A method for stimulating chimeric antigen receptor T cells with hapten-labeled cells and a composition 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] Priority rights and cross-references of related applications This application claims priority to U.S. Provisional Application No. 62 / 714928, filed on 6 August 2018, which is incorporated herein by reference in its entirety.

[0002] Electronic sequence listings This application was filed via EFS-Web along with an electronic sequence listing in ASCII text file format. This electronic sequence listing is filed as SCRI164WOSEQLIST.txt, last updated on August 2, 2019, and is 47,590 bytes in size. The information contained 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 for stimulating chimeric antigen receptor (CAR) T cells. In some embodiments, the CAR T cells may include CARs that specifically bind to haptens. Some embodiments relate to the stimulation of CAR T cells with hapten-labeled cells in vivo or in vitro. [Background technology]

[0004] Immunotherapy using adoptive cell transfer (ACT) of T cells possessing chimeric antigen receptors has been previously reported to be usable in the treatment of cancer, tumors, and even hematological malignancies. The structure of a chimeric antigen receptor (CAR) includes an antigen-binding domain, linker and spacer sequences, a costimulatory activation domain, and a transmembrane region. CAR-expressing cells may be derived from the patient requiring treatment, or from donor cells (related or unrelated). CARs can bind to specific proteins or antigens on cells or tumor cells. When recombinant CAR T cells are injected into a patient, they proliferate further in the patient's body, recognize cancer cells or tumor cells that have specific proteins or antigens on their cell surface, and kill these cells.

[0005] On the other hand, it is important that CAR T cells maintain their efficacy over a long period. When hematological cancers reach the final stage of regression, the number of cancer cells decreases, resulting in a decrease in antigen levels, and consequently, CAR T cells shrink and lose their efficacy. Furthermore, solid tumors exhibit very high immunosuppression within the tumor environment. Therefore, in order to achieve a complete cure, it is necessary to stimulate CAR T cells to eliminate any remaining cancer cells. Moreover, by stimulating and restimulating CAR T cells, it is possible to overcome the immunosuppressive tumor environment.

[0006] Stimulation and restimulation of CAR T cells have been reported in the past. For example, CAR T cell stimulation can be performed in vitro by adding anti-CD3 / CD28 beads before injection into the patient. Embodiments provided herein describe novel approaches for stimulating CAR T cells in vivo and in vitro. [Overview of the project] [Means for solving the problem]

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

[0008] Some embodiments of the methods and compositions provided herein are methods for treating, suppressing, or mitigating cancer in a subject, A process of administering an effective amount of CAR T cells having a chimeric antigen receptor (CAR) that specifically binds to tumor-specific antigens of cancer; and The process involves incubating CAR T cells with hapten antigen-presenting cells (H-APCs), and specifically binding the CAR of the CAR T cells to the hapten bound to the H-APCs, thereby inducing the expansion and proliferation of the CAR T cells. This includes methods that include [specific methods]. In some embodiments, the CAR T cells and H-APCs are derived from the subject, such as humans.

[0009] In some embodiments, the CAR T cells include bispecific CARs.

[0010] In some embodiments, the CAR T cell contains two or more CARs.

[0011] In some embodiments, the CAR T cell includes 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 cells comprise a single-specific CAR. In 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 is performed in vivo.

[0015] In some embodiments, the CAR specifically binds to a tumor-specific antigen. In some embodiments, 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, epidermal 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 contains a binding fragment of an antibody against a hapten listed in Table 1, an antibody listed in Table 2, and an antibody selected from the sequences listed in Table 3, or the CAR contains one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, dinitrophenol, and derivatives thereof. In some embodiments, the hapten is selected from fluorescein, dinitrophenol, and / or derivatives thereof.

[0017] In some embodiments, the hapten is covalently bound to the extracellular surface of the H-APC. In 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, 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+, and CD4+, and also 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 cells selected from a group consisting of T cells and B cells.

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

[0024] Some embodiments of the methods and compositions provided herein are compositions 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 include a first sequence encoding a first CAR and a second sequence encoding a second CAR, The first CAR comprises a tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain. The second CAR comprises a hapten-specific second ligand-binding domain, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. Contains the composition.

[0025] In some embodiments, the first ligand-binding domain specifically binds to antigens selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epidermal 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 contains a binding fragment of an antibody against a hapten listed in Table 1, an antibody listed in Table 2, and an antibody selected from the sequences listed in Table 3, or the CAR contains one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, dinitrophenol, and / or derivatives thereof. In some embodiments, the hapten is selected from fluorescein, dinitrophenol, and / or derivatives thereof.

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

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

[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 includes ligands that specifically bind to the cytoplasmic domains 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 and / or CD3ζ.

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

[0032] Some embodiments further include a sequence that codes for a marker sequence. In 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 sequences encoding cleavable linkers.

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

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

[0037] Some embodiments of the methods and compositions provided herein are compositions 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 tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain. The second CAR comprises a hapten-specific second ligand-binding domain, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. Contains the composition.

[0038] In some embodiments, the first ligand-binding domain specifically binds to antigens selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epidermal 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 contains a binding fragment of an antibody against a hapten listed in Table 1, an antibody listed in Table 2, and an antibody selected from the sequences listed in Table 3, or the CAR contains one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, dinitrophenol, and / or derivatives thereof. In some embodiments, the hapten is selected from fluorescein or dinitrophenol and / or derivatives thereof.

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

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

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

[0043] In some embodiments, the first intracellular signaling domain and / or the second intracellular signaling domain includes ligands that specifically bind to the cytoplasmic domains 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 and / or CD3ζ.

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

[0045] Some embodiments further include a sequence that codes for a marker sequence. In 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 ribosome skipping sequence. In some embodiments, the ribosome 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 containing 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 sequences encoding a tumor antigen-specific first ligand-binding domain, a glycine-serine linker, a hapten-specific second ligand-binding domain, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain.

[0051] In some embodiments, the first ligand-binding domain specifically binds to antigens selected from the group consisting of CD19, CD22, HER2, CD7, CD30, B cell maturation antigen (BCMA), GD2, glypican 3, MUC1, CD70, CD33, epidermal 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 contains a binding fragment of an antibody against a hapten listed in Table 1, an antibody listed in Table 2, and an antibody selected from the sequences listed in Table 3, or the CAR contains one or more sequences listed in Table 4. In some embodiments, the hapten is selected from fluorescein, urushiol, quinone, biotin, dinitrophenol, and / or derivatives thereof. In some embodiments, the hapten is selected from fluorescein or dinitrophenol and / or derivatives thereof.

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

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

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

[0056] In some embodiments, the intracellular signaling domain includes ligands that specifically bind to the cytoplasmic domains 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 and / or the cytoplasmic domain of CD3ζ. In some embodiments, the intracellular signaling domain includes a portion of CD3ζ and a portion of 4-1BB.

[0057] Some embodiments further include a sequence that codes for a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG.

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

[0059] Some embodiments of the methods and compositions provided herein include a bispecific CAR expression vector comprising one or more nucleic acids according to any of the embodiments provided herein.

[0060] Some of the 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 by 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 embodiment provided herein, one or more vectors according to any embodiment provided herein, or a bispecific chimeric antigen receptor according to any embodiment provided 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 these 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+, and CD4+, and also 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 are methods for producing cells that express a first chimeric antigen receptor specific to a hapten and a second chimeric antigen receptor specific to a tumor antigen, A step of introducing one or more nucleic acids according to any embodiment provided herein, or one or more vectors according to any embodiment provided herein, into cells under conditions in which a first chimeric antigen receptor and a second chimeric antigen receptor are expressed. This includes methods that include [specific methods].

[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 these 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+, and CD4+, and also 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 are methods for producing cells that express bispecific chimeric antigen receptors specific to haptens and tumor antigens, A step of introducing one or more nucleic acids according to a specific embodiment provided herein, or one or more vectors according to a specific embodiment provided herein, into cells under conditions in which a first chimeric antigen receptor and a second chimeric antigen receptor are expressed. This includes methods that include [specific methods].

[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 these 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+, and CD4+, and also 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 are methods for stimulating or restimulating T cells having chimeric antigen receptors (CARs) in subjects with diseases such as cancer, A step of providing cells to the subject according to any of the specific embodiments provided herein, A step of monitoring the suppression of the disease in the subject; and The process of providing hapten antigen-presenting cells (H-APCs) to the subject. Includes, The subject may be selected for CAR T cell therapy using CAR T cells that have receptors specific to antigens associated with the disease (such as tumor antigens). Includes methods.

[0074] In some embodiments, the H-APC is a cell produced by labeling the target healthy cells with a hapten in 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 step are repeated.

[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 for cancer therapy. In some embodiments, the subject, such as a human, is subjected to a combination therapy such as chemotherapy or radiotherapy.

[0078] Some embodiments of the methods and compositions provided herein are methods for stimulating or restimulating T cells having a chimeric antigen receptor (CAR) ex vivo, A step of providing cells according to a specific embodiment provided herein; A step of providing hapten antigen-presenting cells (H-APCs) or hapten; A step of mixing the aforementioned cells with the H-APC cells to obtain activated cells; and step of isolating the activated cells This includes methods that include [specific methods]. 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 isolation of the activated cells includes affinity isolation by complexing the hapten with affinity beads. In some embodiments, the isolation of the activated cells includes affinity isolation by complexing EGFRt, CD19t, or Her2tG with affinity beads. [Brief explanation of the drawing]

[0079] [Figure 1A] This is a schematic diagram of three types of chimeric antigen receptors (CARs). Panel (1) shows a second-generation CAR having an antigen-recognizing region (i) presented 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-recognizing region (vi) and a long spacer domain (vii) than the CAR in panel (i). Panel (3) shows a bispecific CAR containing two linked antigen-recognizing domains. This CAR can be activated by recognition of either epitope.

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

[0081] [Figure 1C] This is a schematic diagram of CAR T cells containing a bispecific CAR. Bispecific CAR T cells express a single CAR that can recognize two different epitopes.

[0082] [Figure 2]This is a schematic diagram illustrating an example of a treatment method. Hapten antigen-presenting cells (H-APCs) are produced by loading haptens onto the surface of healthy cells. The produced H-APCs are then injected into the patient. Dual CAR T cells and bispecific CAR T cells (Figure 1C) can be activated either through recognition of tumor cells or via H-APCs. One CAR(i) shown in the figure is designed to target an epitope(ii) on tumor cells, while the other CAR(iii) is recombinant to recognize hapten(iv) on hapten-APCs. Hapten-APCs are produced by loading haptens onto the surface of healthy cells. When hapten-APCs are then reinjected into the patient, they can be recognized and lysed by CAR T cells in the patient's body, thereby activating the CAR T cells. If hapten-APCs are not lysed by CAR T cells, the haptens are metabolized and return to normal healthy cells. It should be noted that in some embodiments, for example, when labeling tumor cells with the same hapten used to label hapten-APCs, a single anti-hapten CAR T cell is used.

[0083] [Figure 3A] The structure of an ether phospholipid (FL-PLE) linked with fluorescein as a hapten is shown. This structure includes (i) a fluorescein portion, (ii) a polyethylene glycol (PEG) portion that functions as a spacer allowing the hapten to extend from the cell surface, (iii) a polar head portion, and (iv) a hydrophobic tail portion that is incorporated 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), which contains 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 incubation of Be2 cells, U87 cells, or daoy cells with 5 μM FL-PLE are shown.

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

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

[0091] [Figure 6A] The following shows the results of measuring signal retention over time after incubation of Be2 cells or U87 cells with 5 μM FL-DHPE.

[0092] [Figure 6B]The results of measuring signal retention over time after incubation of Be2 cells or U87 cells with 5 μM FL-PLE are shown.

[0093] [Figure 7A] This is a series of graphs showing the results of cytotoxic assays. The lytic ability of two types of anti-FL CAR T cells (4M5.3 and FITC-E2) against hapten-labeled cells was tested using a chromium release assay. To produce 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 endogenously activate T cells via the TCR.

[0094] [Figure 7B] This is a series of graphs showing the results of cytokine production measurements using a cytokine release assay. Two types of anti-FL CAR T cells (4M5.3 and FITC-E2) were tested against hapten-labeled cells prepared 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] This is a series of graphs showing the results of cytotoxic assays performed by incubating hapten-labeled cells (K562 cells with 0.5 μM or 5 μM FL-PLE) with anti-FL CAR T cells. K562 parental cells were used as the negative control, and K562+OKT3 cells were used as the positive control.

[0097] [Figure 8C]This is a series of graphs showing the results of cytokine production measurements in a cytokine release assay performed by incubating hapten-labeled cells (K562 cells with 0.5 μM or 5 μM FL-PLE) with anti-FL CAR T cells.

[0098] [Figure 9A] The results of flow cytometry analysis show that anti-FL CAR T cells express similar phenotypic markers regardless of whether they are cultured using the FREP or REP method.

[0099] [Figure 9B] 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) are shown.

[0100] [Figure 9C] This is a series of graphs showing the results of cytotoxicity assays of anti-FL CAR T cells cultured using the FREP or REP method. The assays were performed using cells harvested from the experiment shown in Figure 9B.

[0101] [Figure 9D] This is a series of graphs showing cytokine stimulation by anti-FL CAR T cells cultured using the FREP or REP method. The assay was performed using cells harvested from the experiment in Figure 9B.

[0102] [Figure 10A]The diagram shows the structure of an ether phospholipid (DNP-PLE) linked to 2,4-dinitrophenol [shown in (i)] as a hapten, which is a target of CAR T cells. (ii) represents polyethylene glycol (PEG), which is a spacer to move the target an ideal distance from the cell surface. In the PLE shown in (iii) and (iv), (iii) represents a 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] Figures 11A to 11E show data related to the creation of cells containing haptens (particularly DNP using DNP-PLE) that are anchored to the cell surface and exposed extracellularly.

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

[0106] [Figure 11B] Flow cytometry data of MDA-MB-231 parental 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] Figures 11A to 11D show histogram plots of the flow cytometry data.

[0110] [Figure 12] Figures 12A to 12D show confocal microscopy data regarding the integration of DNP-PLE into cells.

[0111] [Figure 12A] The image shows confocal images of MDA-MB-231 parent cells that do not support DNP-PLE, stained with Alexa Fluor 488-labeled anti-DNP antibody.

[0112] [Figure 12B] The image shows confocal images of MDA-MB-231 parent cells incubated with 5 μM DNP-PLE but not stained with Alexa Fluor 488-labeled anti-DNP antibody.

[0113] [Figure 12C] The image shows confocal images of MDA-MB-231 parent cells incubated with 5 μM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody.

[0114] [Figure 12D] The image shows confocal images of MDA-MB-231 parent cells incubated with 1 μM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody.

[0115] [Figure 13]Figures 13A to 13D show data confirming the extracellular accessibility of haptens supported on cells and confirming that PLEs were supported on the cell membrane.

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

[0118] [Figure 13C] The image shows a confocal image of MDA-MB-231 co-cultured with anti-DNP CAR H9 cells.

[0119] [Figure 13D] The image shows a confocal image of 5 μM DNP-PLE-supported MDA-MB-231 co-cultured with anti-DNP CAR H9 cells.

[0120] [Figure 14] The graph shows data on cytokine production by CD19 CAR T cells co-cultured with various target cells and non-autologous T-APCs.

[0121] [Figure 15] Figures 15A to 15C show data on the activation of autologous T-APC in vitro.

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

[0123] [Figure 15B] The results of flow cytometry detection of EGFRt expression in transduced CD4+CD19 CAR T cells and transduced CD8+CD19 CAR T cells are shown.

[0124] [Figure 15C] The graphs show cytokine production by transduced CD4+ CD19 CAR T cells and transduced CD8+ CD19 CAR T cells.

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

[0126] [Figure 16A] This shows the fluorescence analysis by flow cytometry of K562 leukemia cells incubated overnight in or without 5 μM FL-PLE.

[0127] [Figure 16B] This shows the fluorescence analysis by flow cytometry of primary CD8+ T cells incubated overnight in or without 5 μM FL-PLE.

[0128] [Figure 16C] This graph shows cytokine production by activated anti-FL CAR T cells.

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

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

[0131] [Figure 17B] The graph shows the status of the CAR T cell population, T-APC population, and CD19+ B cell population in the peripheral blood of the second patient after treatment.

[0132] [Figure 17C] Figure 17B shows the results of detecting CAR T cells by flow cytometry on day 1 (C1, T2, D1) in the second patient.

[0133] [Figure 17D] Figure 17B shows the results of detecting CAR T cells by flow cytometry on day 14 (C1.T3.D14) in the second patient. [Modes for carrying out the invention]

[0134] Definition of Terms In this specification, “approximately” may indicate that a particular value includes variations in error inherent to the method used to determine that value, or variations between experiments.

[0135] In this specification, “nucleic acid” or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of monomers made from natural nucleotide monomers (such as DNA or RNA), or analogs of natural nucleotides (for example, enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications to the sugar moiety and / or the pyrimidine base moiety or purine base moiety. Modifications to the sugar moiety include, for example, the substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Furthermore, the entire sugar moiety may be substituted with a structure that is stereochemically similar or electronically similar, such as aza sugars and / or carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Similar bonds to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoranilothioate bonds, phosphoranilidate bonds, and phosphoramidate bonds. "Nucleic acid molecule" also includes "peptide nucleic acid," which includes native nucleic acid bases or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded. In this specification, "encodes" refers to the property that a particular nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA functions as a template for synthesizing another macromolecule, such as a given amino acid sequence.Therefore, when mRNA corresponding to a particular gene is transcribed and translated to produce a protein in a cell or other biological system, that gene encodes this protein. A “nucleotide sequence encoding a polypeptide” includes any degenerate nucleotide sequence encoding the same amino acid sequence. “Specific” or “specificity” may refer to the properties of a ligand to its binding partner, or the properties of a binding partner to a ligand, such as complementary shape, charge, and specificity involved in hydrophobic binding. Specificity involved in binding includes stereospecificity, region selectivity, and / or chemoselectivity. In some embodiments, a method for producing nucleic acids encoding chimeric antigen receptors is provided, which makes it possible to obtain nucleic acids encoding chimeric antigen receptors specific to haptens or tumor antigens.

[0136] A “vector” or “construct” is a nucleic acid used to introduce heterologous nucleic acids into cells and can contain various regulatory elements, thereby enabling the expression of heterologous nucleic acids in cells. Examples of vectors include, but are not limited to, plasmids, minicircles, yeast and / 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.

[0137] The terms “chimeric antigen receptor,” “CAR,” or “chimeric T cell receptor” have the general and ordinary meanings as used herein and include, but are not limited to, synthetically designed receptors in which the ligand-binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with the disease or disorder is ligated via a spacer domain to one or more intracellular signaling domains (e.g., a costimulatory domain) of a T cell receptor or other receptor. Chimeric receptors may also be called artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, and / or chimeric antigen receptors (CARs). Such CARs are recombinant receptors that can be given arbitrary specificity to cells expressing immune receptors. Some researchers understand chimeric antigen receptors, or “CARs,” to include an antibody or antibody fragment, a spacer, a signaling domain, and a transmembrane region. However, the CARs described herein have been modified in various ways, with different components or domains (e.g., epitope-binding regions (e.g., antibody fragments, scFvs, or parts thereof), spacers, transmembrane domains, and / or signaling domains) resulting in remarkable effects, so it is often possible to distinguish the components of the CARs as distinct elements throughout the disclosure herein. In some embodiments, the spacer of the chimeric antigen receptor is selected to confer desired binding properties to the CAR (e.g., so that the spacer has a specific amino acid length). Then, multiple CARs presented with spacers of various lengths, for example on a cell, are screened for their binding affinity or interaction ability with target moieties to which the CAR is directional. Typical target moieties include, but are not limited to, biotin, digoxigenin, dinitrophenol, green fluorescent protein (GFP), yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, far-infrared fluorescent protein, and fluorescein (e.g., fluorescein isothiocyanate (FITC)).The target region to which a CAR binds or interacts can be presented on a substrate such as a membrane, beads, or support (e.g., a well), a binder such as a lipid (e.g., PLE) or a hapten, or on a cell such as a hapten-presenting cell. The CAR may be specific to a hapten on a cell or to an antigen presented on a cancer cell or a pathogen (such as a virus or bacteria). One approach involves contacting a substrate or binder containing a desired target region with a number of cells containing a CAR or TCR specific to this target region, and measuring the amount to which the cells containing the CAR or TCR bound to the target region presented on the substrate or binder. Such evaluation of binding may include staining of cells bound to the target region, or evaluation of fluorescence or disappearance of fluorescence. Modifications made to the structure of the CAR, such as changing the length of a spacer, can also be evaluated in the same manner. In some approaches, the method of the present invention provides hapten-containing cells and performs a step of stimulating T cells having a second CAR or TCR specific to a target region or antigen on a target cell, such as a cancer cell, tumor cell, or target virus, by contacting the hapten-carrying cells.

[0138] "Specific" or "specificity" may refer to the properties of a ligand to its binding partner, or the properties of a binding partner to a ligand, such as complementary shape, charge, and specificity involved in hydrophobic bonding. Specificity involved in binding includes stereospecificity, region selectivity, and / or chemoselectivity. In some embodiments, a method for producing nucleic acids encoding chimeric antigen receptors is provided, which can be used to obtain nucleic acids encoding tumor antigens or haptens.

[0139] In this specification, “antigen” or “Ag” refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immune cells, or both. It is readily apparent that antigens can be produced, synthesized, and manufactured by recombinant technology, and can also be obtained from biological samples. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids (e.g., blood, plasma, or ascites). In this specification, “antitumor effect” refers to a biological effect manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastatic lesions, an extension of life expectancy, or a reduction in various physiological symptoms associated with the cancerous state. “Antitumor effect” may also be manifested by a reduction in recurrence or an extension of the time to recurrence. In some embodiments provided herein, T cells having CARs have an antitumor effect.

[0140] A "bispecific chimeric antigen receptor" refers to a CAR containing two domains: a first domain specific to a first ligand and a second domain specific to 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 contains two scFv domains, the first scFv domain being specific to the tumor-specific ligand and the second scFv domain being specific to the hapten.

[0141] In this specification, “ligand” refers to a substance that specifically binds to another substance to form a complex. Ligands include epitopes on antigens, molecules that bind to receptors, substrates, inhibitors, hormones, and / or activators. In this specification, “ligand-binding domain” refers to a substance or a part thereof that binds to a ligand. Ligand-binding domains include the antigen-binding portion of an antibody, the extracellular domain of a receptor, and / or the active site of an enzyme. The “amino acid sequence identity (%)” identified with respect to the chimeric receptor polypeptide of the present invention is defined as the percentage of amino acid residues in the ligand-binding domain, spacer, transmembrane domain, and / or lymphocyte-activating domain in the candidate sequence that match the amino acid residues in the reference sequence of each domain. This amino acid sequence identity is calculated after aligning the candidate sequence and the reference sequence and inserting gaps as necessary to calculate the maximum value of sequence identity (%), and conservative substitutions are not considered as part of the sequence identity. Alignment for determining amino acid sequence identity (%) can be performed using various methods within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, such as any algorithm necessary to maximize alignment over the full length of the sequences being compared. For example, calculating amino acid sequence identity (%) using the WU-BLAST-2 computer program [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] uses several search parameters, most of which are set to default values. Parameters 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.Amino acid sequence identity (%) is determined by dividing the number of identical amino acid residues that (a) match the amino acid sequence of each polypeptide or all of the reference sequence of the chimeric receptor by the number of identical amino acid residues. In some embodiments, the nucleic acid encoding the CAR or the nucleic acid encoding the polypeptide of the CAR may have sequence identity (%) with the sequences shown in Table 3 or Table 4.

[0142] In some embodiments, cells can be recombined to express two CARs or bispecific CARs using a vector such as a viral vector, such as a gamma retroviral vector or a lentiviral vector, or a CRISPR / CAS9 system. 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.

[0143] The terms “costimulatory domain” or “intracellular signaling domain” have, in light of this specification, a general and ordinary meaning and include, but are not limited to, a signaling portion that provides T cells with signals that mediate T cell responses, such as activation, proliferation, differentiation, and cytokine secretion, in addition to primary signals provided by, for example, the CD3ζ chain of the TCR / CD3 complex. Examples of costimulatory domains include, but are not limited to, whole molecules such as CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or whole ligands that specifically bind to CD83, or parts thereof. In some embodiments, the costimulatory domain is an intracellular signaling domain that mediates cellular responses, including activation, proliferation, differentiation, and / or cytokine secretion, by interacting with other intracellular mediators.

[0144] In some of the embodiments described herein, the CAR of the present invention is specific for a hapten. In some of the embodiments described herein, the second CAR is present on T cells specific for an antigen on cells or tumor cells. In some of the embodiments described herein, the CAR of the present invention comprises a co-stimulatory domain. In some embodiments, the co-stimulatory 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 of any of the foregoing.

[0145] A "transmembrane domain" is a hydrophobic protein region present in the cell membrane bilayer, and functions to anchor the protein embedded in the biological membrane. The topology of the transmembrane domain may be, but is not limited to, a transmembrane α-helix. In some embodiments of the method for producing genetically modified T cells comprising a chimeric antigen receptor, the vector comprises a sequence encoding a transmembrane domain. In some embodiments of the method, the transmembrane domain comprises a CD28 transmembrane sequence or a fragment thereof, and the CD28 transmembrane sequence or fragment thereof is 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, 21 amino acids in length, 22 amino acids in length, 23 amino acids in length, 24 amino acids in length, 25 amino acids in length, 26 amino acids in length, 27 amino acids in length, or 28 amino acids in length, or a length within the range defined by any two of these lengths. In some embodiments of the method, the CD28 transmembrane sequence or fragment thereof is 28 amino acids in length. In some embodiments, the chimeric receptor of the present invention comprises a transmembrane domain. The transmembrane domain functions to anchor the chimeric receptor to the membrane.

[0146] "T cell receptor" or "TCR", as used herein, has its general ordinary meaning, and is, for example, but not limited to, a molecule present on the surface of T lymphocytes, i.e., T cells, which is responsible for the recognition of antigen fragments bound to major histocompatibility complex molecules.

[0147] As used herein, "hapten" has its general ordinary meaning in the context of the present specification, and examples thereof include, but are not limited to, a binding moiety composed 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 the hapten bound to a carrier. In some embodiments, the hapten may be anchored to a carrier (e.g., a cell).

[0148] In some embodiments, the hapten may be an Alexa Fluor fluorescent dye, regardless of its type. In some embodiments, the "hapten" may be any small molecule that can induce an immune response only when bound to a large carrier such as a protein, and the carrier may be one that cannot induce an immune response by itself. In some embodiments, the hapten may be any small molecule that can induce the production of an antibody that specifically binds to the hapten (in free form or in a state bound to a carrier) when combined with a larger carrier such as a protein. In some embodiments, the hapten may be a peptide, other large chemical substance, or an aptamer. In some embodiments, the hapten may be any of the haptens provided in the hapten database accessible at URL: crdd.osdd.net / raghava / haptendb / on the World Wide Web.

[0149] Examples of haptens useful in the embodiments provided herein are listed in Table 1, but are not limited thereto. Table 1: Examples of haptens 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;Alexa Fluor 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); Nicotin (3-(1-methyl-2-pyrrolidyl)pyridine; Blackleaf); 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 (modification: 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); diisopropylatrazine (DIA) (structurally related s-triazines); deethyldiisopropylatrazine (DEDIA) (structurally related s-triazines); deethyldiisopropylatrazine (DEDIA) (structurally related s-triazines); hydroxyatrazine (HA) (structurally related s-triazines); diisopropylhydroxyatrazine (DIHA) (structurally related s-triazines); deethyldiisopropylhydroxyatrazine (DEDIHA) (structurally related s-triazines); simazine (structurally related s-triazines); desmetryn (structurally related s-triazines); prometryn (structurally related s-triazines); 2-hydroxyatrazine (atrazine derivative); 2-hydroxypropazine (structurally related s-triazine); 2-hydroxysimazine; N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (modified: R 1 = NH2, R 2 = NH(CH2)3COOH, R 3= OH); Sulcoflon; 5-chloro-2-{4-chloro-2-[3-(3,4-dichlorophenyl)ureido]phenoxy}benzenesulfonic acid; Fulcoflon (1,3-bis(4-chloro-α,α,α-trifluoro-m-tolyl)urea); Agatha resinol; Sequilin C; Sugiresinol; Hydroxysugiresinol; Hinokiresinol; Coniferyl alcohol; Cinnamyl alcohol; p-coumaric acid; Cinnamic acid; p-coumaric acid; Cinnamic acid; Hinokinin; Guayacylglycerol-β-guaiasyl ether; Morphine-3-glucuronide (M3 G); Codeine; Norcodeine; 6-Monoacetylmorphine; (+)-Methamphetamine; Ceftazidime; Phenobarbital; p-Hydroxyphenobarbital; p-Aminophenobarbital; Cyclobarbital; 3'-Ketocyclobarbital; 3'-Hydroxycyclobarbital; Secobarbital; Barbital; Metalbital; Barbituric acid; Thiopental; Thiobalbituric acid; Primidone; Glutethymide; Pentobarbital; Heroin; Diacetylmorphine; Levalorphan; L-11-Allyl-1,2,3,9,10 ,10a-Hexahydro-4H-10,4a-Iminoethanophenanthrene-6-ol;Petidine (Demerol;Drantin;Meperidin;1-Methyl-4-phenylpiperidine-4-carboxylate ethyl;Isonipecaine);Methamphetamine;d-Desoxyephedrine;Mesedrine;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; desoxymethazone; triamcinolone; 9α-fluoro-11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20-dione; fluocortone; 6α-fluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione; cortisol;11β,17,21-trihydroxypregna-4-ene-3,20-dione; prednisone; 17,21-dihydroxypregna-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 tylcarbamate; BFNP(3-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]amino]propanoic acid); Carbofuran derivatives; 2,3-dihydro-2,2-dimethyl-7-benzofuranol; Benziocarb; Carbaryl; Methiocarb; Propoxul; Aldicarb; Methomyl; Benalaxyl; Methyl N-(phenylacetyl)-N-(2,6-xylyl)-DL-alaninate; Bn-Ba(4-[2-(N-phenylacetyl-N-2,6-xylyl) [Lylamino)propionamide]butyric acid); Bn-COOH (4-[2-(N-phenylacetyl-N-2,6-xylyl-DL-alanine); Benalaxyl derivative; Flalaxyl; Metalaxyl; Acetochlor; Dimetachlor; Metrachlor; 2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide; Dietatylethyl; Benzoylpropetyl; Benzoylpropetyl; 2,4,5-Trichlorophenoxyacetic acid; 2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide; Dietatylethyl Tatyl ethyl; benzoylpropetyl; propachlor; propachlor; 2,4,5-trichlorophenoxyacetic acid; 2,4,5-T; Weedone; 2,4-dichlorophenoxybutyrate (2,4-DB); 2,4-DB; butanoic acid; 4-(2,4-dichlorophenoxy)-; Butoxone; Embutone; MCPA; 2-methyl-4-chlorophenoxyacetic acid; Metaxone; dichlorprop (2,4-DP); 1-[(2-chloro)phenylsulfonyl]monamidosuccinate; chlorsulfuron; chlorbromulon;Amidosulfuron; Chlortoluron; Isoproturon; Diuron; Linuron; O-methyl-O-(4-nitrophenyl)-N-(4-carboxybutyl)-phosphoramidethioate; Parathion methyl; O,O-dimethyl O-4-nitrophenyl phosphorothioate; Metaphos; Wolfatox; Dimethylparathion; Metaside; Parathion ethyl; p-nitrophenylthiophosphate diethyl; O,O-diethyl O-(p-nitrophenyl)phosphorothioate; Fenitrothion; O,O-dimethyl O-4-nitro-m-tolylphosphorothioate; Fenthion, O,O-dimethyl O-4-methylthio-m-tolylphosphorothioate; Bromophos, O-4-bromo-2,5-dichlorophenyl O,O-dimethylphosphorothioate; Chlorpyrifos methyl, O,O-dimethyl O-3,5,6-trichloro-2-pyridyl Phosphothioates; Methyl parathion oxide, paraoxone; Phosphate; O,O-Diethyl O-(4-nitrophenyl) ester, diazinon, O,O-Diethyl O-2-isopropyl-6-methylpyrimidine-4-yl phosphorothioate; Adinphosmethyl; Pyrimphosmethyl; O-2-diethylamino-6-methylpyrimidine-4-yl O,O-dimethyl phosphorothioate; Methidathione; S-2,3-dihydro-5-methoxy-2-oxo-1,3,4-thiadiazole-3-ylmethyl O,O-dimethyl phosphorodithioate; Dimethylchlorothiophosphate; 4-nitrophenol; p-nitrophenol; Phenol derivative (modification 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 biphenyl; Delor 104; polychlorinated biphenyl; Delor 105, polychlorinated biphenyl, 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-dichloropheno 3,5-Dichlorophenol; 2,3,4-Trichlorophenol; 2,3,5-Trichlorophenol; 3-Methylindole; 3-Methylindole derivatives; 4-(3-Methylindole-5-yloxy)butanoic acid; 4-(3-Methylindole-5-yloxy)butanoic acid; 3-Methylindole derivatives; 6-[n-3-Methylindole-5-yloxycarbonyl)amino]hexanoic acid; 6-[n-3-Methylindole-5-yloxycarbonyl)amino]hexanoic acid; 3-Methylindole derivatives; 2-[4-(3-Methylindole-6-yl)buta-1-ylthuro]acetic acid; 2-[4-(3-Methylindole-6-yl)buta-1-ylthuro]acetic acid; 3-Methylindole derivatives; 4-(3-Methylindole-6-yl-4-oxo)butanoic acid;4-(3-methylindole-6-yl-4-oxo)butanoic acid; 3-methylindole derivatives; 6-(3-methylindole-7-yloxy)hexanoic acid; 6-(3-methylindole-7-yloxy)hexanoic acid; indole; indole-3-carboxylic acid; indole-3-acetic acid, an indole derivative; indole-3-acetic acid, an indole derivative; indole-3-propionic acid, an indole derivative; indole-3-propionic acid, an indole derivative Indole-3-carbinol, indole-3-carbinol; tryptophan; tryptamine; 5-methoxyindole-3-carboxyaldehyde, 5-methoxytryptamine; 5-methoxyindole; 6-methoxyindole; 7-methoxyindole, EB1089 (theocalcitol); EB1089 (theocalcitol) 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- Liazines); Irgarol 1051; Fluorescein isothiocyanate; FITC, metanephrine, normetanephrine; Propazine; Terbutyrazine; Terbutyrazine; 6-Chloro-N-(1,1-dimethylethyl)-N'-ethyl-1,3,5-triazine-2,4-diamine; (Structurally related s-triazines); Ametrin (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine (modified: iPr / SCH3 / Et, R; 1 = (CH3)2-CH-NH-, R 2 = -SCH3, R 3 = -NH-CH2-CH3; Irgarol; Cyanazine (Modified: R) 1 = Cl, R 2 = NHCH2CH3, R 3= NHCCN(CH3)2); OH-terbutyrazine; terbutyrazine-2OH; hydroxytriazine (EQ-0027); disopropylatrazine (structurally related s-triazine); desethylterbutyrazine (structurally related s-triazine); desethyl-disopropylatrazine (structurally related s-triazine); atlatone; terbutrin (structurally related s-triazines); atrazine derivatives (modified: R 1 = -NHCH(CH3)2, R 2 = -S(CH2)2COOH, R 3 = -NHC2H5); cyanuryl 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) Procyazin (Modification: R 1 = -Cl, R 2 = -NH-cyclopropyl, R 3 = -NHCCN(CH3)2); (Structurally related s-triazines); prometone (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), desethylatrazine mercapturic acid (desethyl AM) (modification: R 1 = -NAcCys, R2 = -NH2, R 3 = -NHCH(CH3)2); (Structurally related s-triazines); disopropyl atrazine mercapturic acid (disopropyl 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 mercapsule (modified: 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 mercapturate methyl ester (AM methyl ester) (Modification: R 1 = -NAcCysME, R 2 = -NHCH2CH3, R 3 = -NHCH(CH3)2); N-acetylcysteine; S-benzyl mercapturic acid; (structurally related s-triazines); simetryn (modified: R 1 = -SCH3, R 2 = -NHCH2CH3, R 3= -NHCH2CH3); Metrivudine; 4-amino-6-tert-butyl-4,5-dihydro-3-methylthio-1,2,4-triazine-5-one; Sulfonamide; N 4 -Acetylsulfamethazine (modified: N) 4-Acetylsulfamethazine); Sulfonamides; Sulfatiazole; Sulfatiazole; Sulfamerazine; Sulfamerazine; Sulfaquinoxaline; Sulfaquinoxaline; Sulfachlorpyridazine; Sulfachlorpyridazine; Sulfapyridine; Sulfadimethoxine; Sulfadimethoxine; Sulfamethoxazole; Sulfamethoxazole; Sulfisoxazole; Sulfamethazole; Sulfamethazole; Sulfanilamide; Sulfaguanidine; Sulfag Anidine; sulfadiazine; sulfadiazine; sulfamethoxypyrididine; sulfamethoxypyrididine; 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-chloropyridine-3-yl)methyl)imidazolidined-2-imine; pyridine derivatives; 6-chloropyridine-3-carboxylic acid; nicotinic acid; pyridine derivatives; N-((6-chloropyridine-3-yl)methyl )-N-methylacetamide; (6-chloropyridine-3-yl)-N-methylmethanamine; (6-chloropyridine-3-yl)methanol; imidacloprid; 1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidined-2-yrideneamine; 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 (fusarenone X); fluthranil; α,α,α-trifluoro-3'-isopropoxy-o-toluanilide; mepronil; mebenil; Benodanyl; 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)D33-sulfate; 24,25(OH)2D3-hemiglutarate derivative; 11α-hemiglutaryloxy-(24R)-24,25-dihydroxyvitamin D3; 24,25(OH)2D3-hemiglutarate derivative; (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; pe Ntachloroaniline; 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-aminopenicillaneic acid (6-APA); azurocillin; bacampicillin; carbenicillin; epicillin; cloxacillin; dicloxacillin; methanepicillin; methicillin; moxalactam; oxacillin; penicillin G; benzylpenicillin; penicillin V; phenoxymethylpenicillin; pheneticillin; piperacillin; ticarcillin; ampicillin hydrolysate; Penicillin G hydrolysate; 3-phenoxybenzoic acid (3-PBAc); chlorpyrifos; chlorpyrifos derivatives; HCl01; derivatives directly synthesized by substituting the chlorine at position 6 of chlorpyrifos for research purposes with a 3-mercaptopropanoic acid spacer arm; chlorpyrifos derivatives; HTCP (modified: the TCP metabolite HTCP was prepared by hydrolysis of the thiophosphate ester of HCl01); zeatin riboside (trans isomer); zeatin (trans isomer); N; 6 -(2-isopentenyl)-adenosine; IPA; N 6-(2-isopentenyl)-adenine; 2-iP; benzyladenine; kinetin; monulon; monolinuron; fenuron; nebulon; propanil; profam; chloropropam; 4-chloroaniline; methylurea derivative; 1-(3-carboxypropyl)-3-(4-chlorophenyl)-1-methylurea; methylurea derivative; 1-(5-carboxypentyl)-3-(4-chlorophenyl)-1-methylurea; metobromulone; sennoside B; SB; sennoside B with an erythrogenic configuration between C-10 and C-10'; sennoside A ( Modified compounds: Sennoside A (where the C-10 terminals are in a threo configuration); lein; emodin; aloe-emodin; barbaroin; 1,4-dihydroxyanthraquinone; lapontisine; gallic acid; vanillic acid; caffeic acid; homogentisic acid; esculin; cinnamtannin B1; baicalin; naringin hydrate; ougonin; ougonin 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; phenothrin; resmethrin; cyfluthrin; trans-permethrin esfenvalerate; fluvalinate; fenpropatrin; cis-permethrin; 4-phenoxybenzoyl alcohol; diurone derivatives; 1-(3-carboxypropyl)-3-(3,4-dichlorophenyl)-1-methylurea; sidurone; terbutiurone; barban; trifluralin; 2,6-Dinitro-N-propyl-N-(2-carboxyethyl)-4-(trifluoromethyl)benzeneamine; TR-13; 2-Ethyl-7-nitro-1-propyl-5-(trifluoromethyl)-1H-benzimidazole; Benefine; 2,6-Dinitro-N-butyl-N-ethyl-4-(trifluoromethyl)benzeneamine; TR-2; 2,6-Dinitro-N-propyl-4-(trifluoromethyl)benzeneamine; Etalfluralin; 2,6-Dinitro-N-ethyl-N-(2-methyl-2-propenyl)-4-(trifluoromethyl)benzeneamine;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)benzeneamine; 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)benzeneamine; Oryzalin; 3,5-dinitro-4-(dipropylamino)benzenesulfonamide; Pendimethalin; 2,6-dinitro-N-(1-ethylpropyl)-3,4-dimethylbenzeneamine; Pentagalloyl glucose; Pyrene; Pyrene-1-carboxyaldehyde; Phenanthrene; Benzo(a)pyrene; 3,4-benzopyrene; Anthracene; 3,4-benzopyrene; Acenaphthene; Fluorene; Chrysene; 1,2-Benzphen Nanthren; benzo[g,h,i]perylene; benzo[e]pyrene; acenaphthylene; fluorantene; benzo(j,k)fluoren; indeno-1,2,3-cd-pyrene; 1,10-(1,2-phenylene)pyrene; benzo[a]anthracene; 1,2-benzanthracene; benzo(k)fluorantene; naphthalene; benzo[a]fluorantene; dibenzo[ah]anthracene; 1,2,5,6-dibenzanthracene; 2,3-diaminonaphthalene; 2,6-dinitroaniline; 17-β-estradiol (ED); est La-1,3,5(10)-triene-3,17-β-diol; trifluralin derivative; 2,6-dinitro-4-trifluoromethylaniline; trifluralin derivative; N-(2,6-dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid; trifluralin derivative; N-(2,6-dinitro-4-trifluoromethylphenyl)-N-methyl-6-aminohexanoic acid; trifluralin derivative; N-(2,6-dinitro-4-trifluoromethylphenyl)-N-propyl-6-aminohexanoic acid; trifluralin derivative;N-(2,6-dinitro-4-trifluoromethylphenyl)-6-aminohexanoate methyl ester; trifluralin derivative; N-(2,6-dinitro-4-trifluoromethylphenyl)-6-aminohexanoate tert-butyl ester; benfluralin; ethalfluralin; trifluralin derivative; 2,6-dinitro-4-trifluoromethylphenol; isopropaline; aniline; 2-hydroxybenzotrifluoride; N-propyl-6-aminohexanoic acid; N-methyl-6-aminohexanoic acid; MHPG derivative; D-MHPG (D-3-methoxy-4-hydroxyphenyl glycol); MHPG derivative; L-MHPG (L-3-methoxy-4-hydroxyphenyl glycol); MHPG derivative; DL-MHPG (DL-3-methoxy-4-hydroxyphenyl glycol); mixture of isomers of D-MHPG and L-MHPG; MHPG derivative; DL- MHPG-SO4 (DL-3-methoxy-4-hydroxyphenyl glycol sulfate), modified forms may include a mixture of isomers of D-MHPG-SO4 and L-MHPG-SO4; serotonin; 5-HT; 5-hydroxydopamine (5-4HDA); 3,4-dihydroxyphenyl glycol (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; azinephosethyl; S-(3,4-dihydro-4-oxobenzo[d]-[1,2,3]-triazine-3-ylmethyl)O,O-diethylphosphodithioate; phosmet; O,O-dimethyl S-phthalimidomethylphosphodithioate; forpet; N-[(trichloromethyl)thio]phthalimide; tetramethrin; (1-cyclohexen-1,2-dicarboximide)methyl-2,2-dimethyl-3-(2-methylpropenyl)-cyclopropanecarboxylate; N-(bromomethyl)phthalimide; N-(chloromethyl)benzazimide; 6-(N-phthalimidoylmethylthio)hexanoic acid (MFH); bromacyl; 5-bromo-3-sec-br Chil-6-methyluracil; Bromacil derivative; 5-bromo-6-(hydroxymethyl)-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione; Bromacil derivative; 5-bromo-3-(2-methylpropyl-6-methyl-2,4(1H,3H)-pyrimidinedione; Bromacil metabolite; Bromacil derivative; 3-hydroxy-1-methylpropyl-6-methyl-2,4(1H,3H)-pyrimidinedione (modified: Bromacil metabolite); Bromacil derivative; 6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified: bromacyl metabolite); terbacyl derivative; [5-chloro-3-(1,1-dimethylethyl)-6-(hydroxymethyl)-2,4(1H,3H)-pyrimidinedione; terbacyl; 3-tert-butyl-5-chloro-6-methyluracil; bromacyl derivative; ethyl-5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione- 1-yl)hexanoate; bromacyl derivative alkylated at N-1; bromacyl derivative 5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-1-yl)hexanoic acid (modified: bromacyl derivative alkylated at N-1); bromacyl derivative; bromo-6-(bromomethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified: bromacyl derivative with methyl substituted at position 6);Bromacyl derivatives include [5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]-2-carboxylpropanoic acid (modified: a bromacyl derivative with a methyl substituted at position 6); 3-[5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]propanoic acid (modified: a bromacyl derivative with a methyl substituted at position 6); bromo-1,6-dimethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (a bromacyl derivative); and a bromacyl derivative. 5-Bromo-1-butyl-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione; Butachlor; N-Butoxymethyl-2-chloro-2',6'-diethylacetanilide; Amidochlor; N-[(acetylamino)methyl]-2-chloro-N-(2,6-diethylphenyl)acetamide; Nicarbazine; N,N'-bis(4-nitrophenyl) compounds having 4,6-dimethyl-2(1H)-pyrimidinone (modified: DNC+HDP); 2-hydroxy-4,6-dimethylpyrimidine; HDP; Imazalil; [ 1-(β-allyloxy-2,4-dichlorophenethyl)imidazole]; imazalil derivative; EIT-0073 (modified imazalil having an -O(CH2)5-COOH group instead of the original -OCH2CH=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-triazole-1-yl)hexane-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-triazole-1-yl)pentan-3-ol; triflumizole; (E)-4-chloro-α,α,α-trifluoro-N-(1-imidazole-1-yl-2-propoxyethylidene)-o-toluidine; imazalil derivative; EIT-0183; imazalil derivative; EIT-0180; imazalil derivative;EIT-0111; Imazalil derivative; EIT-0158; Imazalil derivative; K-240; Chlorothalonil; Tetrachloroisophthalonitrile(modification on benzene ring: R; 1 = CN, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); 2,4,5,6-tetrachloro-3-cyanobenzamide, which is a chlorothalonil derivative(modification on benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); 2,5,6-trichloro-4-hydroxyisophthalonitrile, which is a chlorothalonil derivative(modification on 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 benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = COOH, R 4 = H, R 5 = Cl, R 6 = Cl); Pentachloronitrobenzene(modification on benzene ring: R 1 = NO2, R 2 = Cl, R 3 = Cl, R 4 = Cl, R 5 = Cl, R 6 = Cl); Benzene hexachloride; Hexachlorobenzene; BHC; Lindane(modification on 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 derivative; ethyl carbamate (modified: R) 1 = OCONHCH2CH3, R 3 = H); 1-Naphthol; 1-Naphthaleneacetamide; -(1-Naphthyl)acetamide; Carbaryl derivative; 1-Methyl carbonate (Modified: R) 1 = OCOOCH3, R 2 = H); Carbaryl derivative; 1-ethyl carbonate (modified: R 1 = OCOOCH2CH3, R 2 = H); 2-ethyl carbonate (modified: R), a carbaryl derivative. 1 = H, R 2 = OCOOCH2CH3); Carbaryl derivative; 1-Ethyl thiocarbonate (modified: R 1 = OCOSCH2CH3, R 2 = H); Carbaryl derivative; 2-ethylthiocarbonate (modified: R 1 = H, R 2 = OCOSCH2CH3); naptalam; N-1-naphthylphthalamiate; carbaryl derivative; 3-hydroxycarbaryl (modified: R 1 = OCONHCH3, R 2 = H, R 3 = OH, R 4 = H, R 5 = H); 4-hydroxycarbaryl (modified: R) is a carbaryl derivative. 1 = OCONHCH3, R 2 = H, R 3 = H, R 4 = OH, R 5 = H); 5-hydroxycarbaryl (modified: R) is a carbaryl derivative. 1 = OCONHCH3, R 2 = H, R 3 = H, R4 = 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-triazine-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 are 2-amino-4-(methylthio)-6-(isopropylamino)triazine (modification: R 1 = -SCH3, R 2 = -NH2, R 3 = -NH-CH(CH3)2); (Structurally related s-triazines are 2-amino-4-methoxy-6-(isopropylamino)triazine (modification: R 1 = -OCH3, R 2 = -NH2, R 3= -NH-CH(CH3)2); TCP derivative (3,5,6-trichloro-2-pyridinol derivative); 3-(3,5-dichloro-6-hydroxy-2-pyridyl)thiopropanoic acid; p-nitrosuccinialidic acid (PNA-S); PNA-S; PNA-C; p-nitro-cis-1,2-cyclohexanedicarboxylic acid; nitroaniline derivative; 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; aromatic alcohols; 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-carbofranphenol; hydroxycarbofran; ketocarbofran; carbosulfan; (dibutylaminothio)methylcarbamate 2,3-dihydro-2,2-dimethylbenzofuran-7-yl; benfuracarb; N-[2,3-dihydro-2,2-dimethylbenzofuran-7-yloxycarbonyl(methyl)aminothio]-N-isopropyl-β-allinate; furathiocarb; 2,3-dihydro-2,2-dimethyl-7-benzofuranyl 2,4-dimethyl-5-oxo-6-oxa-3-thia-2,4-diazadecanoate; carbofuran derivative; 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]amino]butanoic acid (BFNB) (modified: n=3, X=CH2); endrin; nendorin;(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-H Xahydro-4,7-methanoindene; Endosulfan (modified: mixture of α and β isomers); Endosulfan (modified: α isomer); Endosulfan (modified: β 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 isomer (modified: 1S,2R isomer, R:Ph); Fenvalerate isomer (modified: 1R,2S isomer, R:Ph); Fenvalerate isomer (modified: 1R,2R isomer, R:Ph); Fenba lerate isomer (modified: 1S,2R / S isomer, R:Ph); fenvalerate isomer (modified: 1R,2R / S isomer, R:Ph); fenvalerate isomer; fenvalerate (modified: 1R / S,2R / S isomer, R:Ph); thiabendazole; 2-(thiazole-4-yl)benzimidazole; thiabendazole derivative; 5-hydroxythiabendazole (modified: 5-OH-TBZ); thiabendazole derivative; 5-NH2-TBZ; Thiabendazole derivatives; methylbenzimidazole carbamate; albendazole; mebendazole; fenbendazole; thiabendazole derivatives; 2-succinamidhiabendazole; thiabendazole derivatives; 2-succinamidhiabendazole; cambendazole; fenvalerate haptens; (S)-4-chloro-α-(1-methylethyl)benzeneacetate cyano[3-(4-aminophenoxy)phenyl]methyl(4-aminoesfenvalerate); fenvalerate haptens; 4-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]benzenepropanoate benzyl; fenvalerate haptens; 3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetate benzyl; fenvalerate haptens; 3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetate; fenvalerate haptens; 6-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]hexanoate benzyl; 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 mercaptulate (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)phosphoramidethioate (modified form called hapten B); fenthion derivative; fenthion oxide; fenthion derivative; fenthion oxide; pyrimiphos-ethyl; 4-(methylthio)-m-cresol; chlorpyrifos derivative; chlorpyrifos-oxone; 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 derivative (modified form: synthesis of AR1 has been reported); chlorpyrifos derivative; O-ethyl O-(3,5,6-trichloro-2-pyridyl) O-(3-carboxypropyl) phosphorothioate; (PO); a chlorpyrifos derivative, O-ethyl O-(3,5,6-trichloro-2-pyridyl) N-(5-carboxyethyl) phosphoramidothioate; (PN1) (modified form: amide bond of thiophosphate reagent); chlorpyrifos derivative; O-ethyl O-(3,5,6-trichloro-2-pyridyl) N-(2-carboxyethyl)phosphoramidethioate; (PN1) (modified: amide bond of appropriate thiophosphate reagent); triadimephon; (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; (SprAAT (modified: R, a structurally related s-triazine); 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 (modified: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH(CH2CH3); (structurally related s-triazines CDIT (modified: 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]); (Anmeline, a structurally related s-triazine; OAAT (modified: R 1 = OH, R 2 = NH2, R 3 = NH2); (Structurally related s-triazines such as ammerid; OOAT (modification: R 1 = OH, R 2 = OH, R 3 = NH2); (Structurally related s-triazine 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); structurally related s-triazines, N-isopropylammeline; OIAT (modified: R 1 = OH, R2 = NH[CH(CH3)2], R 3 = NH2); structurally related s-triazines, N-ethylammeline; OEAT (modification: R 1 = OH, R 2 = NHCH2CH3, R 3 = NH2); structurally related s-triazines; N-ethyl ammerido; OOET (modification: R 1 = OH, R 2 = OH, R 3 = NHCH2CH3); structurally related s-triazines include cyromazine and CyPAAT (modified: R 1 = NH(C3H5), R 2 = NH2, R 3 = NH2); diamino-s-triazine, a structurally related s-triazine; HAAT (modified: R 1 = H, R 2 = NH2, R 3= NH2); PCBs; 2,5,3',4'-tetrachlorobiphenyl (modified: IUPAC no.: 70); PCBs 2,4,5,3',4'-pentachlorobiphenyl (modified: IUPAC no.: 118); PCBs 2,2',5,5'-tetrachlorobiphenyl (modified: IUPAC no.: 52); PCBs; 6-[3,3',4'-trichlorobiphenyl-4-yl)oxy]hexanoic acid; Methrazone; Trademark name: Mykrox, Zaroxolyn; Furfuryl benzoate; DDT metabolite; 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 (modified: 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'-Tetrabromoviphenyl; 3,4,5,3',4',5'-Hexabromoviphenyl; 2,4,5,2',4',5'-Hexabromoviphenyl; 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 retronesin; 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 Lu; 4,4-dichloro-α-(trichloromethyl)benzhydrol; cyprazine; 6-chloro-N-cyclopropyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine; structurally related s-triazines; dipropetrin; 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-triazole-1-yl)propanol; imazalil; phenalimol; (RS)-2,4'-dichloro-α-(pyrimidine-5-yl)benzhydryl alcohol; rupanine metabolite; (+)-rupanine (modified: R = H); rupanine metabolite; (+)-13-hydroxyrupanine (modified: R = OH); rupanine metabolite; (+)-13-hydroxyrupanine hemysuccinate (modified: R = OCO-(CH2)2COOH); rupanine metabolite; (+)-13-hydroxyrupanine cis-hexahydrophthalate (modified: R = OCOC6H. 10 COOH); Rupanine metabolites; α-Isolupanine; Rupanine metabolites; Hydroxylupanine; Spartein; Cysteine; Multiflolin; Epirupinine; (Structurally related s-triazines); Cyanadic 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); norflurazone; 4-chloro-5-(methylamino)-2-[3-(trifluoromethyl)phenyl]-3(2H)-pyridazinone; norflurazone derivatives; desmethylnorflurazone; metoflurazone; chloro-5-(dimethylamino)-2-[(3-trifluoromethyl)phenyl]-3(2H)-pyridazinone; pyrazone; chloridazone; 5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone (active ingredient); dichlorophenyl-pyridazone; (structurally related s-triazines such as azidoatrazine (modified: R 1 = N3, R 2 = NHCH(CH3)2, R 3 = NHCH2CH3); alachlor, 2-chloro-2',6'-diethyl-N-methoxymethylacetanilide; trichothecolone (modified: R 1 = H, R 2 = OH, R 3 = H, R 4 = O, R 5= H); DON derivative; Acetyl-T-2; DON derivative; T-2 tetraacetate; Chlorpyrifos derivative; Monodechloro-CP; Bromophos derivative; Bromophos methyl; Bromophos derivative; Bromophos ethyl dicapton; 2-Chloro-4-nitrophenyl O,O-dimethyl phosphorothioate; Tetrachlorvinphos; Phosphate (Z)-2-chloro-1-(2,4,5-trichlorophenyl)vinyldimethyl; Triclopyr; 3,5,6-trichloro-2-pyridyloxyacetic acid; Pichloram; 4-amino-3,5,6-trichloropyridine-2-carboxylic acid; Formononetin; Biochanin A; 5,7-dihydroxy-4'-methoxyisoflavones (modified: 4'-methyl ether of genistein); Equol; (7-hydro Xy-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); Angustiphorine; Alodan; 1-Methyl-4-phenyl-4-carboethoxypiperidine 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; Amorolphine; Fenpropidine; (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; cyanazineamide (modified: 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-benzimidazole-2-yl]-methyl ester; Albendazole sulfone; 5(6)-alkylbenzimidazoles; 2-amino-5-(propylthio)benzimidazole; 5(6)-alkylbenzimidazoles; 2-amino-5-(propylsulfonyl)benzimidazole; Oxybendazole; 5-propoxybenzimidazole-2-methylcarbamate; 5(6)-arylbenzimidazoles; Fenbendazole sulfone (modified: sulfone metabolite of fenbendazole); 5(6)-arylbenzimidazoles; 4'-H Droxyfenbendazole; 5(6)-arylbenzimidazoles; oxfendazole (modified: oxfendazole is a 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 Acetylphosphoramidethioate; methamidophos; O,S-dimethylphosphoramidethioate; dichlorvos; 2,2-dichlorovinyldimethyl phosphate; fentoate; S-α-ethoxycarbonylbenzyl O,O-dimethylphosphodithioate; EPN; ethyl p-nitrophenyl thionobenzenephosphonate; bioresmethrin; (1R,3R)-2,2-dimethyl-3-(2-methylpropa-1-enyl)cyclopropanecarboxylic acid 5-benzyl-3-furylmethyl (modification: the ISO general name for the unseparated mixture of isomers of this substance is resmethrin); flufenoxuron; 1-[4-(2-chloro-α,α,α-trifluoro-p-tolyloxy)-2-fluorophenyl]-3-(2,6-difluorobenzoyl)urea; amitorol; 1H-1,2,4-triazole-3-ylamine; molinate; S-ethyl Azepan-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); Pebrate (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 (modified: 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 (modified: R 1 = NHCOCH3, R 2 = CH(CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: R 1 = H, R 2= CH(CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: R 1 = NH2, R 2 = CH(CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: 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) derivative (modified: R 1 = NH2, R 2 = CH2CH3, R 3= CH3); Aminopraoxon; Phosphate; O,O-Diethyl O-(4-aminophenyl) ester, Methylparathion; Phosphothioic 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-Nitrophenyl phosphate; Phosphonic acid; O-(4-nitrophenyl) ester; Phorate; Phosphodithioic acid; O,O-Diethyl S-[(ethylthio)methyl] ester; Ethion; Bis(phosphodithioic acid); S,S'-Methylene O,O,O',O'-Tetraethyl ester; Carbophenothion; Phosphodithioic acid; O,O-Diethyl S-[[(4-chlorophenyl)thio]methyl] ester; Disulfone; Phosphodithioic 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: synthesis of DNP-SL was reported by Balakrishnan et al. (1982); formula is described in the literature by Anglister et al. (1984)); β-ecdysone; benzimidazole derivative; 5(6)-[carboxypentyl)thio]-2-(methoxycarbonyl)amino]-benzimidazole; 2-hydroxybiphenyl; H BP; Atrazine caproic acid; Lysophosphatidic acid (LPA); 1-Acyl-2-hydroxy-sn-glycero-3-phosphate; Berberine; Palmatine; 9-Acetylberberine; Coridaline; Coptisin; Berberbine; 8-Oxoberbein; 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-Methane-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 derivative (modified: hemisuccinate of endosulfandiol); Triazole derivative; 5-(3-hydroxypropyl)-3-amino-2H-1,2,4-triazole; Triazole derivative; 5-(3-hydroxypropyl)-3-(2-nitrophenylsulfenyl)amino-2H-1,2,4-triazole; Triazole derivative; 3-amino-5-[(3-succinyloxy) Propyl]-2H-1,2,4-triazole; triazole derivative; 3-amino-1,2,4-triazole-5-thiol; triazole derivative; 3-[(2-nitrophenylsulfenyl)amino-2H-1,2,4-triazole-5-thiol; triazole derivative; 2H-1,2,4-triazole-5-thiol; triazole derivative; 4-methyl-1,2,4-triazole-3-thiol; triazole derivative; (1,2,4-triazole-2-yl)acetic acid; 1,2,4-triazole; 4-nitrophenyl4'-carboxymethylphenyl Phosphate; 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-triazole-1-yl)-methylhexanoic acid; Succinic acid; Imidazole; L-histidine; L-glutamic acid; Permethrin derivatives; 3-phenoxybenzyl 2,2-Dimethylcyclopropane-1,3-Dicarboxylate; 3-Phenoxybenzaldehyde; Flucitrinate; Chrysanthemum acid; 2,4-Dinitrophenyl; DNP; Thyramhaptens; 4-[Carbodithioate(methyl)-amino]butanoate disodium; Thyramhaptens; 5,11-Dimethyl-6,10-Dithioxo-7,9-Dithia-5,11-Diazadodecanoic acid; Thyramhaptens; 2-{[(dimethylamino)carbotioil]sulfanyl}ethaneic acid; Thyramhaptens; 4-{[(dimethylamino)carbotioil]sulfanyl}butanoic acid; Thyramhaptens;6-{[(dimethylamino)carbotiol]sulfanyl}hexanoic acid; thyram haptens; 11-{[(dimethylamino)carbotiol]sulfanyl}undecanoic acid; thyram haptens; 2-{[(dimethylamino)carbotiol]sulfanyl}ethaneic acid; thyram; tetramethylthiuram monosulfide; tetraethylthiuram disulfide; sodium dimethyldithiocarbamate; zinc dimethyldithiocarbamate; sodium diethyldithiocarbamate; N,N,N',N'-tetramethylthiourea; nabam; zineb; maneb; ethylenethiourea; chlorpyrifoshapten; O,O-diethyl O-[3,5-dichloro-6-[(2-carboxyethyl)thio]-2-pyridyl] Phosphothioates; 2-succinamidebenzimidazole; 2-benzimidazolecarbamate methyl; 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) Phosphothioate; 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-ketotetranorpostan-1,16-diacid;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 derivative;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'-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 (CD A) 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; isoproturone hapten; 3-(4-isopropylphenyl)-1-carboxypropyl-1-methylurea; chlorotolurone; 3-(3-chloro-p-tolyl)-1,1-dimethylurea; metoxurone; 3-(3-chloro (4-Methoxyphenyl)-1,1-dimethylurea; Metamitron; 4-amino-4,5-dihydro-3-methyl-6-phenyl-1,2,4-triazine-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; Methosulfuron;2-[3-(4-methoxy-6-methyl-1,3,5-triazine-2-yl)ureidosulfonyl]methyl benzoate; captoprilhaptens; captopril-4-(maleimidomethyl)-cyclohexanecarboxylic acid (MCC); captoprilhaptens; captopril disulfide modified; mercaptoethanol-MCC; mercaptoethanol-4-(maleimidomethyl)-cyclohexanecarboxylic acid, modified; captoprilhaptens; captopril without MCC; acleaciside A; acleaciside B; solamaldine; solanin; solanin-S; praprin; solasodine; cassianine; tomatine; lycopersicin; tomatidine; 3-O-β-D-glucopyranosyl-solasodine; O-α-L- Munosyl-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-hydroxysolamardin; 12-hydroxysolasonine; isoanguibin; sorabelin I; sorabelin II; xylosyl-β-solamardin; α-solanine; α-kakonine; dioscin; indole derivatives; β-indoleacetic acid; 2-bromo-4,6-dinitroaniline; 2-chloro-4,6-dinitroaniline; tetril; 2,4,6-trinitrophenyl-n-methylnitramine; nitramine; tetralyte; tetril; 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; Nicergoline; Ethylmorphine; 7,8-Didehydro-4,5-Epoxy-3-Ethoxy-17-Methylmorphinan-6- All; dihydromorphine; dihydrocodeine; dihydromorphinone; hydromorphone; dihydrocodeinone; hydrocodone; naltrexone; N-cyclopropylmethyl-14-hydroxydihydromorphinone; dextromethorphan; (±)-3-methoxy-17-methylmorphinane; homatropin; endorphins and their modified derivatives (type: β-endorphin); methoenkephalin; DALEA; D-Ala(2)-D-Leu(5)-enkephalinamide; vincristine; 22-oxovincaleucoblastine; royl Christine; 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 isomer; D-Erythroneopterin; Neopterin isomer; L-Erythroneopterin;Neopterin isomer; D-threoneopterin; Biopterin isomer; L-erythrobiopterin; Biopterin isomer; D-erythrobiopterin; Biopterin isomer; L-threobiopterin; Biopterin isomer; D-threobiopterin; Pterin-6-carboxylic acid; C7H5NiO3; Pterin; Thromboxane B2; (5Z,9α,13E,15S)-9,11,15-trihydroxythromboxa-5,13-dien-1-acid; 15-ketoprostaglandin F; 2α;Fumonicin B1;Macrofusin;FB1;Tyroleverine;TRH;Tyrotropin-releasing factor;Tyrotropin-releasing hormone;TRF;Protirelin;Lopremon;Tyroleverine-OH;TRH-OH;Diketopiperazine;Cyclo(HP);TRH analog;Methylated TRH;TRH analog;TRH elongation peptide;TRH-Gly;TRH elongation peptide;TRH-Gly-Lys-Arg;TRH elongation peptide;TRH-Gly-Lys-Arg-Ala;TRH elongation peptide;P7(modified:QHPGLRF);TRH elongation peptide;P10(modified) :SLRQHPGLRF); TRH elongation peptide; Ps5 (modified: proTRH[178-199]); TRH elongation peptide; TRH-Ps5 (modified: proTRH[172-199]); hypothalamic peptide; LHRH; cyanoginosine LA; cyanoginosine LB; cyanoginosine LR; cyanoginosine LY; cyanoginosine AY; cyanoginosine FR; cyanoginosine YR; Ne-acetyllysine-containing peptide; Gly-Lys(Ac)-e-aminocaproic acid (Aca)-Cys; benzoic acid; benzenecarboxylic acid; phenylformic acid; drasylic 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; Salicylic alcohol; 2-Cyanophenol; 2-Hydroxyphenylacetic acid; p-Hydroxybenzoic acid; p-Aminobenzoic acid Acids; 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-purine-2-ylamino)acetic acid; 7-methylxanthine; 7-methyluric acid; 7-methyladenine; guanine; 2-amino-1,7-dihydro-6H-purine-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-purine-6(7H)-one; 7-(2,3-dihydroxypropyl)guanine; 2-amino-7-(2,3-dihydro Xypropyl)-1H-purine-6(7H)-one; 7-(2-hydroxyethyl)guanine; 2-amino-7-(2-hydroxyethyl)-1H-purine-6(7H)-one; 7-(2-[(2-hydroxyethyl)amino]ethyl)-guanine; 2-amino-7-(2-(2-hydroxyethylamino)ethyl)-1H-purine-6(7H)-one; 7-carboxymethylguanine; 2-(2-amino-6-oxo-1,6-dihydropurine-7-yl)acetic acid; fluorescein; urushiol; quinone; biotin; His tag; FLAG tag; Strep tag; Myc tag; HA tag; Spot tag; and / or NE tag

[0150] In some embodiments, the hapten includes fluorescein or a derivative thereof.

[0151] In some embodiments, the hapten includes DNP or a derivative thereof.

[0152] The term "target moiety" as used herein has a general and ordinary meaning and includes, but is not limited to, a specific group or site on a molecule or chemical that is a binding target for another chemical or target protein. In some embodiments described herein, the target 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 a portion thereof, which comprises, for example, one or more binding domains or one or more CDRs. Examples of antibodies or antigen-binding moieties useful in the embodiments provided herein include, but are not limited to, antibodies against the haptens listed in Table 1 and antibodies listed in Table 2. Table 2: Examples of antibodies 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-triazole-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'-carboxymethylphenyl phosphate antibody; anti-7-(carboxyethyl)guanine (7-CEGua) antibody (antibody group specific to 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-amitrol 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 (4063-21-1MAb 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 mercapturate 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 antibody; Anti-benzo(a)pyrene antibody (10C10 MAb and 4D5 MAb); Anti-(benzoylphenylurea) antibody (mainly anti-diflubenzuron antibody); Anti-berberine antibody; Anti-β-indoleacetic acid antibody; Anti-biopterin (L-erythro) antibody; Anti-brevetoxin PbTx-3 antibody; Anti-bromacil antibody; Anti-bromophos antibody; Anti-bromophosethyl antibody; Anti-butachlor antibody; Anti-captopril-MCC antibody; Anti-carbamazepine (CBZ) antibody; Anti-carbaryl antibody; Anti-carbaryl antibody (LIB-CNH32; LIB-CNH33, LIB-CNH36; LIB-CNH37; LIB-CNH45; LIB-CNA38); Anti-carbaryl antibody (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]ethaneic 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-chlorothalonyl 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-chlortorulon antibody (antiserum); anti-cyanoginosine 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-hydroxyphenyl glycol) 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-deltamethasone antibody; Anti-deltamethasone antibody (Del 01~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~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-flucoflon 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); 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-isoproturone antibody; Anti-KB-6806 antiserum; Anti-(+)rupanin 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-metrachlor antibody; Anti-metrachlor antibody; Anti-metrachlor antibody (MAb 4082-25-4); Anti-morinate antibody; Anti-monulon antibody; Anti-morphine-3-glucuronide (E3 scFv antibody); Anti-morphine 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-erythrotype) antibody; Anti-nicarbazine antibody (Nic 6; Nic 7; Nic 8; and Nic 9); Anti-nicergoline antibody (Nic-1; Nic-2; Nic-3 & BNA-1; BNA-3); anti-norflurazone antibody; anti-normetanephrine antibody; anti-(o-DNCP) antibody; anti-P10 antibody (TRH elongated peptide); anti-paraoxone antibody (BD1 and CE3); anti-paraquat antibody; anti-paraquat antibody; anti-parathion-methyl antibody; anti-PCB antibody (antibody against 3,3',4,4'-tetrachlorobiphenyl) MAb S2B1; anti-pentachlorophenol antibody; 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-propoxul antibody (LIB-PRNP15; LIB-PRNP21; LIB-PRNB21; LIB-PRNB33);Anti-prostaglandin E2 antibody; anti-p-tyramine antibody; anti-pyrene antibody; anti-retronesin antibody; anti-retronesin 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-sulcoflon 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-thyram antibody; anti-THP antibody (7S and 19S); anti-thromboxane B2 antibody; anti-thymidine glycol monophosphate antibody (mAb 2.6F.6B.6C); Anti-tyroliberin (TRH) antibody; Anti-TNT antibody (AB1 antiserum and AB2 antiserum); Anti-triadimephon 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-ametrine); Anti-triazine serum (anti-atrazine); Anti-triazine serum (anti-simazine); Anti-triazine serum (anti-simetrine); Anti-trifluralin antibody; Anti-trifluralin antibody; Anti-vincristine antibody; Anti-zearalenone antibody; Anti-zeatin riboside antibody; E2 G2 and E4 C2; Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7; LIB-BFNP23 Mab; MAb H-7 and H-9 (MAb for 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; MAb Fab fragment K411B (isotype IgG2b with κ light chain) variant from K4E7 (GlnL89Glu); MAb Fab fragment K411B (isotype IgG2b with κ light chain) variant from K4E7 (GlnL89Glu / ValH37Ile / GluL3Val); MAb A variant of the Fab fragment K411B (IgG2b isotype with a κ light chain) derived from K4E7 (GlnL89Glu / ValH37Ile / GluL3Val);Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GlnL89Glu / ValH37Ile); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GlnL89Glu / ValH37Ile); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GluH50Gln); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GluH50X); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GlyH100aAla); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GlyH100aSer); MAb A variant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (HisH95Phe); MAb A variant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (HisH95Tyr); MAb A variant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (PheL32Leu); MAb A variant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (TrpH33Phe,Tyr,Leu); MAb A variant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (Tryl96Phe); MAb A variant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (TryL96Phe); MAb A variant of the Fab fragment K411B (isotype IgG2b with a κ light chain) derived from K4E7 (ValH37Ile); a variant of the Fab fragment K411B (isotype IgG2b with a κ light chain) derived from K4E7 (ValH37Ile); 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 antibody; Dansyl antibody; Fluorescein / Oregon Green dye antibody; Lucifer Yellow dye antibody; Tetramethylrhodamine and Rhodamine Red dye antibodies; Texas Red and Texas Red-X dye antibodies; Biotin antibody; Dinitrophenyl antibody and / or Nitrotyrosine antibody;

[0153] In this specification, “marker sequence” encodes a cell containing the protein of interest or a protein used to select or track the protein of interest. Embodiments described herein provide a fusion protein which may include a marker sequence that can be selected in experiments such as flow cytometry. In some embodiments, the marker is Her2tG, CD19t, or EGFRt.

[0154] In this specification, "scFv" is a fusion protein of the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin, linked by a short linker peptide consisting of 10 to 25 or 10 to approximately 25 amino acids. In some embodiments, we provide CARs containing scFv specific to tumor cell surface molecules or haptens presented on cells.

[0155] In this specification, a “ribosome skip sequence” refers to a sequence that has the function of causing ribosomes to “skip” the ribosome skip sequence during translation, thereby preventing the formation of peptide bonds and causing translation to begin from the region immediately following the ribosome skip sequence. For example, some viruses have ribosome skip sequences, allowing for the sequential translation of multiple proteins from a single nucleic acid, and the translated proteins are obtained as separate proteins without being linked by peptide bonds. In this specification, the ribosome skip sequence is used as a “linker” sequence. In some embodiments of the nucleic acid provided herein, the nucleic acid of the present invention includes a ribosome skip sequence between the sequence encoding a chimeric antigen receptor and the sequence encoding a marker protein, so that the chimeric antigen receptor and the marker protein are co-expressed without being linked by peptide bonds. In some embodiments, the ribosome skip sequence is a P2A sequence, a T2A sequence, an E2A sequence, or an F2A sequence. In some embodiments, the ribosome skip sequence is a T2A sequence. In some embodiments, a ribosome skipping sequence is present between two chimeric antigen receptors, and a second ribosome skipping sequence is present between one of the chimeric antigen receptors and a marker.

[0156] "Biotin," as used herein, has a general and ordinary meaning, and includes, for example, water-soluble vitamin Bs. In embodiments herein, hapten is biotin.

[0157] "Fluorescein" as used herein has a general and ordinary meaning, and includes, for example, synthetic organic compounds soluble in water and alcohol. Fluorescein is widely used in a variety of applications as a fluorescent tracer. In some embodiments of the present invention, fluorescein is a target moiety on a lipid that is recognized by a chimeric antigen receptor and undergoes binding to the chimeric antigen receptor. In some embodiments, the hapten is fluorescein or a derivative thereof. In some embodiments, the lipid is a phospholipid (e.g., an ether phospholipid).

[0158] In this specification, 2,4-dinitrophenol (2,4-DNP or simply DNP) is an organic compound represented by the formula HOC6H3(NO2)2 and has the general and ordinary meaning as used herein. DNP is used, in particular, as a preservative, a non-selective bioaccumulative pesticide, and a herbicide. DNP is also an intermediate chemical in the production of sulfur dyes, wood preservatives, and picric acid. In some embodiments of the present invention, DNP is a target moiety on a lipid that is recognized by a chimeric antigen receptor and undergoes binding to the chimeric antigen receptor. In some embodiments, the hapten is DNP or a derivative thereof. In some embodiments, the lipid is a phospholipid (e.g., an ether phospholipid).

[0159] In this specification, “lipid” has the general and ordinary meaning as used herein, and includes, but is not limited to, a type of organic compound containing a carbon chain, fatty acid, or fatty acid derivative, which is usually insoluble in water but can be miscible or mixed with hydrophobic solvents or organic solvents. Examples of lipids include, but are not limited to, fats, waxes, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, sphingolipids, cerebrosides, ceramides, or phospholipids. This specification describes amphiphilic lipids that have a polar head group and a hydrophobic moiety or may have a hydrophobic group. “Hydrophobic group,” or hydrophobic moiety, has the general and ordinary meaning as used herein, and includes, but is not limited to, a molecule or part thereof that repels water and tends to be nonpolar. Examples of hydrophobic groups include alkanes and fats and oils. Examples of lipids include, but are not limited to, glycerolipids, glycerophospholipids, sphingolipids, sterollipids, prenolipids, saccharolipids, or polyketides.

[0160] In some embodiments, the lipid may be a sphingolipid. Sphingolipids may contain a backbone made of a sphingoid base, such as a series of aliphatic amino alcohols including sphingosine. A sphingolipid in which the R group consists only of hydrogen atoms is a ceramide. Other common R groups include phosphocholine (when the R group is phosphocholine, sphingomyelin is obtained), 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 as sphingoglycolipids in general. In some embodiments, the lipid is a sphingoglycolipid.

[0161] 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 terpenoid lipid such as steroid or cholesterol. In some embodiments, the hydrophobic group comprises an ether bond 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, phosphoethanolamine group, oligosaccharide residue, sugar residue, phosphatidylserine or phosphatidylinositol. In some embodiments, the sugar is glycerol.

[0162] In some embodiments, the lipid is a single-chain alkyl phospholipid.

[0163] In some embodiments, the lipid comprises a synthetic alkylphospholipid structure such as edelhosine, perifosine, or erucylphosphocholine. In some embodiments, the lipid is lysophosphatidylcholine, edelhosine, erucylphosphocholine, D-21805, or perfisone. Such lipids have been reported, for example, by van der Lui et al. ("A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells" Mol Cancer Ther 2007; 6(8), 2007 (this document is incorporated herein by reference in its entirety)). In some embodiments of the lipids described herein, the choline in the polar head group can be substituted with a piperidine moiety. In some embodiments, the lipid is an anticancer alkylphospholipid. Anticancer phospholipids 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 (this document is incorporated herein by reference in its entirety)).

[0164] In some embodiments, the lipids provided herein are structurally related synthetic antitumor agents characterized by their interaction with cell membranes. These synthetic lipids are alkylphospholipids, as reported, for example, by van Blitterswijk et al. ("Anticancer mechanisms and clinical application of alkylphopholipids," Biochimica et Biophysica Acta 1831 (2013) 663-674 (this document is expressly incorporated herein by reference in its entirety)). Examples of such synthetic alkylphospholipids include, but are not limited to, edelhosine, miltefosine, perifosine, erucylphosphocholine, and / or erufosine. In some embodiments, the lipid is edelhosine, miltefosine, perifosine, erucylphosphocholine, or erufosine. In some embodiments, the lipid is a stable analog of lysophosphatidylcholine. In some embodiments, the lipid is a thioether-linked variant of edelfosine, or 1-hexadecylthio-2-methoxymethyl-rac-glycero-3-phosphocholine. In some embodiments, the lipid is LysoPC, edelfosine, ylmofosine, miltefosine, perifosine, erucylphosphocholine, or erufosine.

[0165] In this specification, “polar head group” has a general and ordinary meaning in light of this specification, and includes, for example, the hydrophilic group of a lipid (such as a phospholipid). “Phospholipid” has a general and ordinary meaning in light of this specification, and includes, for example, a special type of lipid that has amphiphilic properties and can form a lipid bilayer, but is not limited to this. A phospholipid molecule comprises at least one hydrophobic fatty acid “tail” and a hydrophilic “head” or “polar head group”. In embodiments of this specification, a phospholipid or ether phospholipid comprises a polar head group. In some embodiments, the polar head group comprises a phosphocholine, piperidine moiety, or trimethylarseno-ethyl-phosphate moiety. In some embodiments, the lipid comprises a target moiety, and the CAR binds to the lipid through interaction with the target moiety. In some embodiments, the lipid comprises a polar head group (e.g., a polar head group comprising an aromatic ring) and an alkyl carbon chain. 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 choline, phosphatidylcholine, sphingomyelin, a phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidylserine, or phosphatidylinositol. In some embodiments, the polar head group comprises phosphocholine, a piperidine moiety, or a 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 sugar 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 a lactosylceramide. In some embodiments, the lipid is a glycolipid. In some embodiments, the glycolipid contains sugar units such as n-glucose, n-galactose, and N-acetyl-n-galactosamine. In some embodiments, the lipid contains a hydrocarbon ring such as a sterol.

[0166] In some embodiments, the polar head group of the lipid includes glycerol. In some embodiments, the polar head group of the lipid includes a phosphate group. In some embodiments, the polar head group of the lipid includes choline. In some embodiments, the lipid is phosphatidylethanolamine. In some embodiments, the lipid is phosphatidylinositol. In some embodiments, the lipid includes a backbone made of a sphingoid base. In some embodiments, the lipid includes sterol lipids such as cholesterol or its derivatives. In some embodiments, the lipid includes saccharolipids. In some embodiments, the polar head group includes choline, phosphate, and / or glycerol.

[0167] In some embodiments, the lipid is a glycolipid. In some embodiments, the lipid contains a sugar. In some embodiments, the lipid is derived from sphingosine. In some embodiments, the lipid is a glyceroglycolipid or a sphingoglycolipid.

[0168] In some embodiments, the lipid is an ether lipid having a hydrophobic branched chain.

[0169] In this specification, “ether phospholipid” has a general and ordinary meaning in light of this specification, and includes, for example, lipids in which one or more carbon atoms of a polar head group are linked to an alkyl chain via an ether bond rather than a more general ester bond. In some embodiments, the polar head group is glycerol.

[0170] In this specification, “antibody” may have the general and ordinary meaning in view of this specification and may refer to a large, Y-shaped protein produced by plasma cells that has the function of identifying and neutralizing foreign substances such as bacteria and viruses in the immune system. An antibody protein may contain four polypeptide chains, i.e., two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain consists of a structural domain called an immunoglobulin domain. These domains can contain 70 to 110 amino acids and are classified into various categories according to their size and function. In some embodiments, the CDR region is found within the antibody region, and based on Kabat numbering, the light chain contains CDRL1 consisting of amino acids 24 to 34; CDRL2 consisting of amino acids 50 to 56; and CDRL3 consisting of amino acids 89 to 97, while the heavy chain contains CDRH1 consisting of amino acids 31 to 35; CDRH2 consisting of amino acids 50 to 65; and CDRH3 consisting of amino acids 95 to 102. The CDR region in the antibody can be easily determined.

[0171] Examples of antibodies or their binding fragments that can be used for binding to a target site include monoclonal antibodies, bispecific antibodies, Fab, Fab2, Fab3, scFv, Bis-scFv, minibodies, triabodies, diabodies, tetrabodies, VhH domains, V-NAR domains, IgNAR, and camel Ig. Other examples of antibodies include IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, IgE, IgD, and IgA. Furthermore, 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.

[0172] The antibody or its binding fragment may be specific to the target site and may, for example, contain an antigen present on a tumor or hapten. Examples of haptens useful in the embodiments provided herein are listed in Table 1.

[0173] Any of the cancer-specific antibodies described herein may bind to antigens on cancer cells, such as tumor cells. Examples of specific tumor cell antigens that can generate antibodies capable of binding to a target site include angiopoietin, 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, cell death receptor 2, D Examples of haptens that may bind to hapten target sites include LL4, EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin 4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, VEGF, or other cancer expression antigens. Furthermore, antibodies that may bind to hapten target sites are also conceivable. Examples of haptens useful in the embodiments provided herein are listed in Table 1.

[0174] In the embodiments described herein, several types of “spacers” are envisioned to be used. Spacers for chimeric antigen receptors refer to polypeptide spacers, the length of which is selected to increase or improve the binding ability of the chimeric antigen receptor to the target. Lipids may also contain spacers to move the target moiety away from the lipid, the spacers being bound to the polar head group of the lipid. Polypeptide spacers selected for use with chimeric antigen receptors may be screened to identify specific spacers that improve desired binding properties to the target moiety (e.g., desired interaction with the receptor, or desired avidity with the receptor). With respect to lipid-specific spacers, lipid spacers may include PEG spacers, hapten spacers, small peptides, or alkane chains. In some embodiments, hapten spacers contain two haptens and are referred to as hapten (2×) spacers. In some embodiments, the lipids contain hydrophobic groups such as alkane chains. In some embodiments, the alkane chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms, or a number of carbon atoms within the 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 the range defined by any two of these numbers.

[0175] In this specification, “cytotoxic T lymphocytes (CTLs)” refers to T lymphocytes that express CD8 on their cell surface (e.g., CD8+ T cells). In some embodiments, such cells are “memory” T cells (T) that have previously experienced an antigen. MPreferably, the cells are cytotoxic T lymphocytes. In some embodiments, cells for secreting fusion proteins are provided. In some embodiments, the cells are cytotoxic T lymphocytes. In this specification, "central memory" T cells (or "T") are used. CM A central memory T cell (T) is a cytotoxic T lymphocyte (CTL) that has experienced an antigen and, compared to naive cells, expresses CD62L, CCR-7, and / or CD45RO on its surface, but does not express CD45RA or has reduced CD45RA expression. In some embodiments, cells are provided for secreting a fusion protein. In some embodiments, the cells are central memory T cells (T). CM In some embodiments, central memory cells may be positive for the expression of CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95, but have reduced expression of CD54RA, compared to naive cells. In this specification, “effector memory” T cells (or “T EM A T cell is an antigen-experienced T cell that, compared to a central memory cell, does not express CD62L on its surface or has reduced CD62L expression, and compared to a naive cell, does not express CD45RA or has reduced CD45RA expression. In some embodiments, cells for secreting a fusion protein are provided. In some embodiments, the cells are effector memory T cells. In some embodiments, the effector memory cells are negative for CD62L and / or CCR7 expression and may or may not be positive for CD28 and / or CD45RA expression compared to a naive cell or a central memory cell.

[0176] In this specification, “naive” T cells refer to T lymphocytes that have not experienced an antigen and 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, such as CD62L, CCR7, CD28, CD127, and / or CD45RA.

[0177] In this specification, "T cells" or "T lymphocytes" may be obtained from any mammal, preferably from primates or other species, including monkeys, dogs, and humans. In some embodiments, the T cells are of the same species as the recipient (same species but from a different donor). In some embodiments, the T cells are autologous T cells (the donor and recipient are the same). In some embodiments, the T cells are syngeneic (the donor and recipient are different, but identical twins).

[0178] In this specification, "progenitor T cell" refers to a lymphocyte progenitor cell that can migrate to the thymus and become a progenitor T cell, and progenitor T cells do not express the T cell receptor. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells (lymphocyte progenitor cells) derived from hematopoietic stem cells settle in the thymus and proliferate through cell division, creating a large population of immature thymocytes. Very early thymocytes do not express either CD4 or CD8, and therefore are not double-negative (CD4 - CD8 - These are classified into double-positive thymocytes (CD4) as they develop. + CD8 + ) and ultimately single positive (CD4 + CD8 - or CD4- CD8 + They mature into thymocytes and are then released from the thymus into peripheral tissues.

[0179] In this specification, “CD8 T cell” or “killer T cell” refers to a T lymphocyte capable of killing cancer cells, virus-infected cells, or damaged cells. CD8 T cells recognize proteins or specific antigens produced by cancer cells or viruses that can stimulate an immune response. When the T cell receptor of a CD8 T cell recognizes an antigen, the CD8 T cell can bind to the presented antigen and destroy the cell.

[0180] In this specification, "central memory T cell (T)" is used. CM A "CTL that has experienced an antigen" refers to a CTL that, compared to naive cells, expresses CD62L or CCR-7 and CD45RO on its surface, but does not express CD45RA or has reduced CD45RA expression. In some embodiments, central memory cells are positive for CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95 expression and have reduced CD54RA expression compared to naive cells.

[0181] In this specification, "effector memory" T cells (or "T EM Effector memory cells are T cells that have experienced an antigen and, compared to central memory cells, do not express CD62L on their surface or have reduced CD62L expression, and compared to naive cells, do not express CD45RA or have reduced CD45RA expression. In some embodiments, effector memory cells may be negative for CD62L and / or CCR7 expression and positive or negative for CD28 and / or CD45RA expression compared to naive cells or central memory cells. In this specification, "effector T cells (T)" refers to T cells that have experienced an antigen and, compared to central memory cells, do not express CD62L or have reduced CD62L expression on their surface, and compared to naive cells or central memory cells. EA "Cell" refers to an antigen-experienced cytotoxic T lymphocyte that, compared to a central memory T cell or naive T cell, does not express CD62L, CCR7, and / or CD28, or has reduced expression of CD62L, CCR7, and / or CD28, and is positive for granzyme B and / or perforin. In some embodiments, cells for secreting fusion proteins are provided. In some embodiments, the cells are effector T cells. In some embodiments, the cells, compared to a central memory T cell or naive T cell, do not express CD62L, CCR7, and / or CD28, or have reduced expression of CD62L, CCR7, and / or CD28, and are positive for granzyme B and / or perforin.

[0182] In this specification, "leader sequence" is also known as a signal sequence that can translocate a protein to the cell surface. The leader sequence contained in a CAR is the first amino acid sequence contained in the CAR, and refers to the amino acid sequence that induces cell surface expression. The leader sequence, or signal sequence, may be required for the cell surface expression of a protein. In some embodiments, the leader sequence includes the signal sequence of a granulocyte-macrophage colony-stimulating factor.

[0183] "Hapten-presenting cells (H-APCs)" have a general and ordinary meaning as used herein, and include, but are 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 prepared from a patient's healthy cells or cells compatible with the patient, and the resulting cells are labeled with a hapten. Table 1 lists examples of haptens useful in the embodiments provided herein. There are various methods for labeling cells with haptens, such as chemicals, peptides, aptamers, lipids, and proteins. One example of a method for loading a hapten onto cells involves incubating the cells of interest with fluorescein lipids overnight. One advantage of using fluorescein as a hapten is that its fluorescence can be utilized. Therefore, hapten incorporation can be monitored by detecting the fluorescence of the fluorescein moiety using flow cytometry. Therefore, after incubation, excess fluorescein lipids can be removed, the cell fraction can be analyzed by flow cytometry to analyze hapten incorporation, and the remaining cells can be used for injection into the patient. This approach is safe in this respect because, after injection into the patient, H-APCs that were not targeted by CAR T cells gradually lose their haptens (due to haptens being metabolized and removed from the cell surface, etc.) and return to their original healthy cell morphology. In some embodiments, cells can be transduced to express haptens on the extracellular surface. In some embodiments, haptens can be covalently bound to the extracellular surface via phospholipids such as ether phospholipids.

[0184] T cell "stimulation," or "activation," refers to a method of inducing T cells to initiate a response, such as a signaling response (e.g., proliferation), while maintaining T cell viability and immune function. Stimulation of T cells may induce the response activity of T cells containing CARs. In some embodiments, the stimulation is carried out using an antibody-conjugated support containing an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the method further includes removing the antibody-conjugated support, such as beads or particles, or a substrate such as a dish or test tube. As described in embodiments herein, T cells containing hapten-specific CARs may be stimulated using hapten antigen-presenting cells (H-APCs), or ex vivo using a support such as hapten-conjugated beads.

[0185] "Chemotherapy drugs" are anticancer drugs that may include chemical substances (e.g., anticancer drugs (chemotherapeutic agents)) that can be administered as part of a standardized chemotherapy regimen. Chemotherapy drugs may be administered for the purpose of curative treatment or for the purpose of extending survival or alleviating symptoms (palliative chemotherapy). Chemotherapy may further include hormone therapy and targeted therapy, which are major categories of medical oncology (cancer drug therapy). These chemotherapy regimens are often used in combination with other cancer therapies such as radiotherapy, surgery, and / or hyperthermia. In a very small number of cases, surgery is known to spread cancer. In some embodiments, recombinant immune cells are administered to the tumor site before or after surgical procedures. In some embodiments of this specification, the subjects receiving treatment with CAR T-cell therapy are subjects selected for administration of chemotherapy drugs or anticancer drugs. Some of the newly developed anticancer drugs (e.g., various monoclonal antibodies, their humanized antibodies, and their conjugate fragments) do not exert cytotoxicity indiscriminately, but rather target proteins that are abnormally expressed in cancer cells and are essential for cancer cell proliferation. Such treatments are often called targeted therapies (to distinguish them from classical chemotherapy) and are frequently used in combination with conventional chemotherapeutic agents in antitumor therapy regimens. In some embodiments, the method of the present invention may further include administering one or more such targeted anticancer therapies (e.g., various monoclonal antibodies, their humanized antibodies and / or their conjugate fragments).

[0186] In chemotherapy, chemotherapy drugs are administered. In a single chemotherapy regimen, one drug may be used (monotherapy), or multiple drugs may be used simultaneously (combination chemotherapy or multi-drug combination chemotherapy). Chemotherapy combined with radiotherapy is called chemoradiotherapy. Chemotherapy using drugs that are converted into substances that exhibit cytotoxic activity when exposed to light is called photochemotherapy or photodynamic therapy. In some embodiments of the method described herein, which involves administering recombinant immune cells, the method may further include administering the recombinant immune cells or recombinant macrophages (GEMs) to a subject having cancer, followed by administering photochemotherapy or photodynamic therapy to the subject.

[0187] Examples of chemotherapeutic agents include, but are not limited to, antibody-drug conjugates (e.g., antibodies bound to a drug via a linker), nanoparticles (e.g., nanoparticles 1-1000 nm in size that enhance tumor selectivity and assist in the delivery of poorly soluble drugs), electrochemotherapy, alkylating agents, antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), phloxuridine, 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 recombinant immune cells containing CAR, or a composition containing said recombinant immune cells containing CAR, are administered in combination with one or more anticancer agents, such as one or more of the compounds or therapeutic agents. In some embodiments, the one or more anticancer agents co-administered or administered in combination with the recombinant immune cells of the present invention include antibody-drug conjugates, nanoparticles, electrochemotherapy, alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators, or checkpoint inhibitors. In some embodiments, the antimetabolites include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), phloxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, or thioguanine.

[0188] "Cancer," as used herein, has a general and ordinary meaning and includes, but is not limited to, a group of diseases involving the proliferation of abnormal cells that may 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, such as, but not limited to, colorectal cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including malignant melanoma), bone cancer, and / or brain tumors. Identification and / or selection of cancer patients can be made by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or tumor-associated molecules are known, such as malignant melanoma, breast cancer, brain tumors, squamous cell carcinoma, colorectal cancer, leukemia, myeloma, and / or prostate cancer. Also, such cancers include, but are not limited to, B-cell lymphoma, breast cancer, brain tumors, prostate cancer, and / or leukemia. In some embodiments, one or more tumorigenic polypeptides are associated with kidney cancer, uterine cancer, colorectal cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer (including malignant melanoma), bone cancer, brain tumor, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, or leukemia. In some embodiments, a method is provided to treat, alleviate, or suppress cancer in a subject. In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colorectal cancer, skin cancer (including malignant melanoma), bone cancer, or brain tumor. In some embodiments, the subject is selected to administer another cancer therapy, which may include cancer drugs, radiotherapy or chemotherapy, or drugs suitable for cancer therapy.In some embodiments, the drug is abiraterone, alemtuzumab, anastrozole, aprepitant, arsenic trioxide, atezolizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, combination of chemotherapy drugs, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil, or fulvest. This includes lanto, gemcitabine, imatinib, imiquimod, ipilimumab, ixabepirone, lapatinib, lenalidomide, letrozole, leuprolide, mesna, methotrexate, nivolumab, oxaliplatin, paclitaxel, palonosetron, pembrolizumab, pemetrexed, prednisone, radium-223, rituximab, Sipuleucel-T, sorafenib, sunitinib, talc suspension for intrapleural injection, tamoxifen, temozolomide, temsirolimus, thalidomide, trastuzumab, vinorelbine, or zoledronic acid.

[0189] The term "tumor microenvironment," as used herein, has a general and ordinary meaning, and includes, for example, the cellular environment in which the tumor resides. The tumor microenvironment may include, but is not limited to, surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and / or the extracellular matrix (ECM). In some embodiments herein, T cells having CAR are administered into the tumor environment and stimulated using H-APC.

[0190] Detailed explanation Some embodiments of the methods and compositions provided herein relate to the use of hapten-labeled cells for stimulating chimeric antigen receptor (CAR) T cells. In some embodiments, the CAR T cells may include CARs that specifically bind to haptens. Some embodiments relate to the stimulation of CAR T cells with hapten-labeled cells in vivo or in vitro.

[0191] Chimeric antigen-expressing cells are immune cells that have been recombinantly modified to confer targeting to biomarkers bound to the surface of malignant cells. By utilizing such cell surface targets or antigens, targeted and specific therapies become possible that can reduce the destruction of healthy tissue and maintain the immune system of patients undergoing treatment. T cells are crucial components of the adaptive immune system because they not only modulate cytotoxicity but also provide long-term cellular "memory" of specific antigens. While the activation of endogenous T cells requires interaction between MHC-presented peptides and their corresponding TCRs, CAR T cells are recombinantly modified to be activated via tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs). Therefore, CAR T cells can be considered "living drugs" containing target-directing domains (single-stranded variable region fragments (scFv), peptides, polypeptides, ligands, mutaines, spacers, and / or linkers) fused to the T cell's signaling domain. When the target-directing domain recognizes and binds to its specific target, the CAR T cell is activated, initiating the killing of the target cell. CAR T-cell therapy has been used as an innovative treatment for hematological malignancies targeting CD19 and CD20. However, CAR T cells have not been able to be clinically applied effectively and efficiently to solid tumors, and further research is needed for their application in the treatment of solid tumors. The embodiments provided herein relate to the stimulation of CAR T cells. Furthermore, stimulating CAR T cells may solve various challenges currently facing CAR T-cell therapy, which are important for the further development and success of CAR T cells, such as persistence in vivo and an immunosuppressive tumor microenvironment.

[0192] In some embodiments of this specification, T cells are transduced, transfected, or transformed so that at least two unique CARs (dual CARs) are expressed in a single cell, where one of the two CARs is specific to a tumor target and the other is specific to a hapten (e.g., fluorescein). In another embodiment, T cells are transduced, transfected, or transformed so that a single CAR (bispecific CAR) containing two target-directing moieties (e.g., two scFvs) is expressed, where one of the target-directing moieties is specific to a tumor and the other is specific to 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 in various ways, for example, by dual transduction with a viral vector, single transduction with a viral vector containing both CARs, or by a non-viral transposon vector. There are also various methods for selecting a pure CAR T cell population or an isolated CAR T cell population. For example, two types of cell surface tags, such as EGFRt, Her2tG, and CD19t, are used to sort cells by each surface marker. In some embodiments of this specification, anti-hapten CARs are sorted using a substrate such as magnetic beads, or a dish or test tube labeled with hapten. A unique feature of this method is that anti-hapten CARs can be constitutively expressed because they do not recognize endogenous epitopes in the patient's body.

[0193] It is desirable to prepare H-APCs (hapten antigen-presenting cells) from the patient's healthy cells or cells compatible with the patient, and to label the resulting cells with a hapten ex vivo. Examples of haptens include, but are not limited to, fluorescein, urushiol, quinone, and biotin. Table 1 lists further examples of haptens useful in the embodiments provided herein. There are various methods for labeling cells with haptens (for example, they can be labeled with chemicals, peptides, aptamers, lipids, or proteins). For example, the cells of interest may be incubated with fluorescein lipids overnight. One advantage of using fluorescein as a hapten is that its fluorescence can be utilized. This method allows monitoring of hapten incorporation by detecting the fluorescence of the fluorescein portion using flow cytometry. Thus, after incubation, excess fluorescein lipids can be removed, the cell fraction can be analyzed by flow cytometry to analyze hapten incorporation, and the remaining cells can be used for injection into the patient. This approach is safe in that, after being injected into a patient, H-APCs that are not targeted by CAR T cells gradually lose their haptens (due to haptens being metabolized and removed from the cell surface, etc.) and return to their original healthy cell form.

[0194] H-APC can be administered at any point in treatment if it is necessary to stimulate CAR T cells in a patient. One example of when CAR T cell stimulation is necessary is when a hematological malignancy reaches the final stage of regression, resulting in a decrease in the number of cancer cells, and consequently, a reduction in the size and effectiveness of CAR T cells. In this case, H-APC can be injected to induce the expansion, proliferation, and activation of CAR T cells, thereby continuing cancer regression and, if possible, achieving complete remission of the tumor.

[0195] Another example of a situation where CAR T cell stimulation is necessary in patients is in the treatment of solid tumors. Solid tumors often exhibit high immunosuppression, and stimulating CAR T cells with H-APCs may help overcome the immunosuppressive tumor environment. An H-APC-based approach allows for safe in vivo stimulation of CAR T cells.

[0196] CAR T cells can also be stimulated in vitro using H-APCs. In certain clinical protocols, CAR T cells are stimulated via the TCR using magnetic beads, and then the CAR T cells are reinjected into the patient. H-APCs can be prepared using hapten-labeled magnetic beads. In this case, the cells are stimulated via the CAR with the H-APC before the CAR T cell injection.

[0197] Furthermore, if rapid expansion culture (REP) is desired before reinfusion into the patient, the use of H-APCs is a safe alternative. Standard REP methods use irradiated TM-LCL and PBMCs as feeder cells. H-APCs can be used as an alternative in various ways within the REP method. Firstly, if H-APCs are prepared from the patient's own cells, the irradiation step can be eliminated, and the culture of TM-LCL and isolation of PBMCs become unnecessary. Secondly, H-APCs can be prepared from irradiated cells obtained from another donor. Thirdly, REP methods using such H-APCs can be used in laboratory operations as an alternative to standard REP methods. These examples provide several approaches to selectively expand and proliferate CAR T cells using hapten-specific stimulation.

[0198] To overcome the clinical hurdles faced by CAR T-cell therapy, particularly in the treatment of solid tumors, supplemental support beyond the activity of a single CAR may be necessary. H-APCs offer a mechanism that enhances CAR T-cell engraftment and persistence beyond what is shown in current clinical protocols and may promote T-cell migration to immunosuppressive solid tumor metastasis sites. In hematological malignancies, primary CAR T-cell engraftment can be difficult due to the low cancer threshold, but early activation can be promoted using H-APCs. In either case, the anti-hapten CAR promotes the activation, proliferation, and dispersion of the injected CAR T-cells, while the other expressed CAR induces tumor removal. This strategy also offers a unique method of expanding CAR T-cell culture using the REP method before injection into the patient.

[0199] The embodiments described herein aim to improve the therapeutic properties of CAR T cell therapy in both solid tumors and hematological malignancies. H-APCs are thought to stimulate CAR T cells in vivo to overcome the immunosuppressive tumor microenvironment, enhance the ability of CAR T cells to find and eradicate minute 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 over time, returning to normal healthy cells. Furthermore, stimulating cells using the REP method with H-APCs offers the advantages of reduced cell culture costs and shorter culture periods.

[0200] Another factor to consider when producing cells using a single viral vector incorporating two types of CARs is the relaxation of size limitations. In some embodiments described herein, another vector is transduced simultaneously. Alternative methods for producing CAR T cells aimed at avoiding the potential problem of size limitations are also envisioned in the present invention.

[0201] The toxicity of haptens must also be taken into consideration. However, those skilled in the art will readily understand that an assay can be performed to determine whether a selected hapten (e.g., fluorescein) is well tolerable in humans. The toxicity of the binding components (e.g., lipids, proteins, peptides, or aptamers) attached to the hapten may also be a concern. In this case as well, toxicity can be controlled by selecting binding components that are metabolized or rapidly eliminated from the body. An example of such a chemical is described in PCT / US2018 / 017126 (this document is explicitly incorporated herein by citation).

[0202] Similarly, autologous T cells transfected to express tROR 1 on their cell surface (ROR 1+T-APC) have been developed in the past (Berger et al. 2015, Cancer Immunology Research, 3(2), 206-216). However, a major difference between Berger's cells and the cells in the embodiments described herein is that Berger et al. required transduction, expansion, culture, and characterization of the ROR1+T-APC preparation over a period of several weeks to several months, which was associated with high costs. On the other hand, in the embodiments described herein, it is only necessary to load the cells with a hapten, which can be done in a very short time (e.g., several hours), and then the cells can be reinjected into the patient. Furthermore, various types of cells can be used in the system of the embodiments described herein. Therefore, it is considered unnecessary to use valuable T cells. In addition, the technique reported by Berger has not been used for solid tumors. Furthermore, Berger produced T-APCs through genetic recombination, which is quite costly and time-consuming. In contrast, the embodiments provided herein allow for the rapid and efficient provision of hapten-labeled cells by directly binding haptens to the extracellular surface. Therefore, the methods described in the embodiments herein will revolutionize the field of T-cell immunotherapy for solid tumors and can significantly improve existing hematological cancer therapies using CAR T cells.

[0203] Induction of the expansion and proliferation of CAR T cells Some embodiments of the methods and compositions provided herein include methods for inducing the expansion and proliferation 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 and proliferation of CAR T cells. In some embodiments, the CAR of the CAR T cells is specifically bound to a hapten bound to the H-APC. Some embodiments include methods for treating, suppressing, or mitigating 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 expansion and proliferation of the CAR T cells is induced by incubating the CAR T cells with hapten antigen-presenting cells (H-APCs) and specifically binding the CAR of the CAR T cells to a hapten bound to the H-APC. 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, livestock, or domestic animal.

[0204] In some embodiments, the CAR T cells may include bispecific CARs. For example, a CAR may have two specific binding domains, consisting of a first binding domain that can specifically bind to a target such as a tumor-specific antigen, and a second binding domain that can specifically bind to a hapten.

[0205] In some embodiments, the CAR T cells may contain two or more CARs. For example, the CAR may include a first CAR having a first binding domain that can specifically bind to a target such as a tumor-specific antigen, and a second CAR having a second binding domain that can specifically bind to a hapten.

[0206] In some embodiments, the CAR T cells may include CARs that can bind to targets such as tumor-specific antigens and also to haptens. In some such embodiments, the target and the hapten may include the same or substantially the same binding portion so that the CAR can bind to the binding portion of the target and the binding portion of the hapten. In some such embodiments, the target and the hapten may be tumor antigens provided herein.

[0207] 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, epidermal 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 / or IL13.

[0208] Examples of haptens that can be used in the embodiments provided herein include those listed in Table 1. In some embodiments, useful haptens in the embodiments provided herein include fluorescein, urushiol, quinone, biotin, or dinitrophenol and / or derivatives thereof.

[0209] In some embodiments, H-APCs are prepared by covalently bonding haptens to the extracellular surface. In some embodiments, the haptens are bound to the H-APCs via ether phospholipids (PLEs).

[0210] In some embodiments, the incubation may be performed in vitro. For example, CAR T cells can be produced by transducing cells with a vector encoding a CAR, and the transduced cells can be incubated with H-APC to induce expansion and proliferation of the transduced cells. In some embodiments, the expanded and proliferated cells can be administered to subjects such as humans. In some embodiments, the incubation may be performed in vivo. For example, CAR T cells can be administered to a subject. The subject may also be further administered H-APC that induces expansion and proliferation of the CAR T cells in vivo.

[0211] 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 these CD8+ cytotoxic T lymphocytes are central memory T cells, and these 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, which are CD45RA+, CD62L+, and CD4+, and also 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.

[0212] In some embodiments, the H-APC is derived from the target healthy cells (such as T cells or B cells).

[0213] 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 of living organism origin. In some embodiments, the healthy cells may be any cells derived from an apheresis preparation. In some embodiments, the healthy cells may be any cells that can be labeled ex vivo.

[0214] In some embodiments, a single CAR cell is used with H-APC. In some embodiments, a multimerized CAR is used with H-APC. In some embodiments, for example, when labeling a tumor with a hapten (e.g., hapten-labeled CD19 antibody, hapten-PLE, hapten-labeled small molecule, hapten-labeled peptide, hapten-labeled aptamer, or other hapten-labeled tumor cells), a single anti-hapten CAR T cell is used, and the anti-hapten CAR T cell is expanded and proliferated in the patient's body using an H-APC made with the same hapten. In some embodiments, dual CAR cells or bispecific CAR cells can be used, in which case one CAR (e.g., CD19, CD22, or ROR1) attacks the cancer, and the other anti-hapten CAR is used to expand and proliferate the dual CAR cells or bispecific CAR cells via H-APC (see, for example, Figure 2). In some embodiments, this concept can be further extended by loading three or more CARs and anti-hapten CARs onto cells (for example, CD19 and CD22 for the treatment of acute lymphoblastic leukemia (ALL)) and using anti-hapten CARs to activate and proliferate CAR T cells.

[0215] In some embodiments, CAR T cells are non-autologous T cells.

[0216] In some embodiments, any type of cell can be expanded and proliferated via CAR and H-APC by using the methods disclosed herein. For example, B cells expressing an anti-hapten CAR can be expanded and proliferated by stimulating them with H-APC. Thus, by using this approach, any type of cell can be expanded and proliferated in vivo.

[0217] In some embodiments, CAR T cells can be produced not only as a treatment for cancer, but also as a treatment for viral infections (such as HIV and hepatitis), and can also be produced as a treatment for autoimmune diseases and associated conditions.

[0218] In some embodiments, tumor-infiltrating lymphocytes (TILs) can be harvested from tumors / cancer cells, transduced with CARs, and expanded in vitro / in vivo using H-APCs.

[0219] nucleic acids encoding CAR or bispecific CAR In some embodiments, one or more nucleic acids are provided for the expression of a first chimeric antigen receptor and a second chimeric antigen receptor. The one or more nucleic acids may be provided incorporated in a single vector or in multiple vectors so as to accommodate the payload sizes of the two CARs. The one or more nucleic acids may include a first sequence encoding the first chimeric antigen receptor and a second sequence encoding the second chimeric antigen receptor, wherein the first chimeric antigen receptor includes a tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain and a first intracellular signaling domain, and the second chimeric antigen receptor includes a hapten-specific second ligand-binding domain, 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 the 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, cell death receptor The CAR comprises body 2, DLL4, EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin 4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF. In some embodiments, the CAR can specifically bind to the haptens listed in Table 1. In some embodiments, the haptens can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol and / or derivatives thereof.In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain comprises an antibody or its binding fragment or scFv. In some embodiments, the second ligand-binding domain comprises an antibody binding fragment, such as an antibody against a hapten listed in Table 1 or an antibody listed in Table 2. Examples of amino acid sequences and nucleic acid sequences of antigen-binding domains (such as svFc) capable of binding to haptens such as fluorescein and dinitrophenol are shown in Table 3 below. Any of these can be incorporated into one or more embodiments described herein. [Table 1] TIFF0007926829000002.tif200164

[0220] In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer are 1-24 amino acid lengths, 25-50 amino acid lengths, 51-75 amino acid lengths, 76-100 amino acid lengths, 101-125 amino acid lengths, 126-150 amino acid lengths, 151-175 amino acid lengths, 176-200 amino acid lengths, 201-225 amino acid lengths, 226-250 amino acid lengths, or 251-275 amino acid lengths. 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 ligands that specifically bind to the cytoplasmic domains 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 and / or 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 ribosome skip sequence. In some embodiments, the ribosome skip sequence is P2A, T2A, E2A, or F2A. The cleavable linker may be located between two sequences encoding each chimeric antigen receptor. Furthermore, the cleavable linker may be used between one of the chimeric antigen receptors and the sequence encoding the marker protein. In some embodiments, one or more vectors are provided, each comprising one or more nucleic acids according to any embodiment of this specification. In some embodiments, chimeric antigen receptors are provided, encoded by nucleic acids according to any embodiment of this specification or by vectors according to any embodiment of this specification.

[0221] In some embodiments, the present invention provides one or more nucleic acids for the expression of a first chimeric antigen receptor and a second chimeric antigen receptor, wherein the one or more nucleic acids comprise 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, the first chimeric antigen receptor comprising a tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain and a first intracellular signaling domain, and the second chimeric antigen receptor comprising a hapten-specific second ligand-binding domain, a second polypeptide spacer, a second transmembrane domain and a second intracellular signaling domain. In some embodiments, the first ligand-binding domain 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, cell death receptor 2 The CARs are specific to DLL4, EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin 4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, and / or VEGF. In some embodiments, the CARs can specifically bind to haptens listed in Table 1. In some embodiments, the haptens can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol and / or derivatives thereof.In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain are 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, cell death receptor The formulation includes antibodies or their conjugated fragments or scFv specific to haptens listed in Table 1 or Table 2. In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer are 1-24 amino acid lengths, 25-50 amino acid lengths, 51-75 amino acid lengths, 76-100 amino acid lengths, 101-125 amino acid lengths, 126-150 amino acid lengths, 151-175 amino acid lengths, 176-200 amino acid lengths, 201-225 amino acid lengths, 226-250 amino acid lengths, or 251-275 amino acid lengths. 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 ligands that specifically bind to the cytoplasmic domains 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 and / or CD3ζ. In some embodiments, the intracellular signaling domain includes 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 ribosome skip sequence. In some embodiments, the ribosome skip sequence is P2A, T2A, E2A, or F2A. In some embodiments, a plurality of vectors comprising nucleic acids according to any embodiment of this specification are provided. In some embodiments, a chimeric antigen receptor encoded by a nucleic acid according to any embodiment of this specification or by a vector according to any embodiment of this specification is provided.

[0222] Bispecific chimeric antigen receptor In some embodiments, one or more nucleic acids are provided for the expression of a bispecific chimeric antigen receptor. In some embodiments, the nucleic acid 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 first ligand-binding domain is encoded by 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, cell death receptor 2, DLL4, The CAR is specific to EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGF2, 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 the haptens listed in Table 1. In some embodiments, the haptens can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol and / or derivatives thereof. In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain comprises an antibody or its binding fragment or scFv. In some embodiments, the second ligand-binding domain includes an antibody-binding fragment, such as an antibody against a hapten listed in Table 1 or an antibody listed in Table 2.In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer are 1-24 amino acid lengths, 25-50 amino acid lengths, 51-75 amino acid lengths, 76-100 amino acid lengths, 101-125 amino acid lengths, 126-150 amino acid lengths, 151-175 amino acid lengths, 176-200 amino acid lengths, 201-225 amino acid lengths, 226-250 amino acid lengths, or 251-275 amino acid lengths. In some embodiments, the nucleic acid further comprises a leader sequence. In some embodiments, the intracellular signaling domain comprises ligands that specifically bind to the cytoplasmic domains 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 and / 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 a transmembrane domain of CD28. In some embodiments, one or more vectors for bispecific CAR expression 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, encoded by a nucleic acid according to any of the embodiments described herein or by a vector according to any of the embodiments described herein.

[0223] Cells containing CAR or bispecific CAR In some embodiments, cells comprising one or more nucleic acids according to any embodiment herein, one or more vectors according to any embodiment herein, or a bispecific chimeric antigen receptor according to any embodiment herein are provided. The one or more nucleic acids may be provided incorporated in a single vector or in multiple vectors so as to accommodate the payload sizes of two CARs. The one or more vectors may contain another nucleic acid provided herein. Alternatively, the nucleic acids may be incorporated 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, the present invention provides a plurality of nucleic acids comprising a first nucleic acid containing a first sequence encoding a first chimeric antigen receptor and a second nucleic acid containing a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a hapten-specific second ligand-binding domain, 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 tumor cell antigens.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, cell death receptor 2 This includes DLL4, EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin 4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF. In some embodiments, the CAR can specifically bind to the haptens listed in Table 1. In some embodiments, the haptens can be selected from fluorescein, urushiol, quinone, biotin, and dinitrophenol and / or derivatives thereof. In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain includes an antibody or its binding fragment or scFv. In some embodiments, the second ligand-binding domain includes an antibody-binding fragment, such as an antibody against a hapten listed in Table 1 or an antibody listed in Table 2. In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer are 1-24 amino acid lengths, 25-50 amino acid lengths, 51-75 amino acid lengths, 76-100 amino acid lengths, 101-125 amino acid lengths, 126-150 amino acid lengths, 151-175 amino acid lengths, 176-200 amino acid lengths, 201-225 amino acid lengths, 226-250 amino acid lengths, or 251-275 amino acid lengths. In some embodiments, the nucleic acid further includes a leader sequence.In some embodiments, the first intracellular signaling domain and / or the second intracellular signaling domain includes 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 and / or CD3ζ. In some embodiments, the intracellular signaling domain includes 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 and / or CD3ζ. In some embodiments, the nucleic acid further includes a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t or Her2tG. In some embodiments, the first and / or second transmembrane domains 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 ribosome skip sequence. In some embodiments, the ribosome skip sequence is P2A, T2A, E2A, or F2A. The cleavable linker may be located between two sequences encoding each chimeric antigen receptor. Furthermore, the 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 bispecific CAR expression are provided, comprising one or more nucleic acids according to any embodiment of this specification. In some embodiments, the bispecific chimeric antigen receptor encoded by the one or more nucleic acids is contained within a cell.The one or more nucleic acids encoding the bispecific chimeric antigen receptor include sequences 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 / or an intracellular signaling domain. In some embodiments, the first ligand-binding domain 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, cell death receptor 2, DLL4, E The hapten is specific to GFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGF2, 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 / or derivatives thereof. In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain comprises an antibody or its binding fragment or scFv. In some embodiments, the second ligand-binding domain includes an antibody-binding fragment, such as an antibody against a hapten listed in Table 1 or an antibody listed in Table 2.In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer are 1-24 amino acid lengths, 25-50 amino acid lengths, 51-75 amino acid lengths, 76-100 amino acid lengths, 101-125 amino acid lengths, 126-150 amino acid lengths, 151-175 amino acid lengths, 176-200 amino acid lengths, 201-225 amino acid lengths, 226-250 amino acid lengths, or 251-275 amino acid lengths. In some embodiments, the nucleic acid further comprises a leader sequence. In some embodiments, the intracellular signaling domain comprises ligands that specifically bind to the cytoplasmic domains 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 and / or the cytoplasmic domain of CD3ζ. In some embodiments, the intracellular signaling domain includes a portion of CD3ζ and a portion of 4-1BB. In some embodiments, the nucleic acid further includes a sequence encoding a marker sequence. In some embodiments, the marker is EGFRt, CD19t, or Her2tG. In some embodiments, the transmembrane domain includes the transmembrane domain of CD28. 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 these central memory T cells 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 cells are naive CD4+ T cells, which are CD45RA+, CD62L+, and CD4+, and CD45RO-. In some embodiments, the cells are precursor T cells.In some embodiments, the cells are hematopoietic stem cells.

[0224] Creation of cells containing two types of CARs or bispecific CARs Embodiments of this specification provide a method for producing cells expressing a first chimeric antigen receptor specific to a hapten and a second chimeric antigen receptor specific to a tumor antigen. In some cases, the method includes introducing one or more nucleic acids according to any embodiment of this specification, or one or more vectors according to any embodiment of this specification, into cells under conditions in which the first and second chimeric antigen receptors are expressed. In some embodiments, a method is provided for producing cells expressing bispecific chimeric antigen receptors specific to a hapten and a tumor antigen. The method includes introducing one or more nucleic acids according to any embodiment of this specification, or one or more vectors according to any embodiment of this specification, into cells under conditions in which the first and second chimeric antigen receptors are expressed. 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, 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+, and CD4+, and also 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 tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a hapten-specific second ligand-binding domain, a second polypeptide spacer, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first ligand-binding domain 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, cell death receptor The hapten is specific to 2, DLL4, EGFL7, EGFR, endosialin, EpCAM, FAP, FRα, fibronectin, frizzled receptor, GD2, GPNMB, HER-1, HER-2, HER-3, IGF-IR, IGF2, LOXL2, mesothelin, MS4A1, mucin 5AC, MUC1, nectin 4, neuropilin, N-glycolyl GM3, PSMA, SLAMF7, TAG-72, TRAIL, TYRP1, or VEGF. 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 / or derivatives thereof.In some embodiments, the first ligand-binding domain and / or the second ligand-binding domain are 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, cell death receptor The formulation includes antibodies or their conjugated fragments or scFv specific to haptens listed in Table 1 or Table 2. In some embodiments, the first polypeptide spacer and / or the second polypeptide spacer are 1-24 amino acid lengths, 25-50 amino acid lengths, 51-75 amino acid lengths, 76-100 amino acid lengths, 101-125 amino acid lengths, 126-150 amino acid lengths, 151-175 amino acid lengths, 176-200 amino acid lengths, 201-225 amino acid lengths, 226-250 amino acid lengths, or 251-275 amino acid lengths. 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 ligands that specifically bind to the cytoplasmic domains 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 and / or CD3ζ. In some embodiments, the intracellular signaling domain includes 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 ribosome skipping sequence. In some embodiments, the ribosome 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 tumor antigen-specific first ligand-binding domain, a glycine-serine linker, a hapten-specific second ligand-binding domain, 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 embodiment of this specification are provided.

[0225] T lymphocytes can be recovered by known techniques and enriched or removed by known techniques such as affinity binding to antibodies, flow cytometry, and / or immunomagnetic selection. After the enrichment and / or removal steps, the desired T lymphocytes can be expanded and proliferated in vitro by known techniques or variations thereof, which are readily understood by those skilled in the art. In some embodiments, the T cells are autologous T cells obtained from a patient.

[0226] For example, a desired T cell population or subpopulation can be expanded by adding an initial T lymphocyte population to an in vitro culture medium before expansion, then adding feeder cells such as non-dividing peripheral blood mononuclear cells (PBMCs) to the medium (for example, adding feeder cells in such a ratio that the post-addition cell population contains at least 5, 10, 20, or 40 or more PBMC feeder cells per T lymphocyte in the initial population before expansion), and incubating the medium (for example, for a time sufficient to sufficiently expand the T cell number). The non-dividing feeder cells may include PBMC feeder cells irradiated with gamma rays. In some embodiments, the PBMCs are irradiated with gamma rays at 3000-3600 rads to prevent cell division of the PBMCs. 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 dose of gamma rays within the range of any two of these values ​​as upper and lower limits. The order in which T cells and feeder cells are added to the culture medium may be changed as needed. Typically, the culture can be incubated under conditions suitable for T lymphocyte proliferation, such as a temperature. The temperature for proliferation of human T lymphocytes is typically at least 25°C, preferably at least 30°C, and more preferably 37°C. In some embodiments, the temperature for proliferation of human T lymphocytes 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 the range of any two of these values ​​as upper and lower limits.

[0227] Examples of expanded and proliferated T lymphocytes include CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T lymphocytes, which may be specific to antigens present on human tumors or pathogens. In some embodiments, the cells are progenitor T cells. In some embodiments, the cells are hematopoietic stem cells.

[0228] In some embodiments, the expansion culture method may further include adding EBV-transformed non-dividing lymphoblastoid cells (LCLs) as feeder cells. The LCLs may be irradiated with gamma rays at 6,000 to 10,000 rads. In some embodiments, the LCLs are irradiated with gamma rays at 6,000 rads, 6,500 rads, 7,000 rads, 7,500 rads, 8,000 rads, 8,500 rads, 9,000 rads, 9,500 rads, or 10,000 rads, or any dose of gamma rays within the range of any two of these values ​​as upper and lower limits. The LCL feeder cells can be provided in any appropriate amount; for example, the ratio of LCL feeder cells to the initial T lymphocytes before expansion culture may be at least 10:1.

[0229] In some embodiments, the expansion culture method may further include adding anti-CD3 antibody and / or anti-CD28 antibody to the culture medium (for example, at a concentration of at least 0.5 ng / ml). In some embodiments, the expansion culture method may further include adding IL-2 and / or IL-15 to the culture medium (the concentration of IL-2 is, for example, at least 10 units / ml). After isolating T lymphocytes and performing expansion culture, or after performing expansion culture, cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive T cell subpopulations, memory T cell subpopulations, and effector T cell subpopulations, respectively.

[0230] CD8+ cells can also be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into these types of CD8+ cells by identifying the cell surface antigens associated with naive CD8+ cells, central memory CD8+ cells, and effector memory CD8+ cells, respectively. In some embodiments, memory T cells are present in both the CD62L+ subset and the CD62L- subset of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ fractions and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, central memory T cells (T CM The expression of phenotypic markers of ) 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 These cells 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 the expression of naive T cell phenotypic markers, such as CD62L, CCR7, CD28, CD3, CD127, and / or CD45RA.

[0231] CD4+ helper T cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations possessing 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-.

[0232] Whether cells or cell populations are positive for a specific cell surface marker can be determined by flow cytometry using staining with an antibody specific to the surface marker and a control antibody matched to its isotype. Negative staining of a specific marker in a cell population means that no cell population stains more strongly with the specific antibody than the isotype control, while positive staining means that a cell population stains more uniformly with the specific antibody than the isotype control. In some embodiments, a decrease in the expression of one or more markers means a decrease of 1 log10 in mean fluorescence intensity compared to a control cell population, and / or a decrease in the percentage of cells expressing the marker to at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or any percentage in the range of 20–100%.In some embodiments, a cell population being positive for one or more markers means that, compared to a control cell population, the percentage of cells expressing the marker is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or any percentage in the range of 50–100%.

[0233] Whether cells or cell populations are positive for a specific cell surface marker can be determined by flow cytometry using staining with an antibody specific to the surface marker and a control antibody matched to its isotype. Negative staining of a specific marker in a cell population means that no cell population stains more strongly with the specific antibody than the isotype control, while positive staining means that a cell population stains more uniformly with the specific antibody than the isotype control. In some embodiments, a decrease in the expression of one or more markers means a decrease of 1 log10 in mean fluorescence intensity compared to a control cell population, and / or a decrease in the percentage of cells expressing the marker to at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or any percentage in the range of 20–100%.In some embodiments, increased expression of one or more markers means an increase in mean fluorescence intensity and / or an increase in the number of cells in a cell population that are positive for one marker or a given marker, for example, that the percentage of cells expressing the marker in a cell population is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or any percentage in the range of 50–100%.

[0234] In some embodiments, antigen-specific CD4+ and CD8+ populations can be obtained by stimulating naive T lymphocytes or antigen-specific T lymphocytes with the antigen. For example, a T cell line or T cell clone specific to cytomegalovirus antigen can be produced by isolating T cells from a subject infected with cytomegalovirus and stimulating those cells in vitro with the same cytomegalovirus antigen. Naive T cells can also be used. The number of antigens derived from tumor cells for use as targets to induce a T cell response is not particularly limited. In some embodiments, the adoptive cell immunotherapy compositions of the present invention are useful for treating diseases or disorders, including solid tumors and / or hematological malignancies.

[0235] Other methods for stimulating cells ex vivo are also possible. CAR T cells can be stimulated before being used as a therapeutic agent using hapten-containing cells or haptens bound to beads. Alternatively, CAR T-expressing cells may be stimulated using hapten-carrying cells prepared by standard known techniques, such as exposure to a hapten-binding support (e.g., beads, wells, or dishes).

[0236] Stimulation of chimeric antigen receptors in vivo This invention provides a method for stimulating or restimulating T cells possessing chimeric antigen receptors (CARs) in subjects with diseases such as cancer. A step of providing cells to the subject according to any embodiment provided herein, A step of monitoring the suppression of the disease in the subject; and The process of providing hapten antigen-presenting cells (H-APCs) to the subject. Includes, The subjects mentioned above may be selected for CAR T cell therapy using CAR T cells that have receptors specific to antigens associated with the disease (such as tumor antigens). The cells may comprise one or more vectors or one or more nucleic acids according to any embodiment of this specification, or a bispecific chimeric antigen receptor according to any embodiment of this specification. The one or more nucleic acids may be provided incorporated in a single vector or in multiple vectors so as to accommodate the payload sizes of two CARs. The one or more vectors may comprise another nucleic acid provided herein. Alternatively, the nucleic acids may be incorporated using a transposon system or an integrase system. The 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 tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a hapten-specific second ligand-binding domain, a second polypeptide spacer, a second transmembrane domain and a second intracellular signaling domain. In some embodiments, the present invention provides a plurality of nucleic acids comprising a first nucleic acid containing a first sequence encoding a first chimeric antigen receptor and a second nucleic acid containing a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a hapten-specific second ligand-binding domain, 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 tumor cell antigens. In some embodiments, the bispecific chimeric antigen receptor encoded by the one or more nucleic acids is contained within a cell. The nucleic acid encoding the bispecific chimeric antigen receptor comprises a sequence encoding a tumor antigen-specific first ligand-binding domain, a glycine-serine linker, a hapten-specific second ligand-binding domain, a polypeptide spacer, a transmembrane domain, and an intracellular signaling domain.In some embodiments, the H-APC is a cell produced by labeling the target healthy cells with a hapten in ex vivo.

[0237] The aforementioned H-APCs are prepared from healthy cells of a patient, such as a human, or cells compatible with the patient, and the cells are labeled with a hapten ex vivo. 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 example of a method for loading haptens onto cells is to incubate the target cells with fluorescein lipids overnight. One advantage of using fluorescein as a hapten is that its fluorescence can be utilized. Therefore, hapten incorporation can be monitored by detecting the fluorescence of the fluorescein portion using flow cytometry. Thus, after incubation, excess fluorescein lipids can be removed, the cell fraction can be analyzed by flow cytometry to analyze hapten incorporation, and the remaining cells can be used for injection into the patient. This approach is safe in that, after injection into the patient, H-APCs that are not targeted by CAR T cells gradually lose their haptens (due to haptens being metabolized and removed from the cell surface, etc.) and return to their original healthy cell form. H-APCs may also be produced by binding haptens and lipids and incorporating them into cells.

[0238] 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 / or derivatives thereof. In some embodiments, the monitoring step and the providing step are repeated. In some embodiments, the subject is a subject having cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the subject is a subject selected for cancer therapy. In some embodiments, the subject is subjected to combination therapy such as chemotherapy or radiotherapy.

[0239] Cellular stimulation in ex vivo In some embodiments, a method is provided for stimulating or restimulating T cells having a chimeric antigen receptor (CAR) ex vivo. In some cases, this method is A step of providing cells according to any embodiment described herein, A step of providing hapten antigen-presenting cells (H-APCs) or hapten; A step of mixing the aforementioned cells with the H-APC cells to obtain activated cells; and step of isolating the activated cells Includes. The cells may comprise one or more vectors or one or more nucleic acids according to any embodiment of this specification, or a bispecific chimeric antigen receptor according to any embodiment of this specification. The one or more nucleic acids may be provided incorporated in a single vector or in multiple vectors so as to accommodate the payload sizes of two CARs. The one or more vectors may comprise another nucleic acid provided herein. Alternatively, the nucleic acids may be incorporated 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 tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a hapten-specific second ligand-binding domain, a second polypeptide spacer, a second transmembrane domain and a second intracellular signaling domain. In some embodiments, the present invention provides a plurality of nucleic acids comprising a first nucleic acid containing a first sequence encoding a first chimeric antigen receptor and a second nucleic acid containing a second sequence encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a tumor antigen-specific first ligand-binding domain, a first polypeptide spacer, a first transmembrane domain, and a first intracellular signaling domain, and the second chimeric antigen receptor comprises a hapten-specific second ligand-binding domain, 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 tumor cell antigens. In some embodiments, the bispecific chimeric antigen receptor encoded by the nucleic acid is contained within a cell. The nucleic acid encoding the bispecific chimeric antigen receptor comprises a sequence encoding a tumor antigen-specific first ligand-binding domain, a glycine-serine linker, a hapten-specific second ligand-binding domain, 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 includes 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 / or derivatives thereof.

[0240] In some embodiments, the isolation of the activated cells includes affinity isolation by complexing a hapten with affinity beads. In some embodiments, the isolation of the activated cells includes affinity isolation by complexing EGFRt, CD19t, or Her2tG with affinity beads.

[0241] In some embodiments, CAR may have an anti-FL(FITC-E2)scFv-IgG4-hinge-CH2(L235D,N297Q)-CH3-CD28tm / 41BB-ζ-T2A-EGFRt structure. Table 4 lists examples of amino acid sequences that can be used in embodiments of the methods and compositions provided herein. [Table 2] TIFF0007926829000004.tif150163 [Examples]

[0242] Example 1 - Preparation of cells having haptens anchored to the cell surface Hapten-labeled cells were prepared by binding fluorescein (FL) as a hapten 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 anti-CD19 antibody labeled with fluorescein isothiocyanate (FITC) for 20 minutes. To confirm the presence of FL, the cells were washed, stained, and analyzed by flow cytometry. Cells prepared by either method showed a positive shift indicating the presence of hapten (FL) compared to untreated control cells. The amount of FL was higher in cells treated with FL-DHPE than in cells treated with anti-CD19 antibody (Figure 4A). This result indicates that the amount of hapten on the cell surface differs depending on the anchoring technique used.

[0243] K562 cells (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 phospholipids incorporated into the cell surface. The amount of FL-PLE incorporated into the cells was analyzed by flow cytometry. The amount of FL detected was higher 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 higher in cells treated with 0.5 μM FL-PLE than in untreated control cells. Thus, by adjusting the concentration of FL-PLE, the amount of FL carried on the cell surface can be varied. By varying the concentration of the agent anchored to the cell surface (such as FL-PLE), the density of haptens (such as FL) on the cell surface can also be varied.

[0244] Be2 cells (neuroblastoma cell line), U87 cells (glioblastoma cell line), or daoy cells (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 cells and daoy cells was greater than that into Be2 cells (Figure 4C). This result indicates that FL-PLE can be incorporated into various cell types, and the amount of FL-PLE incorporated can vary considerably.

[0245] Example 2 - Accessibility of haptens anchored to the cell surface To confirm extracellular accessibility to the hapten supported on the cell surface, 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 portion in the cell. The cell nucleus was stained with DAPI. Green fluorescence staining was observed across the entire cell surface. Therefore, FL-PLE was incorporated across the entire cell surface (Figure 5A). The confocal image, created by overlaying the acquired images, is shown on the left. On the right side of the overlaid confocal image, grayscale images of each layer ((i) nucleus and (ii) FL-PLE) that make up this confocal image are shown.

[0246] To measure the accessibility of the FL region on the cell surface, cells labeled with FL-PLE were stained with Alexa Fluor 647 fluorescent dye-labeled anti-fluorescein antibody. Staining with the anti-fluorescein antibody was observed across the entire cell surface (Figure 5B). This result confirmed that the FL region is accessible via extracellular binding. The confocal image obtained by superimposing the acquired images is shown on the left. On the right side of the superimposed confocal image, grayscale images of each layer constituting this confocal image ((i) nucleus, (ii) FL-PLE, and (iii) anti-fluorescein-Alexa Fluor 647 antibody) are shown.

[0247] Example 3 - Retention of haptens anchored to 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 any remaining FL-DHPE or FL-PLE and 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 higher in cells treated with FL-PLE than in cells treated with FL-DHPE. These results demonstrate that the duration of hapten presence on the cell surface can be altered by varying the agents (such as phospholipids) anchored to the cell surface.

[0248] Example 4 - Recognition of haptens anchored to the cell surface and activation of anti-hapten CAR T cells Hapten-labeled cells were prepared. CD19+K562 cells were incubated overnight with 5 μM FL-DHPE or with FITC-labeled CD19 antibody for 20 minutes. The resulting hapten-labeled cells were then incubated with one of two types of anti-FL CAR T cells (FITC-E2 scFv or 4M5.3 scFv). Cytotoxicity assays, cytokine release assays, and proliferation assays of the CAR T cells were performed using a method substantially similar to that described by 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).

[0249] The lytic ability of anti-FL CAR T cells against hapten-labeled cells was measured using a chromium release assay. Unlabeled control K562 cells could not be lysed by anti-FL(FITC-E2)CAR T cells or anti-FL(4M5.3)CAR T cells (Figure 7A, upper left panel). Positive controls using OKT3 cells, which can activate T cells via the TCR, were shown to be able 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 types of anti-FL CAR T cells (Figure 7A, lower panel).

[0250] The amount of cytokines released by anti-FL CAR T cells was measured. All 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 produced using FL-DHPE tended to induce the release of larger amounts of cytokines than hapten-labeled cells produced using FITC-labeled CD19 antibody.

[0251] Example 5 - Recognition of haptens anchored to the cell surface 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 cells was analyzed by flow cytometry. The resulting hapten-labeled cells were incubated with anti-FL CAR T cells, and the ability of the hapten-labeled cells to induce specific lytic activity and cytokine release activity in anti-FL CAR T cells was measured.

[0252] The amount of FL detected was higher in cells treated with 5 μM FL-PLE than in cells treated with 0.5 μM FL-PLE or untreated control cells (Figure 8A). Furthermore, cell lysis and cytokine release were also higher 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 an extracellular FL moiety anchored to the cell surface can be recognized by anti-FL CAR T cells and activated. The level of anti-FL CAR T cell activation is thought to be related to the amount of FL on the surface of hapten-labeled cells.

[0253] Example 6 - In vitro expansion of anti-hapten CAR T cells CD4+ anti-FL CAR T cells and CD8+ anti-FL CAR T cells were generated by transducing a vector into T cells. After 18 days, the transduced cells underwent their first expansion culture using a standard rapid expansion culture (REP) method with irradiated TM-LCL and PBMCs. The expanded cells were subjected to a second expansion culture using either a standard REP method or a fluorescein-based REP method (FREP). In the FREP method, cells were incubated on feeder cells treated with FL-PLE. Fourteen days after the second expansion culture, cells were analyzed using flow cytometry, specific lysis assays, and cytokine release assays. In the specific lysis assay and cytokine release assay, K562 cells were incubated overnight with FL-PLE, and these K562 cells were incubated with the expanded anti-FL CAR T cells. FL-PLE uptake into cells was analyzed by flow cytometry (Figure 9B).

[0254] Cells grown using the REP method and cells grown using the FREP method both expressed similar phenotypic markers (Figure 9A). Furthermore, CD8+ anti-FL CAR T cells grown using the FREP method had substantially equivalent cytotoxic activity to CD8+ anti-FL CAR T cells grown using the REP method (Figure 9C). Additionally, CD8+ anti-FL CAR T cells grown using the FREP method had substantially equivalent cytokine release activity to CD8+ anti-FL CAR T cells grown using the REP method (Figure 9D). Moreover, CD4+ anti-FL CAR T cells grown using the FREP method had substantially equivalent cytotoxic activity and cytokine release activity to CD4+ anti-FL CAR T cells grown using the REP method. Therefore, cells labeled with haptens (such as FL) can induce the growth of CAR T cells, and these thus grown CAR T cells have substantially equivalent activity to CAR T cells cultured using irradiated TM-LCL and PBMCs.

[0255] Example 7 - Preparation of cells having haptens (especially DNP using DNP-PLE) anchored to the cell surface and exposed extracellularly. MDA-MB-231 (adenocarcinoma) cells were incubated overnight with DNP-PLE in complete medium. Exposed DNP molecules on the cell surface were stained with Alexa Fluor 488-labeled anti-DNP antibody (DNP is nonfluorescent), and the integration 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 MDA-MB-231 parental cells and MDA-MB-231 cells stained with Alexa Fluor 488-labeled anti-DNP antibody. This result was expected, as DNP is not exposed on the surface of MDA-MB-231 cells.

[0256] When MDA-MB-231 parental cells were incubated with 5 μM DNP-PLE and stained with Alexa Fluor 488-labeled anti-DNP antibody, a clear shift from control MDA-MB-231 parental cells was observed (Figure 11B). However, when MDA-MB-231 parental cells were incubated with 50 nM DNP-PLE and stained with Alexa Fluor 488-labeled 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 is 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 parent cells incubated with 500 nM DNP-PLE was between the amount of DNP exposed on the surface of MDA-MB-231 parent cells incubated with 50 nM DNP-PLE and the amount of DNP exposed on the surface of MDA-MB-231 parent cells incubated with 5 μM DNP-PLE (Figure 11C). Histogram plots of the data shown in Figures 11A to 11D are shown in Figure 11E.

[0257] These data demonstrate that we have successfully created cells containing haptens (particularly DNP using DNP-PLE) that are anchored to the cell surface and exposed outside the cell.

[0258] 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 incubated overnight without DNP-PLE loading. After incubation, these cells were washed, and images were acquired using a confocal microscope to examine where DNP-PLE was incorporated into the cells. The cell nucleus was stained with DAPI (i). The cell surface was stained with wheat germ agglutinin (WGA) (ii). As confirmed in Figure 12B, since DNP does not fluoresce, the DNP region was stained with Alexa Fluor 488-labeled anti-DNP antibody (iii). In (iii), fluorescence of the anti-DNP antibody was observed, confirming that DNP-PLE was incorporated throughout the cell surface (Figures 12C and 12D). The fact that the antibody was able to bind to the DNP region indicates from these images that the DNP region is accessible by binding. 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 only MDA-MB-231 parent cells. As can be seen from the lack of staining in the image, the anti-DNP antibody could not bind to the cells, meaning that DNP was not present on the cell surface and therefore could not be stained by the anti-DNP antibody. The left-hand images in Figures 12A to 12D show confocal images obtained by superimposing all the images (i) to (iv) in each figure. To the right of the superimposed confocal image are grayscale images of each layer that makes up this confocal image (nucleus (i), cell surface (ii), and DNP-PLE (iii)).

[0259] Therefore, it was confirmed that anti-DNP CAR cells have the ability to recognize DNP on DNP-PLE supported on their cell surface.

[0260] Example 9 - Confirmation of extracellular accessibility of haptens supported on cells and confirmation that PLE is supported on the cell membrane. Figure 13A shows a schematic diagram of a second-generation CAR cassette with a long spacer for expressing anti-DNP CAR. This cassette contains a gene encoding a double mutant dihydrofolate reductase that enables methotrexate-mediated selection of CAR-positive cells, and a gene encoding EGFRt, a surface marker correlated with CAR positivity.

[0261] The plasmid shown in Figure 13A was transduced into H9 cells (CD4+CD3+ cutaneous T lymphocytes), and a pure anti-DNP CAR population was selected with methotrexate. The purity of anti-DNP CAR H9 cells was measured by staining for the surface marker EGFRt. After cell staining, analysis by flow cytometry showed that the positive rate for the anti-DNP CAR H9 population was 92% in the flow cytometry plot.

[0262] MDA-MB-231 (adenocarcinoma) cells were incubated with 5 μM DNP-PLE to load DNP-PLE, or incubated without loading DNP-PLE. After these cells were washed, they were co-cultured with a pure H9 cell population expressing anti-DNP CAR, and images were acquired using a confocal microscope to investigate whether recognition occurred between DNP exposed on the cell surface and 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). Since DNP does not fluoresce, the DNP portion was stained with Alexa Fluor 488-labeled 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 (nucleus (i), cell surface (ii), DNP-PLE (iii), and anti-DNP CAR-expressing H9 cells (iv)) that make up this superimposed confocal image are shown. Figure 13C shows that no binding occurred between the target and effector. Figure 13D shows the interaction between the target and effector. The upper left image of Figure 13C shows the confocal image obtained by superimposing all images (i) to (iv) of this figure. The upper left image of Figure 13D shows the confocal image obtained by superimposing all images (i) to (iv) of this figure. These images show synapse formation between cells, and therefore confirm that DNP exposed on the surface of target cells was recognized by anti-DNP CAR. This is clearly shown in Figure 13D (iv), where the synapse is observed to extend to the target cell.

[0263] Therefore, we were able to confirm the extracellular accessibility of haptens supported on the cell surface and the presence of PLE on the cell membrane. The experimental data showed that anti-DNP CARs could be produced and that DNP on the cell surface could be accessed using this anti-DNP antibody, thus demonstrating that anti-DNP CARs can bind to DNP exposed on the cell surface.

[0264] Example 10 - Cytokine production by CD19 CAR-transduced T cells against various targets and non-autologous T-APCs in vitro. This paper presents data on the correlation between the induction of CD19 CAR T cell activation and the production of specific cytokines. To analyze cytokine production, a population of pure CD8+CD19 CAR T cells or a population of CD8+ mock T cells [these cells were stimulated with CD3 / CD28 microbeads and used 8 days after rapid expansion culture] (effectors) and a population of CD19-specific target cells were seeded in a 2:1 ratio and incubated for 24 hours. As target cells, K562 parental cells (negative control), K562 OKT3 cells (positive control), K562 CD19 cells, and non-autologous CD4+ / CD8+ mixed transduction antigen-presenting cells (T-APCs) with truncated CD19 (CD19t) prepared from clinical material (positive target cells, the same target as used in Example 11) were used. The presence or absence of cytokines in the supernatant was analyzed. BioPlex assays were performed to measure the production levels of IL-2, TNF-α, and IFN-γ. Significant amounts of cytokines were produced from CD19 CAR T cells when co-cultured with any CD19-specific target cell line, including non-autologous CD4 / CD8 T-APCs. No cytokine production was detected in the K562 parental cell line, which does not express CD19. This experiment demonstrates the production of specific cytokines and therefore shows that CD19 CAR T cells can be activated by non-autologous T-APCs.

[0265] Therefore, it was confirmed that activation by non-autologous T-APCs resulted in cytokine production from CD19 CAR-transduced T cells.

[0266] Example 11 - In vitro activation of autologous T-APC CD4+ / CD8+ mixed antigen-presenting cells (T-APCs) prepared from clinical materials by transduction with truncated CD19 (CD19t) were stained and analyzed by flow cytometry to analyze the expression of CD19t and truncated EGFR (EGFRt) on the cell surface. CD19t T-APCs showed a CD19t positivity rate of 63%, and as expected, EGFRt was not expressed, thus demonstrating CAR negativity (Figure 15A). Transduced autologous CD4+CD19 CAR T cells and transduced autologous CD8+CD19 CAR T cells were prepared from clinical materials and irradiated with CD19 in the presence of rhIL-2 and rhIL-15, with a feeder cell-to-T cell ratio of 7:1. + The cells were cultured using the rapid expansion method (REP) while being stimulated with feeder cells (TM-LCL). On day 7 of the expansion culture, the cells were stained and EGFRt expression was examined by flow cytometry. Both transduced CD4+CD19 CAR T cells and transduced CD8+CD19 CAR T cells showed a 99.9% positive rate for EGFRt expression, which correlates with CAR expression (Figure 15B).

[0267] On day 7 of expansion culture, effector cells and target cells were co-cultured in a 2:1 ratio for 24 hours. Cytokine production was then examined by evaluating 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 co-cultured for 24 hours with CD19t T-APCs, K562-CD19+ cells (K562 parent cells modified to express CD19), K562-OKT3 cells (K562 parent cells modified to express the agonist OKT3 scFv for use as a general-purpose positive control), or K562 parent cells (negative target cells), respectively. The supernatant was collected and cryopreserved until analysis for the presence or absence of cytokines was performed (Figure 15C). Bio-Plex assays showed that CD4+CD19 CAR T cells and CD8+CD19 CAR T cells could only produce cytokines in the presence of K562 CD19+ cells, CD19t T-APCs, or K562 OKT3-positive control cell lines, indicating that they produce cytokines specifically against CD19. As expected, CD4+ / CD8+T-APCs could only produce cytokines in the presence of K562 OKT3 cell lines. Co-culture of CD4+CD19 CAR T cells or CD8+CD19 CAR T cells with CD19t T-APCs resulted in low cytokine production, but this co-culture significantly activated autologous CD19 CAR T cells, leading to significant clinical outcomes (see Example 13 and Figures 17A-17D).

[0268] These data demonstrate that autologous T-APC could be activated in vitro.

[0269] Example 12 - In vitro activation of autologous hapten-APC K562 leukemia cells (Figure 16A) or primary CD8+ T cells (Figure 16B) were incubated overnight in or without 5 μM FL-PLE, and fluorescence was measured by flow cytometry. Fluorescein positivity was observed by flow cytometry, indicating successful fusion of the hapten fluorescein with the cells. The ability of FL-PLE-carrying cells to activate anti-FL CAR T cells was measured by a cytokine release assay (Figure 16C). Autologous anti-FL CAR CD4+ effector T cells or primary autologous CD8+ T cells were co-cultured with FL-PLE-carrying cells for 24 hours, and the supernatant was analyzed for the presence or absence of cytokines as shown in the graph. Autologous CD8+ T cells and autologous anti-FL CAR CD4+ T cells were stimulated with CD3 / CD28 microbeads and used in experiments after 21 days following two expansion culture cycles. Anti-FL CAR T cells produced cytokines when co-cultured with any FL-PLE-carrying K562 cell line, as well as when co-cultured with autologous CD8+ cells (H-APC) carrying FL-PLE. As expected, no cytokine production was detected when co-cultured with K562 parental cell lines that did not carry FL-PLE or with CD8+ T cells lacking CAR expression. However, cytokine production was observed when co-cultured with the positive control cell line, K562 OKT3+ cells (non-CAR expression, TCR-mediated activation). The amount of cytokine produced was comparable to that produced when transduced APC (T-APC) was used in vitro (Figures 15A-15C), demonstrating its effectiveness in patients (see Example 13 and Figures 17A-17D).

[0270] H-APC is expected to demonstrate comparable efficacy to in vivo T-APC in animal models and clinical subjects / patients (for example, during clinical trials and treatments).

[0271] These data demonstrate that autologous hapten-APC could be activated in vitro.

[0272] Example 13 - Persistence of CAR T cells in peripheral blood T-APC was administered sequentially to two pediatric acute lymphoblastic leukemia (ALL) patients, and the persistence of CAR T cells in the peripheral blood of these patients was investigated. 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 the persistence of CAR T cells was longitudinally monitored by cell surface staining of EGFRt, a CAR transduction marker (black circles) (Figures 17A and 17B). The amount of ALL cells was monitored by staining of CD19+ B cells (white diamonds). On day 0, CD19 CAR T cells containing the monitoring cell surface marker EGFRt (black circles) were administered to the patients. ALL cells rapidly regressed to undetectable levels by day 10. CD19 in peripheral blood + B cells became undetectable by day 10 (C1.D10), which was thought to be due to the rapid engraftment of CAR T cells. The persistence of CAR T cells gradually decreased after day 10. Because the persistence of CAR T cells was not sufficiently high, transduction antigen-presenting cells (T-APCs) were continuously administered to the patient at the time points shown in the graph (black inverted triangles) to enhance persistence. These T-APCs are autologous T cells genetically modified to express the CD19 surface antigen. Autologous transduction antigen-presenting T cells (T-APCs) expressing the CD19 surface protein, which is the target of CAR T cells, were administered to the patient. These T-APCs express the CD3 antigen, which is not present in CD19+ B cells, thus distinguishing these two CD19+ populations. This patient received five infusions of T-APCs. After each T-APC administration, CAR T cells proliferated and expanded, thereby preventing ALL relapse. CD19 +T-APCs were monitored over time (half-blacked-out rectangles) and distinguished from CD19+ B cells based on CD3 expression. Temporary expansion and proliferation of CD19 CAR T cells was observed after each T-APC injection, which appeared to correlate with the long-term absence of CD19+ B cell formation. An example of multi-parameter flow cytometry analysis of peripheral blood from a patient, shown in Figure 17B, revealed the detection of CD19+ T-APCs one day after the second T-APC administration (Figure 17C), and the detection of EGFR+ CAR T cells in peripheral blood 14 days after the third T-APC administration (Figure 17D).

[0273] These data showed that CAR T cells could survive in the patients' peripheral blood.

[0274] As used herein, the term “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is open-ended and comprehensive, not excluding any further elements or processes not described herein.

[0275] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention may be modified, as may the manufacturing methods and apparatus. Such modifications will be readily apparent to those skilled in the art, taking into account the practices of the present invention disclosed herein or the present disclosure. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but encompasses all possible modifications and other aspects within the true scope and spirit of the invention.

[0276] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and academic literature, are incorporated herein by reference in their entirety and constitute part of this Specified. If any reference, patent, or patent application cited by reference conflicts with any disclosure herein, the provisions of this Specified shall prevail and / or take precedence over such conflict.

Claims

1. Hapten antigen-presenting cells (H-APCs) for use in treating, suppressing, or mitigating cancer in a subject, wherein the cancer contains a tumor antigen. The aforementioned treatment, suppression, or relief, A step of providing a cell comprising a first nucleic acid encoding a first chimeric antigen receptor (CAR) that can specifically bind to the tumor antigen, and a second nucleic acid encoding a second chimeric antigen receptor (CAR) that can specifically bind to a hapten; and The step of providing the hapten antigen-presenting cells (H-APCs) containing the hapten to the subject. Includes, The H-APC is characterized in that the hapten is bound to the H-APC via an ether phospholipid (PLE), and the hapten is selected from fluorescein and dinitrophenol.

2. The H-APC for use according to claim 1, wherein the hapten is fluorescein.

3. The H-APC for use according to claim 1 or 2, wherein the cell comprises a polynucleotide comprising a first nucleic acid, a second nucleic acid, and a nucleic acid encoding a cleavable linker, the nucleic acid encoding the linker being located between the first nucleic acid and the second nucleic acid.

4. The H-APC for use according to any one of claims 1 to 3, wherein the cell comprises a vector comprising the first nucleic acid and the second nucleic acid.

5. H-APC for use according to claim 1 or 2, wherein the first nucleic acid and the second nucleic acid are located in different vectors.

6. (a) 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; (b) 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; or (c) The cells are T cells, progenitor T cells, or hematopoietic stem cells. H-APC for use according to any one of claims 1 to 5, characterized by the above.

7. H-APC for use according to claim 6, wherein the cells are brought into contact with the hapten antigen-presenting cells (H-APC) to induce the expansion and proliferation of the cells.

8. H-APC for use according to claim 7, wherein the cells and / or the H-APC are the autologous cells of the subject.

9. H-APC for use according to claim 7, wherein the cells and / or the H-APC are of the same species as the subject.

10. H-APC for use according to any one of claims 6 to 9, wherein contact between the cells and the H-APC is performed in vitro.

11. H-APC for use according to any one of claims 6 to 9, comprising co-administering the cells and the hapten antigen-presenting cells (H-APC) to the subject to stimulate or re-stimulate the cells.

12. (i) The CAR T cells and the H-APCs are derived from the subject; (ii) The hapten is covalently bound to the extracellular surface of the H-APC; (iii) The H-APC is derived from cells selected from the group consisting of T cells and B cells; and / or (iv) The H-APC is a cell produced by labeling the target healthy cell with a hapten in ex vivo. An H-APC for use according to claim 1, characterized by the above.

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