;Adenine;6-Aminopurine;6-Amino-1H-purine;6-Amino-3H-purine;6-Amino-9H-purine;7-(2-carboxyethyl)guanine;7-CEGua;7-Ethylguanine;2-Amino-7-ethyl-1H-purin-6(7H)-one;7-(2,3-dihydroxypropyl)guanine;2-Amino-7-(2,3-dihydroxypropyl)guanine )-1H-purin-6(7H)-one; 7-(2-hydroxyethyl)guanine; 2-amino-7-(2-hydroxyethyl)-1H-purin-6(7H)-one; 7-(2-[(2-hydroxyethyl)amino]ethyl)-guanine; 2-amino-7-(2-(2-hydroxyethylamino)ethyl)-1H-purin-6(7H)-one; 7-carboxymethylguanine; 2-(2-amino-6-oxo-1,6-dihydropurin-7-yl)acetic acid; fluorescein; urushiol; quinone; biotin; His tag; FLAG tag; Strep tag; Myc tag; HA tag; Spot tag; or NE tag; or any combination thereof

[0169] In some embodiments, the hapten comprises fluorescein or a derivative thereof.

[0170] In some embodiments, the hapten comprises DNP or a derivative thereof.

[0171] "Targeting moiety," as used herein, has its common and ordinary meaning, including, but not limited to, a specific group or site on a molecule or chemical that is a binding target for another chemical or protein of interest. In some of the embodiments described herein, the targeting moiety is a hapten. Examples of haptens useful in the embodiments provided herein are listed in Table 1. In some embodiments, the CAR comprises an antibody or portion thereof, which is, for example, one or more binding domains or comprises one or more CDRs. Examples of antibodies or antigen-binding portions thereof useful in the embodiments provided herein include, but are not limited to, antibodies against the haptens listed in Table 1 and the antibodies listed in Table 2. Table 2: Examples of antibodies useful in the embodiments described herein Anti-3-methylindole antibody; 3F12; anti-3-methylindole antibody; 4A1G; anti-3-methylindole antibody; 8F2; anti-3-methylindole antibody; 8H1; anti-3-methylindole antibody; anti-fumonisin B1 antibody; anti-1,2-naphthoquinone antibody; anti-15-acetyldeoxynivalenol antibody; anti-(2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propanol) antibody (anti-DTP antibody); anti-22-oxacalcitriol antibody (As-1, As-2 and As-3; anti-(24,25(OH)2D3) antibody (Ab11); anti-(24,25(OH)2D3) antibody (Ab3); anti-(24,25(OH)2D3) antibody (Ab3-4); anti-2,4,5-trichlorophenoxyacetic acid antibody; anti-(2,4,5-trichlorophenoxyacetic acid) antibody; anti-(2,4,6-trichlorophenol) antibody; anti-(2,4,6-trichlorophenol) antibody; anti-2,4,6-trinitrotoluene (TNT) antibody; anti-2,4-dichlorophenoxyacetic acid (MAb B5 / C3; E2 / B5; E2 / G2; F6 / C10; and F6 / E5; anti-(2,4-dichlorophenoxyacetic acid) antibody; anti-2-hydroxybiphenyl antibody; anti-(3,5,6-trichloro-2-pyridinol) antibody (LIB-MC2; LIB-MC3); anti-(3,5,6-trichloro-2-pyridinol) antibody (LIB-MC2 MAb); anti-3-acetyldeoxynivalenol (3-AcDON) antibody; anti-3-phenoxybenzoic acid (3-PBAc) antibody; anti-4-nitrophenol antibody; anti-4-nitrophenyl 4'-carboxymethylphenylphosphate antibody; anti-7-(carboxyethyl)guanine (7-CEGua) antibody (antibody group specific for 7-meGua); anti-7-methylguanine (7-MEGua) antibody; anti-ABA antibody; anti-acephate antibody (antiserum 8377); anti-acetyllysine antibody (mAb AL3D5; AL11; AKL3H6; AKL5C1; anti-Aculaetiside-A antibody; anti-Aflatoxin M1 (AFM1) antibody (mAb A1; N12; R16; FF32); anti-Agatharesinol antibody; anti-Agatharesinol antibody; anti-Amidochlor antibody; anti-Amitrole antibody (anti-1a-BSA antibody); anti-Ampicillin antibody (AMPI I 1D1 and AMPI II 3B5); anti-Anandamide antibody (9C11; C9C;30G8.E6C; 7D2.E2b; 13C2 MAb); anti-atrazine antibody; anti-atrazine antibody; anti-atrazine antibody; anti-atrazine antibody; anti-atrazine antibody; anti-atrazine antibody; anti-atrazine antibody (4063-21-1 MAb cell line mAb and scAb); anti-atrazine antibody (4D8 scAb and 6C8 scAb); anti-atrazine antibody (C193); anti-atrazine antibody (rabbit / sheep); anti-atrazine antibody (K4E7); anti-atrazine antibody (MAb: AM7B2.1); anti-atrazine antibody (scAb); anti-atrazine mercapturic acid antibody; anti-(azinphosmethyl) antibody (MAb: LIB-MFH14; LIB-MFH110); anti-benalaxyl antibody; anti-benzimidazole carboxylic acid; anti-benzimidazole antibody (Ab 587); anti-benzo[a]pyrene antibodies; anti-benzo(a)pyrene antibodies (10C10 MAb and 4D5 MAb); anti-(benzoylphenylurea) antibodies (mainly anti-diflubenzuron antibodies); anti-berberine antibodies; anti-β-indoleacetic acid antibodies; anti-biopterin (L-erythro form) antibodies; anti-brevetoxin PbTx-3 antibodies; anti-bromacil antibodies; anti-bromophos antibodies; anti-bromophosethyl antibodies; anti-butachlor antibodies; anti-captopril-MCC antibodies; anti-carbamazepine (CBZ) antibodies; anti-carbaryl antibodies; anti-carbaryl antibodies (LIB-CNH32; LIB-CNH33, LIB-CNH36; LIB-CNH37; LIB-CNH45; LIB-CNA38); anti-carbaryl antibodies (LIB / CNH-3.6 MAb; anti-carbofuran antibody (LIB-BFNB-52; LIB-BFNB-62; LIB-BFNB-67); anti-carbofuran antibody (LIB-BFNP21); anti-CDA antibody; anti-CDA antibody (anti-2-[2-chloro-(2,6'-diethyl)acetanilide]butanoic acid); anti-CDA antibody (anti-2-[2-chloro-(2',6'-diethyl)acetanilide]ethanoic acid); anti-CDA antibody (anti-5-(4-chloroacetamido-3,5-diethyl)phenoxypentanoic acid); anti-ceftazidime antibody; anti-(chlorodiamino-s-triazine) antibody (anti-CAAT) (PAb1-8); anti-chlorothalonil antibody; anti-chlorpyrifos antibody; anti-chlorpyrifos antibody; anti-chlorpyrifos antibody (LIB-AR1.1 MAb; LIB-AR1.4 MAb); anti-chlorpyrifos antibody (LIB-C4);Anti-(chlorpyrifos) antibody (LIB-C4 MAb); anti-chlorpyrifos antibody (LIB-PN1 MAb); anti-chlorpyrifos antibody (LIB-PN2 MAb); anti-chlorpyrifos antibody (LIB-PO MAb); anti-chlorsulfuron antibody; anti-chlorsulfuron antibody; anti-chlortoluron antibody (antiserum); anti-cyanoginosin LA antibody (mAb 2B2-2; 2B2-7; 2B2-8; 2B2-9; 2B2-10; 2B5-5; 2B5-8; 2B5-14; 2B5-15; 2B5-23); anti-(D-3-methoxy-4-hydroxyphenylglycol) antibody; anti-DDA antibody; anti-DDT antibody (PAb and MAb); anti-DDT MAb (LIB1-11; LIB5-21; LIB5-25; LIB5-28; LIB5-212; LIB5-51; LIB5-52; LIB5-53); anti-DEC antibody (anti-diethylcarbamazine antibody); anti-DEHA antibody; anti-(Delor 103) antibody; anti-deltamethrin antibody; anti-deltamethrin antibody (Del 01 to Del 12 MAb and PAb); anti-deoxynivalenol (DON) antibody; anti-deoxynivalenol (DON) antibody; anti-dexamethasone antibody; anti-dexamethasone antibody; anti-dinitrophenyl (DNP) antibody; spin-labeled anti-dinitrophenyl antibody (AN01 to AN12); anti-diuron antibody (MAb: 21;60;195;202;275;481;488;520); anti-D-MHPG antibody; anti-DNC antibody; anti-EB1089 antibody; anti-ecdysone antibody; anti-endosulfan antibody; anti-endosulfan antibody; anti-esfenvalerate antibody (Ab7588); anti-estradiol antibody; anti-fenitrothion antibody (pAb and mAb); anti-fenpropimorph antibody; anti-fenthion antibody; anti-fenthion antibody; anti-FITC antibody (B13-DEI); anti-flucofuron antibody (F2A8 / 1 / A4B3); anti-flufenoxuron antibody; and anti-(benzoylphenylurea) antibody; anti-formononetin antibody; anti-furosemide antibody (Furo-26 MAb; Furo37 MAb; furo-72 MAb; Furo 73 MAb). MAb); anti-GR151004 antibody; anti-hCG-α-peptide antibody (FA36); anti-hydroxyatrazine antibody (HYB-283-2); anti-hydroxysimazine antibody; anti-imazalil antibody (MoAb) (9C1-1-1; 9C5-1-1; 9C6-1-1; 9C8-1-1;9C9-1-1; 9C12-1-1; 9C14-1-1; 9C16-1-1; 9C18-1-1; 9C19-1-1; 9E1-1; 9G2-1; anti-irgarol antibody; anti-isopentenyladenosine antibody; anti-isoproturon antibody; anti-KB-6806 antiserum; anti-(+) lupanine antibody; anti-lysophosphatidic acid (LPA) antibody; anti-M3G Ab1 and Ab2; anti-M3G Ab1 and Ab2; anti-MBC antibody (anti-2-succinamide benzimidazole antiserum); anti-metanephrine antibody; anti-(+) methamphetamine antibody; anti-methiocarb antibody (LIB-MXNB31; LIB-MXNB-33; LIB-MXNH14 and LIB-MXNH-15 MAb); anti-metolachlor antibody; anti-metolachlor antibody (MAb 4082-25-4); anti-molinate antibody; anti-monuron antibody; anti-morphine-3-glucuronide (E3 scFv antibody); anti-morphine antibody; anti-morphine antibody (mAb 8.2.1; 33.2.9; 35.4.12; 39.3.9; 44.4.1; 76.7F.16; 83.3.10; 115.1.3; 124.2.2; 131.5.13; 158.1.3; 180.2.4); anti-neopterin (D-erythro form) antibody; anti-nicarbazin antibody (Nic 6; Nic 7; Nic 8; and Nic 9); anti-nicergoline antibody (Nic-1; Nic-2; Nic-3 & BNA-1; BNA-3; anti-norflurazon antibody; anti-normetanephrine antibody; anti-(o-DNCP) antibody; anti-P10 antibody (TRH-extended peptide); anti-paraoxon antibody (BD1 and CE3); anti-paraquat antibody; anti-paraquat antibody; anti-parathion-methyl antibody; anti-PCB antibody (antibody to 3,3',4,4'-tetrachlorobiphenyl) MAb S2B1; anti-pentachlorophenol antibody; anti-pentachlorophenol antibody; anti-permethrin antibody (MAb Py-1; Py-3 and Py-4); anti-phencyclidine antibody (MAb 6B5) Fab); anti-phenobarbital antibody; anti-phenobarbital antibody; anti-(p,p'-DDT) antibody (LIB-DDT-35 and LIB-DDT5-52); anti-premethrin antibody (Ab549); anti-propoxur antibody (LIB-PRNP15; LIB-PRNP21; LIB-PRNB21; LIB-PRNB33);Anti-prostaglandin E2 antibody; anti-p-tyramine antibody; anti-pyrene antibody; anti-retronecin antibody; anti-salicylic acid antibody; anti-sennoside A antibody (MAb 6G8); anti-sennoside B antibody (MAb: 7H12; 5G6; 5C7); anti-simazine antibody; anti-sulfonamide antibody (anti-TS); anti-sulcofuron antibody (S2B5 / 1 / C3); anti-sulfamethazine antibody (21C7); anti-synephrine antibody; anti-thiabendazole antibody (antibody 300); anti-thiabendazole antibody (antibody 430 and antibody 448); anti-thiram antibody; anti-THP antibody (7S and 19S); anti-thromboxane B2 antibody; anti-thymidine glycol monophosphate antibody (mAb 2.6F.6B.6C); anti-thyroliberin (TRH) antibody; anti-TNT antibody (AB1 antiserum and AB2 antiserum); anti-triadimefon antibody; anti-triazine antibody (AM1B5.1); anti-triazine antibody (AM5C5.3); anti-triazine antibody (AM5D1.2); anti-triazine antibody (AM7B2.1); anti-triazine antibody (SA5A1.1); anti-triazine serum (anti-ametryne); anti-triazine serum (anti-atrazine); anti-triazine serum (anti-simazine); anti-triazine serum (anti-simetryne); anti-trifluralin antibody; anti-trifluralin antibody; anti-vincristine antibody; anti-zearalenone antibody; anti-zeatin riboside antibody; E2 G2 and E4 C2; Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain); LIB-BFNP23 MAb; MAb H-7 and H-9 (MAbs against O,O-diethyl OP peptide); MoAb 33A7-1-1; MoAb 33B8-1-1; MoAb 33C3-1-1; MoAb 3C10-1-1 and MoAb 3E17-1-1; MoAb 45D6-5-1; MoAb 45E6-1-1; MoAb 45-1-1; a variant (GlnL89Glu) of the Fab fragment K411B (an IgG2b isotype with a kappa light chain) derived from MAb K4E7; a variant (GlnL89Glu / ValH37Ile / GluL3Val) of the Fab fragment K411B (an IgG2b isotype with a kappa light chain) derived from MAb K4E7; MAb Fab fragment K411B (isotype IgG2b with kappa light chain) derived from K4E7, mutant (GlnL89Glu / ValH37Ile / GluL3Val);Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GlnL89Glu / ValH37Ile); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GlnL89Glu / ValH37Ile); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GluH50Gln); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GluH50X); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GlyH100aAla); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GlyH100aSer); MAb Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (HisH95Phe); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (HisH95Tyr); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (PheL32Leu); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (TrpH33Phe,Tyr,Leu); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (Tryl96Phe); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (TryL96Phe); A mutant (ValH37Ile) of the Fab fragment K411B (isotype IgG2b with kappa light chain) derived from MAb K4E7; a mutant (ValH37Ile) of the Fab fragment K411B (isotype IgG2b with kappa light chain) derived from MAb K4E7; P6A7 MAb; PNAS2 6 / 3 56(1)-1-5-1; PNAS2 6 / 3 56(1)-1-5-2; PNAS2 6 / 3 56(1)-1-10-4; PNAS2 6 / 3 56(1)-1-10-5 and PNAS2 6 / 3 56(1)-3-1-5; Alexa Fluor 405 / Cascade Blue dye antibody; Alexa Fluor 488 dye antibody;BODIPY FL dye antibodies; dansyl antibodies; fluorescein / Oregon Green dye antibodies; Lucifer Yellow dye antibodies; tetramethylrhodamine and rhodamine red dye antibodies; Texas Red and Texas Red-X dye antibodies; biotin antibodies; dinitrophenyl antibodies; or nitrotyrosine antibodies; or binding fragments or CDRs of the foregoing antibodies; or any combination of the foregoing antibodies, their binding fragments or their CDR domains;

[0172] As used herein, a "marker sequence" encodes a protein used to select or track cells containing a protein of interest or a protein of interest. In embodiments described herein, fusion proteins are provided that may include a selectable marker sequence in experiments such as flow cytometry. In some embodiments, the marker is Her2tG, CD19t, or EGFRt.

[0173] As used herein, "scFv" refers to a fusion protein of an immunoglobulin heavy chain variable region (VH) and light chain variable region (VL) linked by a short linker peptide consisting of 10 to 25 or 10 to about 25 amino acids. In some embodiments, a CAR is provided that includes an scFv specific for a tumor cell surface molecule or a hapten displayed on a cell.

[0174] As used herein, the term "ribosomal skipping sequence" refers to a sequence that functions to cause translating ribosomes to "skip" the ribosomal skipping sequence, preventing peptide bond formation and allowing translation to begin from the region immediately following the ribosomal skipping sequence. For example, some viruses have ribosomal skipping sequences, which allow multiple proteins to be translated consecutively from a single nucleic acid, and the translated proteins are obtained as separate proteins without being linked by peptide bonds. Herein, the ribosomal skipping sequence is used as a "linker" sequence. In some embodiments of the nucleic acids provided herein, the nucleic acids of the invention contain a ribosomal skipping sequence between the sequence encoding the chimeric antigen receptor and the sequence encoding the marker protein, thereby allowing the chimeric antigen receptor and the marker protein to be co-expressed without being linked by a peptide bond. In some embodiments, the ribosomal skipping sequence is a P2A sequence, a T2A sequence, an E2A sequence, or an F2A sequence. In some embodiments, the ribosomal skipping sequence is a T2A sequence. In some embodiments, there is a ribosomal skipping sequence between two chimeric antigen receptors and a second ribosomal skipping sequence between one of the chimeric antigen receptors and the marker.

[0175] "Biotin" has its common and ordinary meaning in the context of this specification, including, but not limited to, the water-soluble B vitamins. In embodiments herein, the hapten is biotin.

[0176] "Fluorescein" has its common, ordinary meaning in the context of this specification, and includes, but is not limited to, a synthetic organic compound that is soluble in water and alcohol. Fluorescein is widely used as a fluorescent tracer in a variety of applications. In some embodiments of the present invention, fluorescein is a targeting moiety on a lipid that is recognized by and bound by a chimeric antigen receptor. In some embodiments, the hapten is fluorescein or a derivative thereof. In some embodiments, the lipid is a phospholipid (e.g., an ether phospholipid, etc.).

[0177] As used herein, 2,4-dinitrophenol (2,4-DNP or simply DNP) is an organic compound with the formula HOC6H3(NO2)2 and has its common, ordinary meaning in the context of this specification. DNP is used, among other things, as a preservative, a nonselective bioaccumulating pesticide, and a herbicide. DNP is also an intermediate chemical in the manufacture of sulfur dyes, wood preservatives, and picric acid. In some embodiments of the present invention, DNP is a targeting moiety on a lipid and is recognized and bound by a chimeric antigen receptor. In some embodiments, the hapten is DNP or a derivative thereof. In some embodiments, the lipid is a phospholipid (e.g., an ether phospholipid, etc.).

[0178] As used herein, the term "lipid" has its common, ordinary meaning, including, but not limited to, a type of organic compound containing a carbon chain, a fatty acid, or a fatty acid derivative, which is generally insoluble in water but can be miscible or mixed in a hydrophobic or organic solvent. Lipids include, but are not limited to, fats, waxes, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, sphingolipids, cerebrosides, ceramides, or phospholipids. As used herein, amphipathic lipids may have a polar head group and a hydrophobic moiety or portion thereof. The term "hydrophobic group" or hydrophobic portion has its common, ordinary meaning, including, but not limited to, a molecule or portion thereof that tends to repel water and has no polarity. The hydrophobic group may include an alkane or an oil or fat. The lipid may also include, but is not limited to, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, or a polyketide.

[0179] In some embodiments, the lipid may be a sphingolipid. Sphingolipids may contain a backbone consisting of a sphingoid base, such as a series of aliphatic amino alcohols, including sphingosine. A sphingolipid in which the R group consists solely of hydrogen atoms is ceramide. Other common R groups include phosphocholine (when the R group is phosphocholine, sphingomyelin is obtained) and various sugar monomers or sugar dimers (when the R group is a sugar monomer, cerebrosides are obtained, and when the R group is a sugar dimer, globosides are obtained). Cerebrosides and globosides are known collectively as glycosphingolipids. In some embodiments, the lipid is a glycosphingolipid.

[0180] According to the present 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 chain. In some embodiments, the fatty acid is 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 steroid or a terpenoid lipid, such as cholesterol. In some embodiments, the hydrophobic group comprises an ether linkage between the polar head group and the fatty 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.

[0181] In some embodiments, the lipid is a short-chain alkyl phospholipid.

[0182] In some embodiments, the lipid comprises a synthetic alkylphospholipid structure, such as edelfosine, perifosine, or erucylphosphocholine. In some embodiments, the lipid is lysophosphatidylcholine, edelfosine, 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, which is incorporated herein by reference in its entirety). In some embodiments of the lipids described herein, the choline in the polar head group can be replaced with a piperidine moiety. In some embodiments, the lipid is an anticancer alkylphospholipid. Anticancer phospholipids have been 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, which is incorporated herein by reference in its entirety.

[0183] In some embodiments, the lipids provided herein are structurally related synthetic antitumor agents characterized by their ability to interact 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, which is expressly incorporated herein by reference in its entirety). 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, hi some embodiments, the lipid is LysoPC, edelfosine, irmofosine, miltefosine, perifosine, erucylphosphocholine, or erufosine.

[0184] As used herein, the term "polar head group" has its common and ordinary meaning in this specification, including, but not limited to, the hydrophilic group of a lipid (such as a phospholipid). The term "phospholipid" has its common and ordinary meaning in this specification, including, but not limited to, a special type of lipid that has amphipathic properties and is therefore capable of forming lipid bilayers. A phospholipid molecule comprises at least one hydrophobic fatty acid "tail" and a hydrophilic "head" or "polar head group." In embodiments herein, a phospholipid or ether phospholipid comprises a polar head group. In some embodiments, the polar head group comprises a phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the lipid comprises a targeting moiety, and the CAR binds to the lipid through an interaction with the targeting 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. In some embodiments, a complex comprising a lipid is provided. 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 polar head group and an alkyl carbon chain. In some embodiments, the polar head group comprises a choline, phosphatidylcholine, sphingomyelin, a phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidylserine, or phosphatidylinositol. In some embodiments, the polar head group comprises a phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the sugar residue is glycerol. In some embodiments, the polar head group comprises a saccharide group. In some embodiments, the lipid comprises a mannose-containing head group. 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 lactosylceramide. In some embodiments, the lipid is a glycolipid. In some embodiments, the glycolipid comprises a sugar unit such as n-glucose, n-galactose, or N-acetyl-n-galactosamine. In some embodiments, the lipid comprises a hydrocarbon ring such as a sterol.

[0185] In some embodiments, the polar head group of the lipid comprises glycerol. In some embodiments, the polar head group of the lipid comprises a phosphate group. In some embodiments, the polar head group of the lipid comprises a choline. In some embodiments, the lipid is phosphatidylethanolamine. In some embodiments, the lipid is phosphatidylinositol. In some embodiments, the lipid comprises a backbone consisting of a sphingoid base. In some embodiments, the lipid comprises a sterol lipid, such as cholesterol or a derivative thereof. In some embodiments, the lipid comprises a saccharolipid. In some embodiments, the polar head group comprises choline, phosphate, or glycerol.

[0186] In some embodiments, the lipid is a glycolipid. In some embodiments, the lipid comprises a sugar. In some embodiments, the lipid is derived from sphingosine. In some embodiments, the lipid is a glyceroglycolipid or a glycosphingolipid.

[0187] In some embodiments, the lipid is an ether lipid having a hydrophobic branched chain.

[0188] As used herein, "ether phospholipid" has its common and ordinary meaning in this specification, including, but not limited to, a lipid in which one or more carbon atoms of the polar head group are attached to an alkyl chain via an ether bond, rather than the more common ester bond. In some embodiments, the polar head group is glycerol.

[0189] As used herein, the term "antibody" has its common, ordinary meaning, and may refer to a large, Y-shaped protein produced by plasma cells that functions to identify and neutralize foreign substances, such as bacteria and viruses, in the immune system. An antibody protein may contain four polypeptide chains: two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain is composed of structural domains called immunoglobulin domains. These domains can contain 70-110 amino acids and are classified into various categories based on their size and function. In some embodiments, the CDR regions are found within the antibody domains. According to Kabat numbering, the light chain contains CDRL1 (amino acids 24-34), CDRL2 (amino acids 50-56), and CDRL3 (amino acids 89-97). The heavy chain contains CDRH1 (amino acids 31-35), CDRH2 (amino acids 50-65), and CDRH3 (amino acids 95-102). The CDR regions in an antibody can be readily determined.

[0190] Examples of antibodies or binding fragments thereof that can be used to bind to target moieties include monoclonal antibodies, bispecific antibodies, Fab, Fab2, Fab3, scFv, Bis-scFv, minibodies, triabodies, diabodies, tetrabodies, VhH domains, V-NAR domains, IgNAR, and camelid Ig. Other examples of antibodies include IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, IgE, IgD, and IgA. Further examples of antibodies include, but are not limited to, human antibodies, humanized antibodies, or chimeric antibodies. Examples of recombinant antibodies include, but are not limited to, antibodies that specifically bind to NGF.

[0191] The antibody or binding fragment thereof may be specific for a target moiety, for example, an antigen present on a tumor or a hapten. Examples of haptens useful in the embodiments provided herein are listed in Table 1.

[0192] Any of the cancer-specific antibodies described herein may bind to an antigen on a cancer cell, e.g., a tumor cell. Particular tumor cell antigens to which antibodies capable of binding to targeting moieties can be generated include, for example, angiopoietins, transmembrane receptors, cell adhesion molecules, cluster of differentiation molecules, gangliosides, glycoproteins, growth factors, integrins, interleukins, Notch receptors, transmembrane glycoproteins, tumor necrosis factors, and tyrosine kinases. In some embodiments, tumor cell antigens include, for example, 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, DL Examples of hapten-targeting moieties include L4, EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, VEGF, or other cancer-expressed antigens. Additionally, antibodies that may bind to a hapten-targeting moiety are also contemplated. Examples of haptens useful in the embodiments provided herein are listed in Table 1.

[0193] In the embodiments described herein, several types of "spacers" are contemplated for use. A chimeric antigen receptor spacer refers to a polypeptide spacer, the length of which is selected to modulate the binding affinity of the chimeric antigen receptor to the target, e.g., to increase or improve the binding affinity of the chimeric antigen receptor to the target. Lipids may also contain a spacer to distance the targeting moiety from the lipid, and this spacer is attached to the polar head group of the lipid. Polypeptide spacers selected for use in chimeric antigen receptors may be screened to identify specific spacers configured to improve desired binding properties, such as avidity for the targeting moiety (e.g., a desired interaction with the receptor or a desired avidity with the receptor). Regarding lipid-specific spacers, the lipid spacer may comprise a PEG spacer, a hapten spacer, a small peptide, or an alkane chain. In some embodiments, the hapten spacer comprises two haptens and is referred to as a hapten (2x) spacer. In some embodiments, the lipid comprises a hydrophobic group, such as an alkane chain. In some embodiments, the alkane chain can 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 defined by any two of these numbers. In some embodiments, the PEG spacer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 PEG molecules, or a number of PEG molecules within a range defined by any two of these numbers.

[0194] As used herein, "cytotoxic T lymphocytes (CTLs)" refer to T lymphocytes that express CD8 on the cell surface (e.g., CD8+ T cells). In some embodiments, such cells are antigen-experienced "memory" T cells (T MIn some embodiments, the cells are provided for secreting the fusion protein. In some embodiments, the cells are cytotoxic T lymphocytes. As used herein, "central memory" T cells (or "T CM ") refers to antigen-experienced cytotoxic T lymphocytes (CTLs) that express CD62L, CCR-7 and / or CD45RO on their surface, but do not express CD45RA or have reduced expression of CD45RA compared to naive cells. In some embodiments, cells are provided for secreting the fusion protein. In some embodiments, the cells are central memory T cells (T CM In some embodiments, central memory cells may express positively CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95, but may have reduced expression of CD54RA, compared to naive cells. As used herein, "effector memory" T cells (or "T EM ") refers to an antigen-experienced T cell that does not express CD62L or has reduced expression of CD62L on its surface compared to central memory cells, and does not express CD45RA or has reduced expression of CD45RA compared to naive cells. In some embodiments, cells for secreting fusion proteins are provided. In some embodiments, the cells are effector memory T cells. In some embodiments, effector memory cells are negative for CD62L and / or CCR7 expression and may be positive or negative for CD28 and / or CD45RA expression compared to naive or central memory cells.

[0195] As used herein, "naive" T cells refer to T lymphocytes that have not experienced an antigen and that express CD62L and / or CD45RA but not CD45RO compared to central memory cells or effector memory cells. In some embodiments, cells for secreting fusion proteins are provided. In some embodiments, the cells are naive T cells. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers, including CD62L, CCR7, CD28, CD127, and / or CD45RA.

[0196] As used herein, "T cells" or "T lymphocytes" may be obtained from any mammal, preferably from a primate or other species, including monkeys, dogs, and humans. In some embodiments, the T cells are allogeneic (from the same species but from a different donor) to the recipient subject. In some embodiments, the T cells are autologous T cells (donor and recipient are the same). In some embodiments, the T cells are syngeneic (donor and recipient are different but identical twins).

[0197] As used herein, "precursor T cells" refers to lymphocyte precursor cells that can migrate to the thymus and become precursor T cells, and precursor T cells do not express T cell receptors. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic precursor cells (lymphoid precursor cells) derived from hematopoietic stem cells settle in the thymus and expand by cell division, generating a large population of immature thymocytes. Very early thymocytes do not express either CD4 or CD8, and are therefore double-negative (CD4 - CD8 - ) cells. As their development progresses, they are classified as double-positive thymocytes (CD4 + CD8 + ) and finally became single positive (CD4 + CD8 - or CD4- CD8 + ) They mature into thymocytes and are then released from the thymus into peripheral tissues.

[0198] As used herein, "CD8 T cells" or "killer T cells" are T lymphocytes that can kill cancer cells, virus-infected cells, or damaged cells. CD8 T cells recognize specific antigens, or proteins 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 binds to the presented antigen and can destroy the cell.

[0199] As used herein, "central memory T cells (T CM ")" refers to an antigen-experienced CTL that expresses CD62L or CCR-7, and CD45RO on its surface, but does not express CD45RA or has reduced CD45RA expression, compared to naive cells. In some embodiments, central memory cells are positive for CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95, and have reduced CD54RA expression, compared to naive cells.

[0200] As used herein, "effector memory" T cells (or "T EM ") refers to T cells that have experienced an antigen and do not express CD62L or have reduced CD62L expression on their surface compared to central memory cells, and do not express CD45RA or have reduced CD45RA expression compared to naive cells. In some embodiments, effector memory cells are negative for CD62L and / or CCR7 expression, and may be positive or negative for CD28 and / or CD45RA expression compared to naive or central memory cells. As used herein, the term "effector T cells (T E"T lymphocytes" refer to antigen-experienced cytotoxic T lymphocytes that 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, compared to central memory T cells or naive T cells. In some embodiments, cells are provided for secreting fusion proteins. In some embodiments, the cells are effector T cells. In some embodiments, the cells 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, compared to central memory T cells or naive T cells.

[0201] As used herein, the term "leader sequence" is also known as a signal sequence that enables a protein to be translocated to the cell surface. The leader sequence contained in a CAR refers to the first amino acid sequence contained in the CAR, which is an amino acid sequence that induces cell surface expression. A leader sequence, or signal sequence, may be required for cell surface expression of a protein. In some embodiments, the leader sequence comprises the signal sequence of granulocyte-macrophage colony-stimulating factor.

[0202] The term "hapten-presenting cells (H-APCs)" has its general, ordinary meaning in the context of this specification, including, but not limited to, cells labeled with a hapten. In some embodiments, the hapten is bound to the extracellular surface. In some embodiments, H-APCs can be generated from healthy cells of a patient or cells compatible with the patient, and the resulting cells are labeled with a hapten. Examples of haptens useful in the embodiments provided herein are listed in Table 1. There are various methods for labeling cells with haptens, such as chemicals, peptides, aptamers, lipids, and proteins. One method for loading cells with a hapten involves incubating the cells of interest with fluorescein lipid overnight. One advantage of using fluorescein as a hapten is that its fluorescence can be exploited. Therefore, hapten incorporation can be monitored by detecting the fluorescence of the fluorescein moiety using flow cytometry. Thus, after incubation, excess fluorescein lipid is removed, the cell fraction is analyzed by flow cytometry to analyze hapten incorporation, and the remaining cells can be used for infusion into the patient. After infusion into the patient, H-APCs that are not targeted by CAR T cells gradually lose the hapten (e.g., by being metabolized and removed from the cell surface) and return to their original, healthy cell morphology, making this approach safe. In some embodiments, cells can be transduced to express the hapten on their extracellular surface. In some embodiments, the hapten can be covalently bound to the extracellular surface. In some embodiments, the hapten can be covalently bound to the extracellular surface via a phospholipid, such as an ether phospholipid.

[0203] "Stimulation" or "activation" of T cells refers to a method of inducing T cells to mount a response, such as a signaling response (e.g., proliferation), while maintaining T cell viability and immune function. Stimulating T cells may induce the activity of a CAR-containing T cell response. In some embodiments, the stimulation is carried out using a support to which an antibody is bound, including an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the method further includes removing the support to which the antibody is bound, such as a bead or particle, or a substrate such as a dish or test tube. As described in embodiments herein, T cells containing a CAR specific for a hapten may be stimulated using a hapten-antigen-presenting cell (H-APC), e.g., stimulated ex vivo using a support such as a hapten-bound bead.

[0204] A "chemotherapeutic agent" refers to a class of anti-cancer pharmaceuticals, including chemicals (e.g., anti-cancer drugs (chemotherapeutic agents)) that can be administered as part of a standardized chemotherapy regimen. Chemotherapeutic agents may be administered with curative intent or with the intent of prolonging survival or alleviating symptoms (palliative chemotherapy). Chemotherapy may also include hormonal therapy and targeted therapy, which are major categories of oncology (cancer chemotherapy). These chemotherapeutics are often combined with other cancer therapies, such as radiation therapy, surgery, and / or hyperthermia. Surgery is known to cause cancer spread in a small percentage of cases. In some embodiments, genetically modified immune cells are administered to the tumor site before or after the surgical procedure. In some embodiments herein, the subject receiving CAR T cell therapy treatment is a subject selected for administration of a chemotherapeutic or anti-cancer agent. Some of the newly developed anti-cancer agents (e.g., various monoclonal antibodies, their humanized antibodies, and their binding fragments) are not indiscriminately cytotoxic but target proteins that are aberrantly expressed in cancer cells and are essential for cancer cell proliferation. Such treatments are often referred to as targeted therapies (as distinct from classical chemotherapy) and are often combined with conventional chemotherapeutic agents in antitumor treatment regimens. In some embodiments, the methods of the present invention may further comprise administering one or more of such targeted anticancer therapies (e.g., various monoclonal antibodies, humanized antibodies thereof, and / or binding fragments thereof).

[0205] Chemotherapy involves the administration of chemotherapeutic agents, and one type of agent may be used in a single chemotherapy session (single-agent chemotherapy) or multiple agents may be used simultaneously (combination chemotherapy or polychemotherapy). Chemotherapy combined with radiation therapy is called chemoradiotherapy. Chemotherapy using agents that are converted into cytotoxic substances upon exposure to light is called photochemotherapy or photodynamic therapy. In some embodiments of the methods comprising administering genetically modified immune cells described herein, the methods can further include administering photochemotherapy or photodynamic therapy to a subject with cancer after administering the genetically modified immune cells or genetically modified macrophages (GEMs) to the subject.

[0206] Chemotherapeutic agents include, but are not limited to, antibody-drug conjugates (e.g., antibodies attached to a drug via a linker), nanoparticles (which may be, for example, nanoparticles of 1-1000 nm in size, which improve tumor selectivity and aid in the delivery of low-solubility drugs), electrochemotherapy, alkylating agents, antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, and thioguanine), antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators, or checkpoint inhibitors (e.g., checkpoint kinases CHK1 or CHK2). In some embodiments of the methods described herein, the genetically modified immune cells comprising a CAR, or compositions comprising the genetically modified immune cells comprising a CAR, are administered in combination with one or more anti-cancer agents, such as one or more of the compounds or therapies described above. In some embodiments, the one or more anti-cancer agents co-administered or administered in combination with the genetically modified immune cells of the invention comprise antibody-drug conjugates, nanoparticles, electrochemotherapy, alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators, or checkpoint inhibitors. In some embodiments, the antimetabolite comprises 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, or thioguanine.

[0207] "Cancer" has its common, ordinary meaning in this specification, and includes, but is not limited to, a group of diseases involving the proliferation of abnormal cells that can invade or spread to other parts of the body. Subjects that can be treated using the methods described herein include subjects identified or selected to have cancer, including, but not limited to, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, and / or brain cancer. Identification and / or selection of cancer patients can be performed by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or tumor-associated molecules are known, and the tumors include, but are not limited to, melanoma, breast cancer, brain cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma, prostate cancer, or any combination thereof. The cancer also includes, but is not limited to, B-cell lymphoma, breast cancer, brain cancer, prostate cancer, or leukemia. In some embodiments, the one or more tumorigenic polypeptides are associated with kidney cancer, uterine cancer, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, brain cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, or leukemia. In some embodiments, methods are provided for treating, alleviating, or inhibiting cancer in a subject. In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, kidney cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colon cancer, skin cancer (including melanoma), bone cancer, or brain cancer. In some embodiments, the subject is selected for administration of another cancer therapy, including a cancer therapeutic agent, radiation therapy, chemotherapy, or a drug suitable for cancer therapy.In some embodiments, the agent is abiraterone, alemtuzumab, anastrozole, aprepitant, arsenic trioxide, atezolizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, chemotherapy combinations, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil, fulvest These include lantozole, gemcitabine, imatinib, imiquimod, ipilimumab, ixabepilone, 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.

[0208] "Tumor microenvironment" has its general, ordinary meaning in the present specification, including, but not limited to, the cellular environment in which a tumor exists. The tumor microenvironment may include, but is not limited to, surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and / or extracellular matrix (ECM). In some embodiments herein, CAR-bearing T cells are administered into the tumor environment and stimulated using H-APC.

[0209] Detailed Description Some embodiments of the methods and compositions provided herein relate to the use of hapten-labeled cells to stimulate chimeric antigen receptor (CAR) T cells. In some embodiments, the CAR T cells may comprise a CAR that specifically binds to a hapten. Some embodiments relate to the stimulation of CAR T cells with hapten-labeled cells in vivo or in vitro.

[0210] Chimeric antigen-expressing cells are immune cells engineered to direct T cells toward biomarkers bound to the surface of malignant cells. Utilizing these cell surface targets or antigens allows for targeted and specific therapies that can reduce destruction of healthy tissue and preserve a patient's immune system during treatment. T cells are a critical component of the adaptive immune system, not only because they can orchestrate cytotoxicity but also because they can provide long-term cellular "memory" for specific antigens. While activation of endogenous T cells requires interaction between an MHC-presented peptide and its corresponding TCR, CAR T cells are engineered to activate through tumor-associated antigens (TAA) or tumor-specific antigens (TSA). Thus, CAR T cells can be thought of as "living drugs" containing a targeting domain (single-chain variable fragment (scFv), peptide, polypeptide, ligand, mutein, spacer, and / or linker) fused to a T cell signaling domain. When the targeting domain recognizes and binds to its specific target, the CAR T cell is activated and begins killing the target cell. CAR T-cell therapy has been used as an innovative treatment for hematological malignancies by targeting CD19 and CD20. However, CAR T cells have not been effectively and efficiently applied clinically to solid tumors, and further research is needed to apply them to the treatment of solid tumors. Embodiments provided herein relate to the stimulation of CAR T cells. Furthermore, stimulating CAR T cells may address various challenges currently facing CAR T-cell therapy, such as in vivo persistence and an immunosuppressive tumor microenvironment, which are important for the further development and success of CAR T cells.

[0211] In some embodiments herein, T cells are transduced, transfected, or transformed to express at least two unique CARs (dual CARs) in a single cell, where one of the at least two CARs is specific for a tumor target and the other is specific for a hapten (e.g., fluorescein). In another aspect, T cells are transduced, transfected, or transformed to express a single CAR (bispecific CAR) containing two targeting moieties (e.g., two scFvs), where one targeting moiety is specific for a tumor and the other is specific for a hapten. However, if the tumor is labeled with a hapten, only an anti-hapten CAR is required. Dual CAR T cells and bispecific CAR T cells can be produced by various methods, such as dual transduction with a viral vector, single transduction with a viral vector containing both CARs, or non-viral transposon vectors. There are also various methods for selecting pure or isolated CAR T cell populations. For example, two cell surface tags, such as EGFRt, Her2tG, and CD19t, are used to sort cells by each surface marker. In some embodiments of the present specification, anti-hapten CARs are sorted using a substrate such as magnetic beads or a dish or test tube labeled with a hapten. A unique feature of this method is that the anti-hapten CAR can be constitutively expressed because it does not recognize endogenous epitopes in the patient's body.

[0212] H-APCs (hapten antigen-presenting cells) are preferably generated by ex vivo labeling of healthy cells of a patient or cells compatible with the patient with a hapten. Examples of haptens include, but are not limited to, fluorescein, urushiol, quinone, or biotin. Further examples of haptens useful in the embodiments provided herein are listed in Table 1. There are various methods for labeling cells with haptens (e.g., labeling can be performed with chemicals, peptides, aptamers, lipids, or proteins). For example, the cells of interest may be incubated overnight with fluorescein lipids. One advantage of using fluorescein as a hapten is that its fluorescence can be utilized. This method allows for monitoring hapten incorporation by detecting the fluorescence of the fluorescein moiety using flow cytometry. Thus, after incubation, excess fluorescein lipids can be removed, and a cell fraction can be analyzed by flow cytometry to analyze hapten incorporation, while the remaining cells can be used for infusion into the patient. After infusion into patients, H-APCs that are not targeted by CAR T cells gradually lose the hapten (e.g., by being metabolized and removed from the cell surface) and revert to their original healthy cell morphology, making this approach safe in this respect.

[0213] H-APCs can be administered at any time during treatment if stimulation of CAR T cells is required in a patient. One example of when stimulation of CAR T cells is required in a patient is when a blood cancer reaches the final stage of regression, reducing the number of cancer cells and causing the CAR T cells to shrink and lose their effectiveness. In this case, H-APCs can be infused to induce the expansion and activation of CAR T cells, thereby continuing the regression of the cancer and, hopefully, achieving complete tumor remission.

[0214] Another example of when CAR T cell stimulation is required in patients is in the treatment of solid tumors. Solid tumors are often highly immunosuppressive, and stimulating CAR T cells with the addition of H-APCs may overcome the immunosuppressive tumor environment. This approach using H-APCs allows for safe stimulation of CAR T cells in vivo.

[0215] H-APC can also be used to stimulate CAR T cells in vitro. In certain clinical protocols, CAR T cells are stimulated via TCR by magnetic beads, and then the CAR T cells are reinfused into patients. H-APC can be made using hapten-labeled magnetic beads. In this case, before injecting CAR T cells, H-APC stimulates cells via CAR.

[0216] Furthermore, if rapid expansion (REP) is desired before reinfusion into the patient, the use of H-APCs offers a safe alternative. In the standard REP method, irradiated TM-LCLs and PBMCs are used as feeder cells. H-APCs can be used as an alternative in the REP method in various ways. First, if H-APCs are generated from the patient's own cells, the irradiation step can be omitted, eliminating the need for TM-LCL culture and PBMC isolation. Second, H-APCs can be generated from irradiated cells obtained from another donor. Third, such REP methods using H-APCs can be used in laboratory experiments as an alternative to the standard REP method. These examples provide several approaches for selectively expanding CAR T cells using hapten-specific stimulation.

[0217] To overcome the clinical hurdles facing CAR T-cell therapy, particularly for the treatment of solid tumors, additional support beyond the activity of a single CAR may be required. H-APCs offer a mechanism to improve CAR T-cell engraftment and persistence beyond that demonstrated in current clinical protocols and may facilitate T-cell migration to immunosuppressive solid tumor metastases. In hematologic cancers, where primary CAR T-cell engraftment can be challenging due to the low tumor threshold, H-APCs can facilitate initial activation. In both cases, the anti-hapten CAR promotes the activation, proliferation, and dispersal of infused CAR T cells, while the other expressed CAR induces tumor elimination. This strategy also offers a unique method for expanding CAR T cells in the REP method prior to infusion into patients.

[0218] The embodiments described herein aim to improve the therapeutic properties of CAR T cell therapy in both solid and hematological cancers. H-APCs may stimulate CAR T cells in vivo to overcome the immunosuppressive tumor microenvironment, enhance the ability of CAR T cells to detect and eradicate trace amounts of cancer, or simply support CAR T cells. H-APCs are safe to use, and H-APCs that are not lysed by CAR T cells safely degrade their haptens over time, reverting to normal, healthy cells. Furthermore, stimulating cells using the REP method with H-APCs offers the advantages of lower cell culture costs and shorter culture periods.

[0219] Another factor to consider when using a single viral vector incorporating two CARs to produce cells is the alleviation of size limitations.In some embodiments described herein, another vector is simultaneously transduced.Alternative methods for producing CAR T cells are also envisioned in the present invention, which aim to avoid the potential problem of size limitations.

[0220] The toxicity of the hapten must also be taken into consideration. However, those skilled in the art will readily understand that assays can be performed to determine whether a selected hapten (e.g., fluorescein) is well tolerated in humans. The toxicity of the binding moiety (e.g., lipid, protein, peptide, or aptamer) attached to the hapten may also be an issue. Again, toxicity can be controlled by selecting a binding moiety that is metabolized or rapidly eliminated from the body. An example of such a chemical is described in PCT / US2018 / 017126, which is expressly incorporated herein by reference in its entirety.

[0221] Similarly, autologous T cells transfected to express tROR1 on their cell surface (ROR1+ T-APCs) have been previously developed (Berger et al. 2015, Cancer Immunology Research, 3(2), 206-216). However, a key difference between the cells described by Berger and those described in the present embodiment is that Berger et al. required transduction, expansion, culture, and characterization of the ROR1+ T-APC preparation over a period of weeks to months, with attendant high costs. In contrast, the present embodiment requires only loading the cells with a hapten, which can be accomplished in a very short time (e.g., a few hours), and then reinjected into the patient. Furthermore, various cell types can be used in the system described in the present embodiment. Therefore, it is believed that precious T cells are not necessary. Furthermore, the technology reported by Berger has not been used for solid tumors. Furthermore, Berger generated T-APCs through genetic engineering, which is costly and time-consuming. In contrast, the embodiments provided herein allow for rapid and efficient delivery of hapten-labeled cells by directly attaching haptens to the extracellular surface. Thus, the methods described in the embodiments herein revolutionize the field of T cell immunotherapy for solid tumors and significantly improve upon existing CAR T cell-based hematological cancer therapies.

[0222] Some embodiments of the methods and compositions provided herein include aspects disclosed in WO2018 / 148224; WO2019 / 156795; WO2019 / 144095; US2019 / 0224237; and PCT / US2019 / 044981, published as WO2020 / 033272, all of which are expressly incorporated by reference in their entirety.

[0223] Induction of CAR T cell expansion Some embodiments of the methods and compositions provided herein include methods of inducing the expansion of chimeric antigen receptor (CAR) T cells. In some such embodiments, CAR T cells are incubated with hapten antigen-presenting cells (H-APCs) under conditions that induce the expansion of CAR T cells. In some embodiments, the CAR of the CAR T cells is specifically bound to the hapten bound to the H-APCs. Some embodiments include methods of treating, suppressing, or alleviating cancer in a subject. In some embodiments, an effective amount of CAR T cells having a CAR that specifically binds to a tumor-specific antigen of cancer is administered to a subject, and the CAR T cells are incubated with hapten antigen-presenting cells (H-APCs) to induce the expansion of the CAR T cells by specifically binding the CAR of the CAR T cells to the hapten bound to the H-APCs. In some embodiments, the CAR T cells and the H-APCs are derived from a single subject, such as a human. In some embodiments, the subject is a mammal, such as a human, a livestock animal, or a domestic animal.

[0224] In some embodiments, the CAR T cells may comprise a bispecific CAR, for example, a CAR may have two specific binding domains: a first binding domain capable of specifically binding to a target, such as a tumor-specific antigen, and a second binding domain capable of specifically binding to a hapten.

[0225] In some embodiments, the CAR T cells may comprise two or more CARs, for example, a first CAR comprising a first binding domain capable of specifically binding to a target, such as a tumor-specific antigen, and a second CAR comprising a second binding domain capable of specifically binding to a hapten.

[0226] In some embodiments, the CAR T cells may comprise a CAR that can bind to a target, such as a tumor-specific antigen, and also bind to a hapten. In some of these embodiments, the target and the hapten may comprise the same or substantially the same binding moiety, such that the CAR can bind to the binding moiety of the target and the binding moiety of the hapten. In some of these embodiments, the target and the hapten may be tumor antigens provided herein.

[0227] Examples of target antigens that can be used in the embodiments provided herein include CD19, CD22, HER2, CD7, CD30, B-cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epithelial cell adhesion molecule (EpCAM), epidermal growth factor variant III, receptor tyrosine kinase-like orphan receptor 1 (ROR1), CD123, prostate stem cell antigen (PSCA), CD5, Lewis Y antigen, B7H3, CD20, CD43, HSP90, or IL13, or any combination thereof.

[0228] Examples of haptens that can be used in the embodiments provided herein include those listed in Table 1. In some embodiments, haptens useful in the embodiments provided herein include fluorescein, urushiol, quinone, biotin, or dinitrophenol and / or derivatives thereof.

[0229] In some embodiments, H-APCs are prepared by covalently attaching a hapten to the extracellular surface, hi some embodiments, the hapten is attached to the H-APC via an ether phospholipid (PLE).

[0230] In some embodiments, the incubation may be performed in vitro. For example, CAR T cells can be generated by transducing cells with a vector encoding a CAR, and the transduced cells can be incubated with H-APCs to induce the expansion of the transduced cells. In some embodiments, the expanded cells can be administered to a subject, such as a human. In some embodiments, the incubation may be performed in vivo. For example, CAR T cells can be administered to a subject. H-APCs, which induce the expansion of the CAR T cells in vivo, can also be administered to the subject.

[0231] In some embodiments, the CAR T cells are derived from CD4+ cells or CD8+ cells. In some embodiments, the CD8+ 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 CD8+ cells are CD8+ cytotoxic T lymphocytes, and the CD8+ cytotoxic T lymphocytes are central memory T cells, and the central memory T cells are CD45RO+, CD62L+, and CD8+. In some embodiments, the CD4+ 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 CD4+ helper lymphocytes are naive CD4+ T cells, and the naive CD4+ T cells are CD45RA+, CD62L+, CD4+, and CD45RO-. In some embodiments, the CAR T cells are derived from progenitor T cells. In some embodiments, the CAR T cells are derived from hematopoietic stem cells. In some embodiments, the H-APCs are derived from healthy cells (such as T cells or B cells) of the subject.

[0232] In some embodiments, the healthy cells may be T cells, B cells, monocytes, macrophages, dendritic cells, NK cells, or erythrocytes. In some embodiments, the healthy cells may be any peripheral blood mononuclear cells. In some embodiments, the healthy cells may be any healthy cells derived from a living organism. In some embodiments, the healthy cells may be any cells derived from an apheresis product. In some embodiments, the healthy cells may be any cells that can be labeled ex vivo.

[0233] In some embodiments, a single CAR cell is used with an H-APC. In some embodiments, a multimeric CAR is used with an H-APC. In some embodiments, for example, when a tumor is labeled with a hapten (e.g., a hapten-labeled CD19 antibody, a hapten-PLE, a hapten-labeled small molecule, a hapten-labeled peptide, a hapten-labeled aptamer, or other hapten-labeled tumor cells), a single anti-hapten CAR T cell is used, and an H-APC generated using the same hapten is used to expand the anti-hapten CAR T cell in the patient's body. In some embodiments, dual CAR cells or bispecific CAR cells can be used, in which one CAR (e.g., CD19, CD22, or ROR1) attacks the cancer, and the other anti-hapten CAR is used to expand the dual CAR cells or bispecific CAR cells via an H-APC (see, e.g., Figure 2). In some embodiments, this concept can be further extended to load cells with three or more CARs and an anti-hapten CAR (e.g., loading cells with CD19 and CD22 for the treatment of acute lymphoblastic leukemia (ALL)), and use the anti-hapten CAR to activate and expand CAR T cells.

[0234] In some embodiments, the CAR T cells are non-autologous T cells.

[0235] In some embodiments, the methods disclosed herein can be used to expand any type of cell via CAR and H-APC. For example, B cells expressing anti-hapten CAR can be used and stimulated with H-APC to expand these cells. Thus, this approach can be used to expand any type of cell in vivo.

[0236] In some embodiments, CAR T cells can be generated not only as a treatment for cancer, but also as a treatment for viral infections (such as HIV and hepatitis), and can also be generated as a potential treatment for autoimmune diseases and associated conditions.

[0237] In some embodiments, tumor infiltrating lymphocytes (TILs) can be collected from tumors / cancers, transduced with a CAR, and expanded in vitro / in vivo using H-APCs.

[0238] Nucleic acid encoding a CAR or bispecific CAR In some embodiments, one or more nucleic acids are provided for expressing a first chimeric antigen receptor and a second chimeric antigen receptor. The one or more nucleic acids may be provided in a single vector or multiple vectors to accommodate the payload sizes of two CARs. The one or more nucleic acids may comprise a first sequence encoding the first chimeric antigen receptor and a second sequence encoding the second chimeric antigen receptor, where the first chimeric antigen receptor comprises a first ligand-binding domain specific to a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a second ligand-binding domain specific to a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first ligand-binding domain is specific to a tumor cell antigen. In some embodiments, the tumor cell antigen is 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, or cell death receptor. In some embodiments, the CAR can specifically bind to a hapten listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof.In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain comprises an antibody or binding fragment thereof, or an scFv. In some embodiments, the second ligand-binding domain comprises an antibody to a hapten listed in Table 1 or a binding fragment of an antibody, such as an antibody listed in Table 2. Examples of amino acid and nucleic acid sequences encoding antigen-binding domains (e.g., svFc) capable of binding to haptens such as fluorescein or dinitrophenol are provided in Table 3 below, any of which may be incorporated into one or more embodiments described herein. [Table 1] TIFF0007744914000002.tif200163

[0239] In some embodiments, the first polypeptide spacer, the second polypeptide spacer, or both are 1 to 24 amino acids, 25 to 50 amino acids, 51 to 75 amino acids, 76 to 100 amino acids, 101 to 125 amino acids, 126 to 150 amino acids, 151 to 175 amino acids, 176 to 200 amino acids, 201 to 225 amino acids, 226 to 250 amino acids, or 251 to 275 amino acids in length. In some embodiments, the nucleic acid further comprises a leader sequence. In some embodiments, the first intracellular signaling domain and / or the second intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ, or both. In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB. In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG. In some embodiments, the first transmembrane domain, the second transmembrane domain, or both, comprise the transmembrane domain of CD28. In some embodiments, the nucleic acid further comprises a sequence encoding a cleavable linker. In some embodiments, the linker is a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is P2A, T2A, E2A, or F2A. The cleavable linker may be located between two sequences encoding each chimeric antigen receptor. Additionally, a cleavable linker may be used between one of the chimeric antigen receptors and a sequence encoding a marker protein. In some embodiments, one or more vectors are provided comprising one or more nucleic acids according to any of the embodiments herein. In some embodiments, a chimeric antigen receptor encoded by a nucleic acid according to any of the embodiments herein or a vector according to any of the embodiments herein is provided.

[0240] In some embodiments, one or more nucleic acids are provided for expressing a first chimeric antigen receptor and a second chimeric antigen receptor, the one or more nucleic acids comprising: a first nucleic acid comprising a first sequence encoding the first chimeric antigen receptor; and a second nucleic acid comprising a second sequence encoding the second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific for a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain; and the second chimeric antigen receptor comprises a second ligand-binding domain specific for a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first ligand binding domain is selected from the group consisting of 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, and DLL4. , EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF, or any combination thereof. In some embodiments, the CAR is capable of specifically binding to a hapten listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof.In some embodiments, the first ligand-binding domain or the second ligand-binding domain is selected from the group consisting of 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, DLL4, E In some embodiments, the second ligand-binding domain comprises an antibody or binding fragment thereof or scFv specific for GFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF, or any combination thereof. In some embodiments, the second ligand-binding domain comprises an antibody against a hapten listed in Table 1 or a binding fragment of an antibody, such as an antibody listed in Table 2. In some embodiments, the first polypeptide spacer, the second polypeptide spacer, or both are 1 to 24 amino acids, 25 to 50 amino acids, 51 to 75 amino acids, 76 to 100 amino acids, 101 to 125 amino acids, 126 to 150 amino acids, 151 to 175 amino acids, 176 to 200 amino acids, 201 to 225 amino acids, 226 to 250 amino acids, or 251 to 275 amino acids in length. In some embodiments, the nucleic acid further comprises a leader sequence. In some embodiments, the first intracellular signaling domain and / or the second intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ, or both.In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB. In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG. In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises the transmembrane domain of CD28. In some embodiments, the nucleic acid further comprises a sequence encoding a cleavable linker. In some embodiments, the linker is a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is P2A, T2A, E2A, or F2A. In some embodiments, a plurality of vectors comprising the nucleic acid according to any of the embodiments herein are provided. In some embodiments, a chimeric antigen receptor encoded by a nucleic acid according to any of the embodiments herein or a vector according to any of the embodiments herein is provided.

[0241] Bispecific Chimeric Antigen Receptors In some embodiments, one or more nucleic acids are provided for expression of a bispecific chimeric antigen receptor. In some embodiments, the nucleic acid comprises a sequence encoding a first ligand-binding domain specific for a tumor antigen, a glycine-serine linker, a second ligand-binding domain specific for a hapten, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the first ligand-binding domain is selected from the group consisting of 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, DLL4, The CAR is specific for EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF, or other antigens expressed by cancer. In some embodiments, the CAR can specifically bind to a hapten listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the first ligand-binding domain, the second ligand-binding domain, or both, comprise an antibody or binding fragment thereof, or an scFv. In some embodiments, the second ligand binding domain comprises an antibody against a hapten listed in Table 1 or a binding fragment of an antibody, such as an antibody listed in Table 2.In some embodiments, the first polypeptide spacer, the second polypeptide spacer, or both are 1 to 24 amino acids, 25 to 50 amino acids, 51 to 75 amino acids, 76 to 100 amino acids, 101 to 125 amino acids, 126 to 150 amino acids, 151 to 175 amino acids, 176 to 200 amino acids, 201 to 225 amino acids, 226 to 250 amino acids, or 251 to 275 amino acids in length. In some embodiments, the nucleic acid further comprises a leader sequence. In some embodiments, the intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ, or both. In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB. In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG. In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28. In some embodiments, one or more vectors for expressing a bispecific CAR are provided, comprising one or more nucleic acids according to any of the embodiments described herein. In some embodiments, a bispecific chimeric antigen receptor is provided, the bispecific chimeric antigen receptor being encoded by a nucleic acid according to any of the embodiments herein or a vector according to any of the embodiments herein.

[0242] Cells containing a CAR or bispecific CAR In some embodiments, a cell is provided comprising one or more nucleic acids according to any of the embodiments herein, one or more vectors according to any of the embodiments herein, or a bispecific chimeric antigen receptor according to any of the embodiments herein. The one or more nucleic acids may be provided in a single vector or multiple vectors to accommodate the payload sizes of two CARs. The one or more vectors may contain another nucleic acid provided herein. Alternatively, the nucleic acid may be integrated using a transposon system or an integrase system. The one or more nucleic acids may comprise a first sequence encoding a first chimeric antigen receptor and a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific to a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a second ligand-binding domain specific to a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, a plurality of nucleic acids is provided, the plurality comprising: a first nucleic acid comprising a first sequence encoding a first chimeric antigen receptor; and a second nucleic acid comprising a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific for a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain; and the second chimeric antigen receptor comprises a second ligand-binding domain specific for a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first ligand-binding domain is specific for a tumor cell antigen.In some embodiments, the antigen is 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, DLL4, EGFR The CAR may include FL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF, or any combination thereof. In some embodiments, the CAR can specifically bind to a hapten listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the first ligand-binding domain, the second ligand-binding domain, or both, comprise an antibody or binding fragment thereof, or an scFv. In some embodiments, the second ligand-binding domain comprises an antibody against a hapten listed in Table 1 or a binding fragment of an antibody, such as an antibody listed in Table 2. In some embodiments, the first polypeptide spacer or the second polypeptide spacer, or both, are 1 to 24 amino acids in length, 25 to 50 amino acids in length, 51 to 75 amino acids in length, 76 to 100 amino acids in length, 101 to 125 amino acids in length, 126 to 150 amino acids in length, 151 to 175 amino acids in length, 176 to 200 amino acids in length, 201 to 225 amino acids in length, 226 to 250 amino acids in length, or 251 to 275 amino acids in length. In some embodiments, the nucleic acid further comprises a leader sequence.In some embodiments, the first intracellular signaling domain, the second intracellular signaling domain, or both, comprise a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ. In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG. In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the nucleic acid further comprises a sequence encoding a cleavable linker. In some embodiments, the linker is a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is P2A, T2A, E2A, or F2A. The cleavable linker may be located between two sequences encoding each chimeric antigen receptor. Additionally, a cleavable linker may be used between one of the chimeric antigen receptors and a sequence encoding a marker protein. In some embodiments, one or more vectors for expressing a bispecific CAR are provided, comprising one or more nucleic acids according to any of the embodiments herein. In some embodiments, the bispecific chimeric antigen receptors encoded by the one or more nucleic acids are contained in a cell.The one or more nucleic acids encoding the bispecific chimeric antigen receptor comprise sequences encoding a first ligand-binding domain specific for a tumor antigen, a glycine-serine linker, a second ligand-binding domain specific for a hapten, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the first ligand-binding domain is selected from the group consisting of 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, DLL4, E The hapten is specific for GFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF, or another antigen expressed on cancer cells. In some embodiments, the hapten is selected from the haptens listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the first ligand-binding domain, the second ligand-binding domain, or both, comprise an antibody or binding fragment thereof, or an scFv. In some embodiments, the second ligand binding domain comprises an antibody against a hapten listed in Table 1 or a binding fragment of an antibody, such as an antibody listed in Table 2.In some embodiments, the first polypeptide spacer, the second polypeptide spacer, or both are 1 to 24 amino acids, 25 to 50 amino acids, 51 to 75 amino acids, 76 to 100 amino acids, 101 to 125 amino acids, 126 to 150 amino acids, 151 to 175 amino acids, 176 to 200 amino acids, 201 to 225 amino acids, 226 to 250 amino acids, or 251 to 275 amino acids in length. In some embodiments, the nucleic acid further comprises a leader sequence. In some embodiments, the intracellular signaling domain comprises CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or a ligand that specifically binds to the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB. In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG. In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28. In some embodiments, the 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. In some embodiments, the CD8+ cytotoxic T lymphocyte is a central memory T cell, and the central memory T cell is CD45RO+, CD62L+, and CD8+. In some embodiments, the 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. In some embodiments, the cells are naive CD4+ T cells, which are CD45RA+, CD62L+, CD4+, and CD45RO-. In some embodiments, the cells are progenitor T cells.In some embodiments, the cells are hematopoietic stem cells.

[0243] Generation of cells containing two CARs or bispecific CARs In some embodiments, a method for producing a cell expressing a first chimeric antigen receptor specific to a hapten and a second chimeric antigen receptor specific to a tumor antigen is provided. In some cases, the method comprises introducing one or more nucleic acids according to any of the embodiments herein or one or more vectors according to any of the embodiments herein into the cell under conditions in which the first chimeric antigen receptor and the second chimeric antigen receptor are expressed. In some embodiments, a method for producing a cell expressing a bispecific chimeric antigen receptor specific to a hapten and a tumor antigen is provided. The method comprises introducing one or more nucleic acids according to any of the embodiments herein or one or more vectors according to any of the embodiments herein into the cell under conditions in which the first chimeric antigen receptor and the second chimeric antigen receptor are expressed. In some embodiments, the 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. In some embodiments, the CD8+ cytotoxic T lymphocytes are central memory T cells, which are CD45RO+, CD62L+, and CD8+. 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. In some embodiments, the CD4+ helper lymphocytes are naive CD4+ T cells, which are CD45RA+, CD62L+, CD4+, and CD45RO-. In some embodiments, the cells are progenitor T cells. In some embodiments, the cells are hematopoietic stem cells.The one or more nucleic acids include a first nucleic acid comprising a first sequence encoding a first chimeric antigen receptor and a second nucleic acid comprising a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific to a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a second ligand-binding domain specific to a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first ligand binding domain is selected from the group consisting of 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, and DLL4. , EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF, or any combination thereof. In some embodiments, the hapten is selected from the haptens listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof.In some embodiments, the first ligand binding domain or the second ligand binding domain, or both, is selected from the group consisting of 5T4, B7-H3, carbonic anhydrase IX, carcinoembryonic antigen, CA-125, CD-3, CD-19, CD-20, CD-22, CD-30, CD-33, CD-38, CD-40, CD-51, CD-52, CD-56, CD-70, CD-74, CD-79b, CD-138, CD-221, CD-319, CD-326, cell adhesion molecule 5, CTLA-4, cytokeratin polypeptide, death receptor 2, DL In some embodiments, the second ligand-binding domain comprises an antibody or binding fragment thereof or scFv specific for L4, EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGLF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin-4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF, or any combination thereof. In some embodiments, the second ligand-binding domain comprises an antibody to a hapten listed in Table 1 or a binding fragment of an antibody, such as an antibody listed in Table 2. In some embodiments, the first polypeptide spacer, the second polypeptide spacer, or both are 1 to 24 amino acids, 25 to 50 amino acids, 51 to 75 amino acids, 76 to 100 amino acids, 101 to 125 amino acids, 126 to 150 amino acids, 151 to 175 amino acids, 176 to 200 amino acids, 201 to 225 amino acids, 226 to 250 amino acids, or 251 to 275 amino acids in length. In some embodiments, the nucleic acid further comprises a leader sequence. In some embodiments, the first intracellular signaling domain and / or the second intracellular signaling domain comprises a ligand that specifically binds to the cytoplasmic domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; or the cytoplasmic domain of CD83 or the cytoplasmic domain of CD3ζ.In some embodiments, the intracellular signaling domain comprises a portion of CD3ζ and a portion of 4-1BB. In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG. In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises the transmembrane domain of CD28. In some embodiments, the nucleic acid further comprises a sequence encoding a cleavable linker. In some embodiments, the linker is a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is P2A, T2A, E2A, or F2A. In some embodiments, the nucleic acid encoding the bispecific chimeric antigen receptor comprises a sequence encoding a first ligand-binding domain specific for a tumor antigen, a glycine-serine linker, a second ligand-binding domain specific for a hapten, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a plurality of vectors comprising one or more nucleic acids according to any of the embodiments herein are provided.

[0244] T lymphocytes can be collected by known techniques and enriched or depleted by known techniques such as affinity binding to antibodies, flow cytometry, and / or immunomagnetic selection. After the enrichment and / or depletion steps, the desired T lymphocytes can be expanded in vitro by known techniques or modifications thereof readily apparent to those skilled in the art. In some embodiments, the T cells are autologous T cells obtained from the patient.

[0245] For example, a desired T cell population or subpopulation can be expanded by adding an initial pre-expansion T lymphocyte population to in vitro culture medium, then adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture medium (e.g., so that the resulting cell population contains at least 5, 10, 20, or 40 or more PBMC feeder cells for every T lymphocyte in the initial pre-expansion population), and incubating the culture medium (e.g., for a period of time sufficient to expand the T cell population). The non-dividing feeder cells may include γ-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with 3000-3600 rad of γ-rays to prevent cell division. In some embodiments, to prevent cell division of the PBMCs, the PBMCs are irradiated with gamma rays at 3000 rad, 3100 rad, 3200 rad, 3300 rad, 3400 rad, 3500 rad, or 3600 rad, or any radiation dose within a range defined by any two of these values. The order in which T cells and feeder cells are added to the culture medium may be reversed as needed. Typically, the culture is incubated under conditions, including temperature, suitable for T lymphocyte proliferation. For example, the temperature for human T lymphocyte proliferation is typically at least 25°C, preferably at least 30°C, and more preferably 37°C. In some embodiments, the temperature for human T lymphocyte proliferation is 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, or 37°C, or any other temperature within a range defined by any two of these values.

[0246] The expanded T lymphocytes can include CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T lymphocytes, which may be specific for antigens present on human tumors or pathogens. In some embodiments, the cells include precursor T cells. In some embodiments, the cells are hematopoietic stem cells.

[0247] In some embodiments, the expansion method may further include adding EBV-transformed non-dividing lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with 6,000 to 10,000 rad of gamma rays. In some embodiments, the LCL are irradiated with gamma rays at 6,000 rad, 6,500 rad, 7,000 rad, 7,500 rad, 8,000 rad, 8,500 rad, 9,000 rad, 9,500 rad, or 10,000 rad, or any dose within a range defined by any two of these values. The LCL feeder cells may be provided in any suitable amount; for example, the ratio of LCL feeder cells to the initial T lymphocytes before expansion may be at least 10:1.

[0248] In some embodiments, the expansion method may further comprise adding an anti-CD3 antibody and / or an anti-CD28 antibody to the culture medium (e.g., at a concentration of at least 0.5 ng / ml). In some embodiments, the expansion method may further comprise adding IL-2 and / or IL-15 to the culture medium (e.g., the concentration of IL-2 is at least 10 units / ml). Before or after isolating and expanding T lymphocytes, cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, respectively.

[0249] CD8+ cells can also be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive CD8+ cells, central memory CD8+ cells, and effector memory CD8+ cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, central memory T cells (T CM Expression of phenotypic markers of T cells (T) includes CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and Granzyme B is negative or shows low expression. In some embodiments, central memory T cells are CD45RO+, CD62L+ and / or CD8+ T cells. In some embodiments, effector T cells (T E ) are negative for CD62L, CCR7, CD28, and / or CD127 and positive for granzyme B and / or perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and / or CD45RA.

[0250] CD4+ helper T cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations bearing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, and / or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and / or CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and / or CD45RO-.

[0251] Whether a cell or cell population is positive for a particular cell surface marker can be determined by flow cytometry using staining with an antibody specific for that surface marker and an isotype-matched control antibody. Negativity for a particular marker in a cell population refers to the absence of a cell population that stains more intensely with the specific antibody than the isotype control, while positivity refers to the presence of a cell population that stains more uniformly with the specific antibody than the isotype control.

[0252] In some embodiments, a decrease in expression of one or more markers refers to a 1 log decrease in mean fluorescence intensity and / or at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, at least a 95% decrease, or at least a 100% decrease, or any percentage decrease in the range of 20-100%, in the percentage of cells expressing the marker compared to a control cell population. In some embodiments, a cell population is positive for one or more markers when, compared to a control cell population, the percentage of cells expressing the marker is at least 50% of the total cells, at least 55% of the total cells, at least 60% of the total cells, at least 65% of the total cells, at least 70% of the total cells, at least 75% of the total cells, at least 80% of the total cells, at least 85% of the total cells, at least 90% of the total cells, at least 95% of the total cells, or at least 100% of the total cells, or any percentage in the range of 50-100%.

[0253] In some embodiments, antigen-specific CD4+ and CD8+ populations can be obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, T cell lines or T cell clones specific for a cytomegalovirus antigen can be generated by isolating T cells from a subject infected with cytomegalovirus and stimulating the cells in vitro with the same cytomegalovirus antigen. Naive T cells can also be used. The number of antigens derived from tumor cells that can be used as targets for inducing T cell responses is not particularly limited. In some embodiments, the adoptive cellular immunotherapy compositions of the present invention are useful for treating diseases or disorders including solid tumors and / or hematological malignancies.

[0254] Other methods of ex vivo cell stimulation are also conceivable.Hapten-containing cells or beads can be bound to hapten to stimulate CAR T cells before using the CAR T cells as a therapeutic method.Hapten-bearing cells can also be prepared by standard known techniques, such as exposing to hapten-bound support (for example, beads, wells or dishes), to stimulate CAR T-expressing cells.

[0255] Stimulation of chimeric antigen receptors in vivo Provided is a method for stimulating or restimulating T cells bearing a chimeric antigen receptor (CAR) in a subject with a disease, such as cancer, comprising: providing a cell according to any of the embodiments provided herein to said subject; monitoring said subject for suppression of said disease; and providing hapten antigen-presenting cells (H-APCs) to said subject. Including, The subject may be a subject selected for CAR T cell therapy using CAR T cells having a receptor specific to an antigen associated with the disease (such as a tumor antigen). The cells may contain one or more vectors or one or more nucleic acids according to any of the embodiments herein, or a bispecific chimeric antigen receptor according to any of the embodiments herein. The one or more nucleic acids may be provided in a single vector or in multiple vectors to accommodate the payload sizes of two CARs. The one or more vectors may contain another nucleic acid provided herein. Alternatively, the nucleic acid may be integrated using a transposon system or an integrase system. The nucleic acid may comprise a first sequence encoding a first chimeric antigen receptor and a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific to a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a second ligand-binding domain specific to a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, a plurality of nucleic acids is provided, the plurality comprising a first nucleic acid comprising a first sequence encoding a first chimeric antigen receptor and a second nucleic acid comprising a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific to a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a second ligand-binding domain specific to a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first ligand-binding domain is specific to a tumor cell antigen. In some embodiments, the bispecific chimeric antigen receptor encoded by the one or more nucleic acids is contained in a cell. The nucleic acid encoding the bispecific chimeric antigen receptor comprises a sequence encoding a first ligand-binding domain specific to a tumor antigen, a glycine-serine linker, a second ligand-binding domain specific to a hapten, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain.In some embodiments, the H-APCs are cells generated by labeling healthy cells of the subject with a hapten ex vivo.

[0256] The H-APCs are generated by ex vivo labeling of healthy cells from a patient, such as a human, or cells compatible with the patient with a hapten. Examples of haptens include fluorescein, urushiol, quinone, and biotin. There are various methods for labeling cells with haptens, such as chemicals, peptides, aptamers, lipids, and proteins. One method for loading cells with a hapten is to incubate the cells of interest with fluorescein lipid overnight. One advantage of using fluorescein as a hapten is its fluorescence. Therefore, hapten incorporation can be monitored by detecting the fluorescence of the fluorescein moiety using flow cytometry. After incubation, excess fluorescein lipid is removed, and a cell fraction is analyzed by flow cytometry to analyze hapten incorporation, while the remaining cells can be used for infusion into the patient. After infusion into patients, H-APCs that are not targeted by CAR T cells gradually lose the hapten (e.g., by being metabolized and removed from the cell surface) and revert to their original, healthy cell morphology, making this approach safe. H-APCs can also be generated by conjugating a hapten to a lipid and incorporating it into cells.

[0257] In some embodiments, the hapten is selected from the haptens listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof. In some embodiments, the monitoring and providing steps are repeated. In some embodiments, the subject has cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the subject has been selected or identified for cancer therapy, for example, by conventional clinical or diagnostic evaluation tests, or both. In some embodiments, the subject is administered a combination therapy, such as chemotherapy or radiation therapy.

[0258] Ex vivo cell stimulation In some embodiments, methods are provided for stimulating or restimulating T cells bearing a chimeric antigen receptor (CAR) ex vivo. In some cases, the methods include: Providing a cell according to any of the embodiments described herein; providing hapten antigen-presenting cells (H-APCs) or haptens; mixing the cells with the H-APC cells to obtain activated cells; and isolating the activated cells Includes. The cells may comprise one or more vectors or one or more nucleic acids according to any of the embodiments herein, or a bispecific chimeric antigen receptor according to any of the embodiments herein. The one or more nucleic acids may be provided in a single vector or in multiple vectors to accommodate two different CAR payload sizes. The one or more vectors may comprise another nucleic acid provided herein. Alternatively, the nucleic acid may be integrated using a transposon system or an integrase system. The one or more nucleic acids may comprise a first sequence encoding a first chimeric antigen receptor and a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific to a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a second ligand-binding domain specific to a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, a plurality of nucleic acids is provided, the plurality comprising a first nucleic acid comprising a first sequence encoding a first chimeric antigen receptor and a second nucleic acid comprising a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a first ligand-binding domain specific to a tumor antigen, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a second ligand-binding domain specific to a hapten, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first ligand-binding domain is specific to a tumor cell antigen. In some embodiments, the bispecific chimeric antigen receptor encoded by the nucleic acid is contained in a cell. The nucleic acid encoding the bispecific chimeric antigen receptor comprises a sequence encoding a first ligand-binding domain specific to a tumor antigen, a glycine-serine linker, a second ligand-binding domain specific to a hapten, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain.In some embodiments, the hapten is selected from the haptens listed in Table 1. In some embodiments, the H-APC comprises a hapten selected from the haptens listed in Table 1. In some embodiments, the hapten can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol, and derivatives thereof.

[0259] In some embodiments, isolating the activated cells comprises affinity isolation by conjugating a hapten to affinity beads, hi some embodiments, isolating the activated cells comprises affinity isolation by conjugating EGFRt, CD19t, or Her2tG to affinity beads.

[0260] In some embodiments, the CAR may have the structure anti-FL(FITC-E2)scFv-IgG4hinge-CH2(L235D,N297Q)-CH3-CD28tm / 41BB-ζ-T2A-EGFRt. An example of an amino acid sequence that can be used in embodiments of the methods and compositions provided herein is listed in Table 4. [Table 2] TIFF0007744914000004.tif149161 [Example]

[0261] Example 1 - Generation of cells with haptens tethered to the cell surface Hapten-labeled cells were generated by conjugating the hapten fluorescein (FL) to cells via phospholipids incorporated into the cell membrane or via antibodies. CD19+ Raji cells (lymphoma cell line) were incubated overnight with 5 μM FL-DHPE (Figure 3B) or with fluorescein isothiocyanate (FITC)-conjugated anti-CD19 antibodies for 20 minutes. To confirm the presence of FL, cells were washed, stained, and analyzed by flow cytometry. Both methods of cell labeling showed a positive shift in response to the presence of FL compared with untreated control cells. FL levels were higher in FL-DHPE-treated cells than in anti-CD19 antibody-treated cells (Figure 4A). These results indicate that the different anchoring techniques result in different amounts of hapten on the cell surface.

[0262] K562 cells (a leukemia cell line) were incubated overnight with 0.5 μM or 5 μM FL-PLE (Figure 3A) in the presence of FBS, which can reduce the amount of phospholipid incorporation onto the cell surface. The amount of FL-PLE incorporation into the cells was analyzed by flow cytometry. The amount of FL detected was greater in cells treated with 5 μM FL-PLE than in cells treated with 0.5 μM FL-PLE (Figure 4B). Furthermore, the amount of FL detected was greater in cells treated with 0.5 μM FL-PLE than in untreated control cells. Thus, adjusting the concentration of FL-PLE can vary the amount of FL loaded onto the cell surface. The density of haptens (e.g., FL) on the cell surface can also be varied by varying the concentration of the tethering agent (e.g., FL-PLE).

[0263] Be2 cells (a neuroblastoma cell line), U87 cells (a glioblastoma cell line), or Daoy cells (a medulloblastoma cell line) were incubated overnight with 5 μM FL-PLE and analyzed by flow cytometry. FL-PLE was incorporated into each cell line, and the amount of FL incorporated into U87 and Daoy cells was greater than that into Be2 cells (Figure 4C). These results demonstrate that FL-PLE can be incorporated into various cell types, and the amount of FL-PLE incorporated can vary.

[0264] Example 2 - Accessibility of cell surface-tethered haptens To confirm the extracellular accessibility of the hapten loaded onto the cells, U87 cells were incubated overnight with 5 μM FL-PLE and images were acquired using a confocal microscope to confirm the location of the FL moiety in the cells. The cell nuclei were stained with DAPI. Green fluorescent staining was observed throughout the cell surface. Therefore, FL-PLE was incorporated throughout the cell surface (Figure 5A). The confocal image is an overlay of the acquired images shown on the left. The grayscale images of each layer ((i) nucleus and (ii) FL-PLE) that make up this confocal image are shown on the right.

[0265] To measure the accessibility of the FL moiety on the cell surface, cells labeled with FL-PLE were stained with an anti-fluorescein antibody conjugated to an Alexa Fluor 647 fluorescent dye. Anti-fluorescein antibody staining was observed across the entire cell surface (Figure 5B). This result confirmed that the FL moiety was accessible via extracellular binding. The left side shows a confocal image obtained by overlaying the acquired images. The right side of the overlaid confocal image shows grayscale images of each layer (i) nucleus, (ii) FL-PLE, and (iii) anti-fluorescein-Alexa Fluor 647 antibody) that make up this confocal image.

[0266] Example 3 - Retention of tethered haptens on the cell surface Be2 or U87 cells were incubated overnight in the presence of 5 μM FL-DHPE or 5 μM FL-PLE. The cells were washed to remove residual FL-DHPE or FL-PLE and then cultured in fresh medium for 4 days. The cells were analyzed by flow cytometry. Cells treated with FL-DHPE or FL-PLE retained FL for at least 4 days (Figures 6A and 6B). After 4 days, the amount of FL was greater in cells treated with FL-PLE than in cells treated with FL-DHPE. These results demonstrate that the duration of hapten retention on the cell surface can be altered by varying the tethering agent (e.g., phospholipid) used.

[0267] Example 4 - Recognition of cell surface-tethered haptens and activation of anti-hapten CAR T cells Hapten-labeled cells were prepared. CD19+K562 cells were incubated with 5 μM FL-DHPE overnight or with FITC-labeled CD19 antibody for 20 minutes. The resulting hapten-labeled cells were then incubated with either of two anti-FL CAR T cells (FITC-E2 scFv or 4M5.3 scFv). The cytotoxicity assay, cytokine release assay, and proliferation assay of the CAR T cells were performed using a method substantially similar to that described in Hudecek M et al. (2013) (Hudecek M, et al., (2013) Clin Cancer Res. 19:3153-64; this document is incorporated herein by reference in its entirety).

[0268] A chromium release assay was used to measure the lytic ability of anti-FL CAR T cells against hapten-labeled cells. Control K562 cells, not labeled with hapten, were unable to induce lysis by anti-FL (FITC-E2) CAR T cells or anti-FL (4M5.3) CAR T cells (Figure 7A, upper left panel). A positive control using OKT3 cells, which can activate T cells via TCR, demonstrated the ability to induce lysis by anti-FL (FITC-E2) CAR T cells or anti-FL (4M5.3) CAR T cells (Figure 7A, upper right panel). Both hapten-labeled cells were able to induce lysis by each of the two anti-FL CAR T cells (Figure 7A, lower panel).

[0269] The amount of cytokines released by anti-FL CAR T cells was measured. Both hapten-labeled cells induced the release of IFN-γ, IL-2, and TNF-α upon contact with anti-FL (FITC-E2) CAR T cells (Figure 7B). The amount of IFN-γ and TNF-α released by hapten-labeled cells was lower when contacted with anti-FL (4M5.3) CAR T cells. Furthermore, hapten-labeled cells generated using FL-DHPE tended to induce greater cytokine release than hapten-labeled cells generated using FITC-labeled CD19 antibody.

[0270] Example 5 - Recognition of cell surface-tethered haptens and activation of anti-hapten CAR T cells Hapten-labeled cells were prepared. K562 cells were incubated overnight with 0.5 μM or 5 μM FL-PLE. FL-PLE uptake into the cells was analyzed by flow cytometry. The resulting hapten-labeled cells were then incubated with anti-FL CAR T cells, and the ability of the hapten-labeled cells to induce the specific lytic and cytokine-releasing activity of the anti-FL CAR T cells was measured.

[0271] The amount of FL detected was greater in cells treated with 5 μM FL-PLE than in cells treated with 0.5 μM FL-PLE or untreated control cells (Figure 8A). Cell lysis and cytokine release were also greater in cells treated with 5 μM FL-PLE than in cells treated with 0.5 μM FL-PLE or untreated control cells (Figures 8B and 8C). Therefore, FL-PLE-treated cells with extracellular FL moieties tethered to their cell surface can be recognized by and activate anti-FL CAR T cells. The amount of activation of anti-FL CAR T cells is thought to be related to the amount of FL on the surface of hapten-labeled cells.

[0272] Example 6 - Expansion of anti-hapten CAR T cells in vitro CD4+ and CD8+ anti-FL CAR T cells were generated by transducing T cells with vectors. After 18 days, the transduced cells were expanded for the first time using the standard rapid expansion method (REP) with irradiated TM-LCL and PBMCs. The expanded cells were then subjected to a second round of expansion using either the standard REP method or the fluorescein-assisted REP method (FREP). For the FREP method, cells were incubated on feeder cells treated with FL-PLE. After 14 days of the second round of expansion, cells were analyzed by flow cytometry, specific lysis, and cytokine release assays. For the specific lysis and cytokine release assays, K562 cells were incubated overnight with FL-PLE, and the expanded anti-FL CAR T cells were then incubated with these K562 cells. FL-PLE uptake into cells was analyzed by flow cytometry (Figure 9B).

[0273] Both the cells expanded using the REP method and the cells expanded using the FREP method expressed similar phenotypic markers (Figure 9A). Furthermore, CD8+ anti-FL CAR T cells expanded using the FREP method had substantially equivalent cytotoxic activity to CD8+ anti-FL CAR T cells expanded using the REP method (Figure 9C). Furthermore, CD8+ anti-FL CAR T cells expanded using the FREP method had substantially equivalent cytokine release activity to CD8+ anti-FL CAR T cells expanded using the REP method (Figure 9D). Furthermore, CD4+ anti-FL CAR T cells expanded using the FREP method had substantially equivalent cytotoxic activity and cytokine release activity to CD4+ anti-FL CAR T cells expanded using the REP method. Thus, cells labeled with haptens (such as FL) can induce the expansion of CAR T cells, and CAR T cells expanded in this manner have substantially equivalent activity to CAR T cells expanded using irradiated TM-LCL and PBMC.

[0274] Example 7 - Creation of cells with extracellularly exposed haptens (specifically DNP using DNP-PLE) tethered to the cell surface MDA-MB-231 (adenocarcinoma) cells were incubated overnight with DNP-PLE in the presence of complete medium. Cells were stained for DNP molecules exposed on the cell surface with Alexa Fluor 488-conjugated anti-DNP antibody (DNP is non-fluorescent), and the incorporation of DNP-PLE into the cells was analyzed by flow cytometry. As shown in the control data in Figure 11A, almost no shift was observed between parental MDA-MB-231 cells and MDA-MB-231 cells stained with Alexa Fluor 488-conjugated anti-DNP antibody. This result was expected, since DNP is not exposed on the cell surface of MDA-MB-231 cells.

[0275] When parental MDA-MB-231 cells were incubated with 5 μM DNP-PLE and stained with Alexa Fluor 488-conjugated anti-DNP antibody, a clear shift from control parental MDA-MB-231 cells was observed (Figure 11B). However, when parental MDA-MB-231 cells were incubated with 50 nM DNP-PLE and stained with Alexa Fluor 488-conjugated anti-DNP antibody, the shift from control was smaller (Figure 11D). This difference in shift indicates a difference in the amount of DNP exposed on the cell surface that was recognized by CAR T cells. By varying the concentration of this chemical (DNP), the density of this hapten (DNP) on the cell surface could also be varied. The amount of DNP exposed on the surface of MDA-MB-231 parental cells incubated with 500 nM DNP-PLE was between that of MDA-MB-231 parental cells incubated with 50 nM DNP-PLE and that of MDA-MB-231 parental cells incubated with 5 μM DNP-PLE (Figure 11C). A histogram plot of the data shown in Figures 11A-11D is shown in Figure 11E.

[0276] These data demonstrated that we successfully generated cells with haptens (especially DNP using DNP-PLE) tethered to the cell surface and exposed extracellularly.

[0277] Example 8 - Confirmation of whether anti-DNP CAR has the ability to recognize DNP on DNP-PLE supported on the cell surface MDA-MB-231 (adenocarcinoma) cells were incubated overnight with 5 μM DNP-PLE or 1 μM DNP-PLE to load DNP-PLE, or without DNP-PLE. After incubation overnight, the cells were washed and images were captured by confocal microscopy to determine where DNP-PLE was incorporated into the cells. Cell nuclei were stained with DAPI (i). The cell surface was stained with wheat germ agglutinin (WGA) (ii). As shown in Figure 12B, because DNP is non-fluorescent, the DNP moiety was stained with Alexa Fluor 488-conjugated anti-DNP antibody (iii). Anti-DNP antibody fluorescence was observed in (iii), confirming that DNP-PLE was incorporated throughout the cell surface (Figures 12C and 12D). These images indicated that the DNP moiety was accessible for binding, as the antibody was able to bind to it. The image in Figure 12C is brighter than that in Figure 12D, which correlates with the amount of DNP exposed on the cell surface. Figure 12A shows a control image of parental MDA-MB-231 cells alone. As can be seen from the lack of staining in the image, the anti-DNP antibody was unable to bind to the cells; that is, DNP was not present on the cell surface, resulting in no staining by the anti-DNP antibody. The left images in Figures 12A-12D show confocal images obtained by overlaying all of the images (i)-(iv) in each figure. The right side of the overlaid confocal image shows grayscale images of each layer (nucleus (i), cell surface (ii), and DNP-PLE (iii)) that make up this confocal image.

[0278] Therefore, it was confirmed that anti-DNP CAR cells have the ability to recognize DNP on DNP-PLE carried on the cell surface.

[0279] Example 9 - Confirmation of extracellular accessibility of haptens loaded onto cells and confirmation of loading of PLE onto cell membranes Figure 13A shows a schematic of a second-generation CAR cassette with a long spacer for expressing an anti-DNP CAR. This cassette contains a gene encoding a double-mutated dihydrofolate reductase, which allows for selection of CAR-positive cells with methotrexate, and a gene encoding EGFRt, a surface marker that correlates with CAR positivity.

[0280] The plasmids shown in Figure 13A were transduced into H9 cells (CD4+CD3+ skin T lymphocytes), and a pure anti-DNP CAR population was selected with methotrexate. The purity of the anti-DNP CAR H9 cells was measured by staining for the surface marker EGFRt. After cell staining, analysis by flow cytometry showed a 92% positive rate in the flow cytometry plot, indicating the anti-DNP CAR H9 population.

[0281] MDA-MB-231 (adenocarcinoma) cells were incubated with or without 5 μM DNP-PLE, then washed and co-cultured with a pure population of H9 cells expressing anti-DNP CAR. Images were acquired by confocal microscopy to examine whether recognition occurred between the cell surface-exposed DNP and the anti-DNP CAR (Figures 13C and 13D). Two groups were used in this experiment: MDA-MB-231 cells co-cultured with anti-DNP CAR-expressing H9 cells (Figure 13C), and MDA-MB-231 cells co-cultured with anti-DNP CAR-expressing H9 cells after loading with 5 μM DNP-PLE (Figure 13D). Cell nuclei were stained with DAPI (i). Cell surfaces were stained with wheat germ agglutinin (WGA) (ii). Because DNP is not fluorescent, the DNP moiety was stained with Alexa Fluor 488-conjugated anti-DNP antibody ((iii) and (iv)). To distinguish between MDA-MB-231 cells and CAR H9 cells, CAR H9 cells were stained with anti-CD3 antibody (red). Below each color image, grayscale images of each layer constituting the overlapped confocal image (nucleus (i), cell surface (ii), DNP-PLE (iii), and anti-DNP CAR-expressing H9 cells (iv)) are shown. Figure 13C shows no binding between the target and effector. Figure 13D shows the interaction between the target and effector. The top left image in Figure 13C shows the overlapped confocal image of all images (i) to (iv) in this figure. The top left image in Figure 13D shows the overlapped confocal image of all images (i) to (iv) in this figure. These images demonstrate synapse formation between the cells, thus confirming that DNP exposed on the surface of the target cell was recognized by the anti-DNP CAR. This is clearly shown in Figure 13D (iv), where synapses are observed extending to the target cells.

[0282] Therefore, we confirmed the extracellular accessibility of the hapten carried on the cells and the presence of PLE on the cell membrane. The experimental data demonstrated that the anti-DNP CAR was successfully prepared and that DNP on the cell surface was accessible using this anti-DNP antibody, thus demonstrating that the anti-DNP CAR can bind to DNP exposed on the cell surface.

[0283] Example 10 - Cytokine production by CD19 CAR-transduced T cells against various targets and non-autologous T-APCs in vitro This figure shows the correlation between the induction of CD19 CAR T cell activation and the production of specific cytokines. To analyze cytokine production, pure CD8+ CD19 CAR T cell populations or CD8+ mock T cell populations (effectors) [these cells were stimulated with CD3 / CD28 microbeads and used 8 days after rapid expansion] were seeded at a 2:1 ratio with CD19-specific target cells and incubated for 24 hours. Target cells used were K562 parental cells (negative control), K562 OKT3 cells (positive control), K562 CD19 cells, and non-autologous CD4+ / CD8+ mixed transduced antigen-presenting cells (T-APCs) with truncated CD19 (CD19t) generated from clinical samples (positive target cells, the same target as used in Example 11). The supernatants were analyzed for the presence of cytokines. BioPlex assays were performed to measure the production of IL-2, TNF-α, and IFN-γ. When co-cultured with any CD19-specific target cells, including non-autologous CD4 / CD8 T-APCs, CD19 CAR T cells produced significant amounts of cytokines. Cytokine production was not detected in the parental K562 cell line, which does not express CD19. This experiment demonstrated the production of specific cytokines and therefore demonstrated that CD19 CAR T cells can be activated by non-autologous T-APCs.

[0284] Therefore, it was confirmed that cytokines were produced by CD19 CAR-transduced T cells upon activation with non-autologous T-APCs.

[0285] Example 11 - Activation of autologous T-APCs in vitro CD4+ / CD8+ mixed antigen-presenting cells (T-APCs) generated from clinical specimens by transduction with cleaved CD19 (CD19t) were stained and analyzed by flow cytometry for cell surface expression of CD19t and cleaved EGFR (EGFRt). CD19t T-APCs were 63% CD19t positive and, as expected, lacked EGFRt expression, demonstrating their CAR-negative status (Figure 15A). Transduced autologous CD4+CD19 CAR T cells and transduced autologous CD8+CD19 CAR T cells were generated from clinical specimens and transduced with irradiated CD19 T cells at a feeder cell to T cell ratio of 7:1 in the presence of rhIL-2 and rhIL-15. + The cells were expanded by rapid expansion (REP) while stimulating with feeder cells (TM-LCL). On day 7 of expansion, the cells were stained and examined for EGFRt expression by flow cytometry. Both transduced CD4+CD19 CAR T cells and transduced CD8+CD19 CAR T cells had a 99.9% positive rate for EGFRt expression, which correlated with CAR expression (Figure 15B).

[0286] On day 7 of the expansion culture, effector and target cells were cocultured at a 2:1 ratio for 24 hours, and then cytokine production was examined by assessing the supernatants of CD19t T-APCs (Figure 15A) and CD4+CD19 CAR T cells and CD8+CD19 CAR T cells (Figure 15B) using a Bio-Plex assay kit manufactured by Bio-Rad. CD4+CD19 CAR T cells, CD8+CD19 CAR T cells, and CD4+ / CD8+CD19t T-APCs were cocultured for 24 hours with CD19t T-APCs, K562-CD19+ cells (parental K562 cells engineered to express CD19), K562-OKT3 cells (parental K562 cells engineered to express the agonist OKT3 scFv to serve as a universal positive control), or parental K562 cells (negative target cells), respectively. Supernatants were collected and frozen until analysis for the presence of cytokines (Figure 15C). Bio-Plex assays demonstrated that CD4+CD19 and CD8+CD19 CAR T cells were able to produce cytokines only in the presence of K562 CD19+ cells, CD19t T-APCs, or the K562 OKT3 positive control cell line, demonstrating anti-CD19-specific cytokine production. As expected, CD4+ / CD8+ T-APCs were able to produce cytokines only in the presence of the K562 OKT3 cell line. Although coculture of CD4+CD19 or CD8+CD19 CAR T cells with CD19t T-APCs resulted in low cytokine production, this coculture significantly activated autologous CD19 CAR T cells and resulted in significant clinical outcomes (see Example 13 and Figures 17A-17D).

[0287] These data demonstrated that autologous T-APCs could be activated in vitro.

[0288] Example 12 - Activation of autologous hapten-APCs in vitro K562 leukemia cells (Figure 16A) or primary CD8+ T cells (Figure 16B) were incubated overnight in the presence or absence of 5 μM FL-PLE, and fluorescence was measured by flow cytometry. Flow cytometry analysis showed fluorescein positivity, indicating successful cell loading of the hapten fluorescein. The ability of FL-PLE-loaded cells to activate anti-FL CAR T cells was measured by cytokine release assay (Figure 16C). FL-PLE-loaded cells were cocultured with autologous anti-FL CAR CD4+ effector T cells or primary autologous CD8+ T cells for 24 hours, and the supernatants were analyzed for the presence of the indicated cytokines. Autologous CD8+ T cells and autologous anti-FL CAR CD4+ T cells were stimulated with CD3 / CD28 microbeads and used for experiments 21 days after two rounds of expansion. Anti-FL CAR T cells produced cytokines when cocultured with either FL-PLE-bearing K562 cells or with autologous CD8+ cells bearing FL-PLE (H-APC). As expected, cytokine production was not detected when cocultured with the parental K562 cell line (not bearing FL-PLE) or CD8+ T cells lacking CAR expression, but was detected when cocultured with the positive control cell line, K562 OKT3+ cells (non-CAR-expressing, activated via TCR). The cytokine production levels were comparable to those observed with transduced APCs (T-APCs) in vitro (Figures 15A-C), demonstrating efficacy in patients (see Example 13 and Figures 17A-D).

[0289] H-APCs are believed to be as effective as T-APCs in vivo in animal models and clinical subjects / patients (eg, during clinical trials or treatment).

[0290] These data demonstrated that autologous hapten-APCs could be activated in vitro.

[0291] Example 13 - Persistence of CAR T cells in peripheral blood Two pediatric acute lymphoblastic leukemia (ALL) patients were serially administered T-APCs, and the persistence of CAR T cells in their peripheral blood was examined. Values ​​are shown as lymphocyte percentage (%) (Figure 17A) or cell density (cells / μl) (Figure 17B). On day 0, CD19 CAR T cells were infused into the patients (white inverted triangles), and CAR T cell persistence was monitored longitudinally by cell surface staining for EGFRt, a CAR transduction marker (black circles) (Figures 17A and 17B). ALL cell abundance was monitored by staining for CD19+ B cells (white diamonds). On day 0, patients were administered CD19 CAR T cells containing the monitoring cell surface marker EGFRt (black circles). ALL cells rapidly regressed to undetectable levels by day 10. CD19 in peripheral blood + B cells were no longer detectable by day 10 (C1.D10), likely due to rapid engraftment of CAR T cells. CAR T cell persistence gradually declined after day 10. Because CAR T cell persistence was not sufficiently high, the patient received serial transduced antigen-presenting cells (T-APCs) at the time points indicated on the graph (black inverted triangles) to enhance persistence. These T-APCs were autologous T cells genetically engineered to express the CD19 surface antigen. The patient received autologous transduced antigen-presenting T cells (T-APCs) expressing the CD19 surface protein, the target of CAR T cells. These T-APCs express the CD3 antigen, which is not present on CD19+ B cells, allowing differentiation between these two CD19+ populations. This patient received five T-APC infusions. After each T-APC infusion, CAR T cells expanded, preventing relapse of ALL. CD19 +T-APCs were monitored over time (filled boxes) and distinguished from CD19+ B cells by CD3 expression. A transient expansion of CD19 CAR T cells was observed after each T-APC infusion, which appeared to correlate with the lack of long-term CD19+ B cell formation. An example of multiparameter flow cytometry analysis of peripheral blood from a patient shown in Figure 17B demonstrates the detection of CD19+ T-APCs 1 day after the second T-APC infusion (Figure 17C) and the detection of EGFR+ CAR T cells in the peripheral blood 14 days after the third T-APC infusion (Figure 17D). These data demonstrate the persistence of CAR T cells in the patient's peripheral blood.

[0292] Example 14 - Labeling of peripheral blood mononuclear cells with FL-PLE Peripheral blood mononuclear cells (PBMCs) were isolated from blood using a conical leukapheresis chamber. T cell depletion was performed by sequentially separating T cells from PBMCs using CD8+ and CD4+ magnetic beads. T cell-depleted PBMCs are shown in Figures 18A and 18D. Specifically, Figure 18A shows the PBMC cell population, and Figure 18D shows the amount of FL-PLE loaded on these cells. A portion of the PBMCs obtained by the separation procedure to obtain "(T cell-depleted) PBMCs" was stained with 5 μM FL-PLE (Figures 18B and 18E). After staining, a portion of the FL-PLE-loaded PBMCs (T cell-depleted) was analyzed by flow cytometry, and the remaining portion was frozen in fresh freezing medium. The frozen PBMCs were thawed and analyzed by flow cytometry (Figures 18C and 18F). Compared with unstained PBMCs (T cell-depleted) (Figure 18D), which showed no blue shift, both FL-PLE stained samples (Figures 18E and 18F) showed a complete blue shift, indicating FL-PLE incorporation into T cell-depleted PBMCs. These results demonstrate that FL-PLE-incorporated cells can undergo freeze-thaw cycles while maintaining FL-PLE, and that any PBMC cell population (T cell-depleted) can be stained with FL-PLE. Therefore, cells can be labeled with FL-PLE, and FL-PLE-labeled cells can be frozen and thawed, maintaining FL-PLE labeling.

[0293] Example 15 - Expansion of hapten-specific CAR T cells in vitro T cells isolated from PBMCs in Example 14 were transduced with polynucleotide cassettes encoding second-generation anti-fluorescein (FL) CARs containing long spacers. Two types of anti-FL CARs were used: FITC-E2 and FITC-E2 Tyr100gAla. Each polynucleotide cassette contained a selectable marker gene encoding a double-mutated dihydrofolate reductase, which allows for methotrexate-mediated selection of CAR-positive cells, and a gene encoding a truncated CD19 polypeptide (CD19t), a cell surface selectable marker.

[0294] Transduced cells were selected with methotrexate to obtain a homogenous CAR-positive cell population. Cells were expanded using standard rapid expansion (REP) methods with irradiated TM-LCLs and PBMCs (Figure 19A). Fluorescein-assisted REP (FREP) was performed using irradiated TM-LCLs loaded with 5 μM FL-PLE at a target-to-effector cell ratio of 7:1 (Figure 19B). As shown in Figures 19C and 19D, FREP was also performed using irradiated autologous PBMCs loaded with 5 μM FL-PLE at a target-to-effector cell ratio of 7:1 or 14:1. As shown in Figure 19E, FREP was also performed using irradiated autologous PBMCs loaded with 5 μM FL-PLE and then frozen and thawed (T cell-depleted) at a target-to-effector cell ratio of 7:1. Two types of anti-FL CAR T cells and mock T cells were also expanded using the standard REP method. Large-scale expansion using the FREP method enabled only the expansion of anti-FL CAR T cells. In particular, the expansion of anti-FL CAR T cells was significant when autologous PBMCs (T cell-depleted) loaded with 5 μM FL-PLE were used. These data demonstrate that anti-FL CAR T cells can be expanded in vitro using fluorescein-labeled autologous cells.

[0295] Example 16 - In vivo expansion of hapten-specific CAR T cells using hapten-APC On day 0, 20 NSG mice were intravenously (IV) injected with a homogenous population of anti-fluorescein (anti-FL) CAR T cells. Approximately 40% of this CAR T cell population contained a gene encoding a fusion protein of mCherry and firefly luciferase (mCherryffLuc). This fusion protein allows quantitative tracking of T cells by bioluminescence imaging. An increase in bioluminescence signal indicates expansion of anti-FL CAR T cells. Mice were divided into four groups: (A) a group receiving only anti-FL CAR T cells (control); (B) a group receiving anti-FL CAR T cells and irradiated 20 × 10 CAR T cells on days 1, 4, and 10. 6 (C) 5 × 10 TM-LCLs intravenously injected; (D) 5 × 10 TM-LCLs intravenously injected; (E) 5 × 10 TM-LCLs intravenously injected; (F) 5 × 10 TM-LCLs intravenously injected; (G) 5 × 10 TM-LCLs intravenously injected; (H) 5 × 10 TM-LCLs intravenously injected; (I) 5 × 6 (D) a group of 20 × 10 TM-LCLs (hapten-APC) intravenously injected with hapten-APC; and (E) a group of 20 × 10 TM-LCLs (hapten-APC) intravenously injected with hapten-APC on days 1, 4, and 10, which were irradiated and then loaded with 5 μM FL-PLE. 6 Patients were divided into four groups: one group receiving intravenous injections of TM-LCLs (hapten-APCs), the other group receiving intravenous injections of TM-LCLs (hapten-APCs). Both groups A and B showed the least expansion of anti-FL CAR T cells. The lack of significant expansion in group B indicated that TM-LCL cells alone were insufficient to expand CAR T cells. In groups C and D, CAR T cell expansion was observed with each intravenous injection of hapten-APCs. In groups C and D, anti-FL CAR T cells expanded after the second injection of hapten-APCs, followed by an initial decline in anti-FL CAR T cells, and then again after the third injection of hapten-APCs. The results are shown in Figures 20A-20E. These data demonstrate that hapten-specific CAR T cells can be repeatedly expanded in vivo using hapten-APCs.

[0296] As used herein, the term "comprising" is synonymous with the terms "including," "containing," or "characterized by," and is open-ended and inclusive, not excluding additional elements or steps not described herein.

[0297] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention are susceptible to modification, as are the manufacturing methods and equipment. Such modifications will be readily apparent to those skilled in the art from practice of the invention disclosed herein or from consideration of this disclosure. Therefore, the present invention is not limited to the particular embodiments disclosed herein, but rather encompasses all modifications and other aspects which fall within the true scope and spirit of the invention.

[0298] All references cited herein, including, but not limited to, published patent applications, unpublished patent applications, patents, and scientific literature, are incorporated herein by reference in their entirety and made a part of this specification. In the event that any reference, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or take precedence over such conflicting matter.

Claims

1. 1. A pharmaceutical composition for treating, inhibiting, or alleviating cancer in a subject, comprising: hapten-containing antigen-presenting cells (H-APCs), the subject comprises chimeric antigen receptor (CAR) T cells; the CAR T cell comprises at least two CARs, one of which is a CAR that specifically binds to a tumor-specific antigen of the cancer, and the other is a CAR that specifically binds to the hapten; The H-APC is used so as to be repeatedly administered, A pharmaceutical composition, wherein administration of the H-APC induces expansion of the CAR T cells.

2. The pharmaceutical composition of claim 1, wherein the CAR T cells and the H-APCs are derived from the subject.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the CAR T cells comprise a bispecific CAR.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the CAR T cells comprise two or more types of CARs.

5. 5. The pharmaceutical composition of claim 1, wherein the CAR T cell comprises a first ligand-binding domain that specifically binds to a tumor-specific antigen and a second ligand-binding domain that specifically binds to the hapten.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the CAR T cells comprise a monospecific CAR.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the tumor-specific antigen is selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B-cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epithelial cell adhesion molecule (EpCAM), epidermal growth factor variant III, receptor tyrosine kinase-like orphan receptor 1 (ROR1), CD123, prostate stem cell antigen (PSCA), CD5, Lewis Y antigen, B7H3, CD20, CD43, HSP90, and IL13.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the hapten is selected from the haptens listed in Table 1.

9. the hapten is selected from fluorescein, urushiol, quinone, biotin and dinitrophenol and derivatives thereof; and / or The CAR that specifically binds to the hapten comprises an scFv comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 to 6, 8 and 10; The pharmaceutical composition according to any one of claims 1 to 8.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the hapten is covalently bound to the extracellular surface of the H-APC.

11. The pharmaceutical composition of claim 10, wherein the hapten is bound to the H-APC via an ether phospholipid (PLE).

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the CAR T cells are derived from CD4+ cells, CD8+ cells, progenitor T cells, or hematopoietic stem cells.

13. The method of claim 12, wherein the CD8+ 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; or The pharmaceutical composition of claim 12, wherein the CD4+ 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.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the H-APC is derived from a T cell or a B cell.

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