Activation-inducible fusion proteins containing heat shock protein 90-binding domains

Abstract

We describe an activity-inducible fusion protein that utilizes an hsp90-binding domain and a drug molecule, whose activity can be post-translationally regulated. The activity-inducible fusion protein is inactivated in the absence of the drug molecule, but can be activated in the presence of the drug molecule by a physiological parameter of interest. An example of an activity-inducible fusion protein is the chimeric antigen receptor (CAR), where the physiological parameter of interest is antigen binding.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 149,131, filed February 12, 2021, and U.S. Provisional Patent Application No. 63 / 226,554, filed July 28, 2021, the contents of both of which are incorporated by reference in their entireties herein.

[0002] The present disclosure provides an activity-inducible fusion protein having a heat shock protein 90 (hsp90) binding domain. The activity of the fusion protein is post-translationally regulated using a drug molecule that can bind to the hsp90 binding domain with higher affinity than hsp90. The fusion protein is inactive in the absence of the drug molecule, but can be activated in the presence of the drug molecule. [Background technology]

[0003] Genetic engineering has made great advances in activating immune system cells to kill cancer cells or infected cells. For example, T cells have been engineered to express molecules that have an extracellular portion that binds to a specific target antigen and an intracellular portion that induces the action of the T cell when the extracellular portion binds to the target antigen. In one example, the extracellular portion can be designed to bind to a target antigen found on cancer cells or infected cells, and when the target antigen of the cancer cell or infected cell binds to the extracellular portion, the intracellular portion activates the T cell and the cell that binds to the extracellular portion is destroyed by the T cell. An example of such a molecule is the chimeric antigen receptor (CAR).

[0004] Although CAR-expressing T cells can exhibit potent antitumor activity, they can also exhibit significant toxicity, including cytokine release syndrome (cytokine release syndrome), tumor lysis syndrome (TLS), and chronic B cell depletion induced by transplanted cells, all of which are due to the unregulated function of constitutively expressed and active CARs. Such toxicity may limit the application of CAR-based therapy.

[0005] Although methods to eliminate CAR-T cells, such as the use of suicide genes, can reduce such toxicity, these methods may reduce the antitumor activity before it is fully exerted, which may have a significant impact on therapeutic efficacy. Therefore, there is a need to find a way to control the activity of CARs after they are expressed by genetically modified immune cells.

[0006] In addition to CARs, there are many other proteins whose activation state can be controlled for which it would be beneficial, such as transmembrane receptors that transmit costimulatory or inhibitory signals in immune cells. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides a fusion protein whose activation state can be controlled by administration of a drug molecule, the fusion protein comprising a heat shock protein 90 (hsp90) binding domain that binds to the drug molecule.

[0008] An example of the fusion protein of the present disclosure is a CAR that can be activated by binding to an antigen in vivo and can be controlled by administering a drug molecule. If activation can be controlled after expression in vivo, safety, which is important for immune cell therapy using CAR, can be improved.

[0009] The present disclosure achieves this advancement by incorporating an hsp90 binding domain into the intracellular portion of the CAR, which in the absence of drug molecules can bind hsp90 and prevent other important intracellular molecules required for CAR activation following antigen binding from interacting with the CAR.

[0010] When the drug molecule is present, it displaces the bound hsp90 from the hsp90-binding domain site and / or induces a conformational change such that upon antigen binding, intracellular signaling occurs.

[0011] In certain embodiments disclosed herein, a hormone binding domain is utilized as the hsp90 binding domain. The hormone binding domain may be an estrogen receptor binding domain (EBD). The EBD may be derived from the natural estrogen receptor, but must contain at least one mutation such that the EBD does not bind estrogen but binds to a drug molecule with higher affinity than hsp90. Examples of drug molecules include tamoxifen or its derivatives or metabolites with reduced side effects, such as 4-hydroxytamoxifen (4-OHT), CMP8, ES8, etc.

[0012] In the absence of tamoxifen or its derivatives or metabolites, hsp90 binds to the EBD and CAR is in the "off" state. Upon entry of nanomolar concentrations of tamoxifen into the cytosol, tamoxifen actively acts to outcompete hsp90 for binding to the EBD and / or induces a conformational change that results in an activation signal being transmitted by CAR upon antigen binding.

[0013] This strategy can be used to control in a similar way the activity of other proteins that interact with hsp90 with lower affinity than drug molecules. Further examples include transmembrane receptors, such as those that transmit costimulatory or inhibitory signals in immune cells.

[0014] One advantage of the present disclosure is that the activity of a single-chain protein can be controlled without relying on dimerization or multimerization with another protein, nor on stabilization / destabilization of the protein, such as by incorporation of a degron sequence. [Brief description of the drawings]

[0015] Some of the drawings submitted in this application may be more easily understood in color, and applicants hereby contemplate color versions of these drawings as part of the original application and reserve the right to submit color images of such drawings in subsequent proceedings.

[0016] [Figure 1A-1C] We visually demonstrate that EBDs (e.g., ERT2, EBD(CMP8) or EBD(ES8)) play a role in CAR signaling (Figure 1A). In the absence of drug molecules, EBDs are bound by heat shock protein 90 (hsp90) and sterically inhibit the interaction of the signaling domains of CD3ζ and 4-1BB with lymphocyte-specific protein tyrosine kinase (LCK), an SRC family kinase, resulting in the inhibition of LCK-mediated phosphorylation of CD3ζ and TCRζ chain-associated protein kinase (ZAP70). (Figure 1B, Figure 1C) However, upon addition of drug molecules (e.g., 4-hydroxytamoxifen (4-OHT), CMP8 or ES8), hsp90 dissociates, allowing the binding domains of CAR (e.g., scFv) to interact with the relevant antigen, allowing the two signaling domains (CD3ζ and 4-1BB) to carry out normal signaling.

[0017] [Figure 2A-2B]A schematic diagram of a CAR is shown. (FIG. 2A) A schematic diagram of a CAR disclosed herein is shown. The CAR includes a ligand binding domain, a spacer, a transmembrane domain, a signaling domain, an estrogen binding domain (EBD) and a selection / transduction marker. (FIG. 2B) A schematic diagram of a second generation 806-ERT2 CAR is shown. This CAR incorporates an IgG4 hinge between the scFv and the transmembrane region. In this CAR, a Gly3 linker consisting of three glycines links the signaling domain to the ERT2 EBD. This ERT2 EBD has the sequence shown in SEQ ID NO: 13 and is referred to herein as ERT2, EBD(4-OHT), or EBD or ERT2 with G400V, M543A and L544A mutations. The ERT2 domain is followed by P2A-DHFRdm as a selection marker and T2A-CD19t as a transduction marker. The 806-ERT2 CAR and the 806EGFR CAR differ in that the 806EGFR CAR does not contain EBD or DHFRdm and uses EGFRt as a transduction marker.

[0018] [Diagram 3] Jurkat-Dual NF-kB-Luc reporter T cells are shown. Jurkat-Dual cells feature a promoter-driven Lucia luciferase gene (secreted luciferase reporter gene) fused to five copies of the NF-kB consensus transcription response element. Secreted Lucia luciferase is measured as an indicator of NF-kB response via CAR activation.

[0019] [Figure 4A-4B]Jurkat-Dual NF-kB reporter cells were transduced with 806EGFR CAR or 806EGFR-ERT2 CAR and selected with MTX when available. Transduced cells were co-cultured with K562+EGFRvIII cells in the presence of various concentrations of 4-OHT (0-16,000nM) 24 hours prior to various assays. At the 24 hour time point, 10μl of supernatant was harvested and mixed with 50μl of QUANTI-Luc and read immediately on SpectraMax. Transduced Jurkat-DUAL cells were harvested by flow cytometry prior to various assays. (Figure 4A) Flow cytometry of CD19t (CD19 APC) and (Figure 4B) flow cytometry of EGFRt (cetuximab APC) confirmed the expression of 806EGFR CAR or 806EGFR-ERT2 CAR, respectively, in Jurkat-DUAL cells.

[0020] [Figure 5A-5B] Jurkat Dual NF-kB-Luc reporter assay. Secreted Lucia luciferase was measured by end-point luminescence assay. The assay was performed with an effector-to-target ratio of 2:1 (Figure 5A) or 1:1 (Figure 5B). Prior to the assay, Jurkat-DUAL cells transduced with 806EGFR CAR or 806EGFR-ERT2 CAR were stimulated with K562+EGFRvIII cells and the indicated concentrations of 4-OHT for 24 hours. Both cell lines showed 80% CAR positivity. 4-OHT was added 24 hours before the assay. Selection of cells insensitive to MTX was performed the day before the assay. Jurkat-DUAL cells transduced with the 806EGFR-ER2T CAR were detected to secrete increasing levels of Lucia luciferase with increasing concentrations of 4-OHT, indicating that the CAR T cells were activated in a dose-dependent manner. As mentioned above, ERT2 is also referred to herein as EBD(4-OHT), and its sequence is set forth in SEQ ID NO:13.

[0021] [Figure 6A-6B] Cells are shown on day 6 of rapid expansion culture after stimulation (S1R1D6). Post-stimulation culture (S1): Mock cells, 806EGFR CAR T cells (Fig. 6B) and 806EGFR-ERT2 CAR T cells (Fig. 6A) were expanded by rapid expansion procedure (REP) stimulated with irradiated feeder cells (20x106 TM-LCL and 100x106 PBMC) in the presence of rhIL-2, rhIL-15 and exogenous OKT3 antibody. On day 6 of REP, CAR positivity and percentage of CD4 T cells and CD8 T cells were measured by flow cytometry. Mock (40% CD4 / 60% CD8); 806 CAR (30% CD4 / 70% CD8); 806-ERT2 (46% CD4 / 53% CD8).

[0022] [Figure 7A-7B] Intracellular cytokine staining (ICCS) is shown. IL-2, IFN-γ, and TNFα are shown. A 4-hour intracellular cytokine staining (ICCS) assay was performed using primary T cells expressing the EGFR806 CAR (Figure 7A) or primary T cells expressing the EGFR806-ERT2 CAR (Figure 7B). Gating was performed on lymphocytes / single cells / live cells / CD4 / CD19t to evaluate CD4 T cells containing CAR. 4-OHT was added 24 hours prior to the assay and incubated for 4 hours. The EGFR806-ERT2 CAR was only able to induce a cytokine response against EGFRvIII+ target cells in the presence of 500 nM 4-OHT or (Z)-endoxifen (on state).

[0023] [Figure 7C]Intracellular cytokine staining (ICCS) is shown. CD107a degranulation assay is shown. A 4-hour intracellular cytokine staining (ICCS) assay was performed using primary T cells expressing EGFR806 CAR or primary T cells expressing EGFR806-ERT2 CAR. Gating was performed on lymphocytes / single cells / live cells / CD8 / CD19t to evaluate CD8 T cells containing CAR. 24 hours prior to the assay, 4-OHT was added at each dose level (20nM, 100nM, and 500nM) and incubated for 4 hours. In EGFR806-ERT2 CAR, the expression of CD107a marker increased with increasing 4-OHT concentration, indicating increased degranulation.

[0024] [Figure 7D] Intracellular cytokine staining (ICCS) is shown. The early activation marker Nur77 is shown. A 4-hour intracellular cytokine staining (ICCS) assay was performed with primary T cells expressing EGFR806 CAR or primary T cells expressing EGFR806-ERT2 CAR. Gating was performed on lymphocytes / single cells / live cells / CD8 / CD19t to assess CD8 T cells containing CAR. 4-OHT was added 24 hours prior to the assay and incubated for 4 hours. EGFR806-ERT2 CAR expresses the early activation marker Nur77 on EGFRvIII+ target cells only in the presence of 500 nM 4-OHT or (Z)-endoxifen (on state).

[0025] [Figure 8A-8D]Chromium release assay. Primary T cells expressing EGFR806 CAR or EGFR806-ERT2 CAR were used in a 4-hour chromium release assay. EGFR806-ERT2 CAR T cells were added to achieve 78% CAR positivity to be consistent with 806EGFR CAR T cells. 4-OHT was added 18 hours prior to the assay and allowed to remain present for 4 hours of incubation. EGFR806-ERT2 CAR was only able to lyse EGFRvIII+ target cells in the presence of 4-OHT or (Z)-endoxifen. 4-OHT or (Z)-endoxifen was added 24 hours prior to the assay and during the incubation. Figure 8A shows K562 parental cells; Figure 8B shows K562+OKT3 (positive control); Figure 8C shows K562+EGFRvIII; and Figure 8D shows chromium release assays at day 10 (S1R1D10) of rapid expansion after stimulation. Figure 8D shows a graph of the grouping of cells with various doses of 4-OHT or (Z)-endoxifen.

[0026] [Figure 9] A schematic diagram of the Jurkat iSynPro:GFP-ffluc model (top) and results related to this model (bottom) are shown.

[0027] [Figure 10] FIG. 1 shows activation curves for several huCD19-EBDs (ERT2, also referred to herein as EBD(4-OHT) (SEQ ID NO: 13)) with different glycine linkers.

[0028] [Figure 11]The design of the B7H3 CAR EBD mutant study is shown. This study was conducted to investigate the EBD incorporated into B7H3CAR, as well as to investigate the effectiveness of the activation of two EBD mutants (EBD(4-OHT) and EBD(CMP8)) when interacting with each estrogen analog (4-OHT or CMP8) and the difference between the two EBD mutants in this regard. As Jurkat iSynPro lines, cJ10792 (Jurkat iSynPro-GFP:ffluc); cJ13093 (Jurkat iSynPro-GFP:ffluc+B7H3 CAR); cJ13094 (Jurkat iSynPro-GFP:ffluc+B7H3 CAR EBD(4-OHT)); and cJ13095 (Jurkat iSynPro-GFP:ffluc+B7H3 CAR EBD(CMP8)) were used, and K562 parental cells were used as target cells. Each cell line was tested at 4-OHT and CMP8 concentrations of 0 nM, 500 nM, or 1000 nM. EBD(4-OHT) is ERT2 (SEQ ID NO: 13), and EBD(CMP8) is shown in SEQ ID NO: 11.

[0029] [Figure 12] GFP was induced in B7H3 CAR with EBD(4-OHT (SEQ ID NO: 13)) or B7H3 CAR with EBD(CMP8) (with L384M, M421G and G521R mutations (SEQ ID NO: 11)), indicating regulated activation. In this figure, EBD(CMP8) is shown as B7H3 ERT2-(L384M|M421G|G521R(CMP8)).

[0030] [Figure 13] Activation curves for the B7H3 CAR ERT2 mutant (SEQ ID NO: 13, shown in medium grey and diamonds) and the B7H3 CAR EBD (CMP8) mutant (SEQ ID NO: 11, shown in light grey and squares or dark grey and circles).

[0031] [Figure 14]The design of the activation curve study is shown. This study aimed to investigate the activation curve of CAR EBD Jurkat iSynPro lines with increasing concentrations of 4-OHT or (Z)-endoxifen. The Jurkat iSynPro lines used were cJ10792 (Jurkat iSynPro-GFP:ffluc); cJ13227 (Jurkat iSynPro-GFP:ffluc+huCD19 CAR); cJ13097 (Jurkat iSynPro-GFP:ffluc+huCD19 CAR 1GLY EBD); cJ13098 (Jurkat iSynPro-GFP:ffluc+huCD19 CAR 2GLY EBD); and cJ13096 (Jurkat iSynPro-GFP:ffluc+huCD19 CAR 3GLY EBD), with K562 and CD19 as targets. This study was performed using a 1:2.5 drug dilution series. The highest concentration of all drugs tested was 2500 nM. The EBD used in this study was ERT2 (also referred to herein as EBD(4-OHT); SEQ ID NO: 13).

[0032] [Figure 15] Dose response and comparison of different huCD19 CAR-EBD linker variants are shown.

[0033] [Figure 16]The design of the activation curve study is shown. This study aimed to investigate the activation curve of CAR EBD Jurkat iSynPro lines when the concentration of 4-OHT, (Z)-endoxifen or CMP8 was increased stepwise. The Jurkat iSynPro lines used were cJ10792 (Jurkat iSynPro-GFP:ffluc); cJ13093 (Jurkat iSynPro-GFP:ffluc+B7H3 CAR); cJ13094 (Jurkat iSynPro-GFP:ffluc+B7H3 CAR ERT2(4-OHT) (SEQ ID NO: 13); and cJ13095 (Jurkat iSynPro-GFP:ffluc+B7H3 CAR EBD(CMP8) (SEQ ID NO: 11)), and K562 was used as the target. Drug dilution series (1:2.5 curves) were generated starting at a concentration of 1000 nM for 4-OHT and CMP8, and 6250 nM for (Z)-endoxifen.

[0034] [Figure 17] Dose response and comparison of various B7H3 CAR-EBD linker variants (EBD(4-OHT) (SEQ ID NO: 13) and EBD(CMP8) (SEQ ID NO: 11)) are shown.

[0035] [Figure 18]Drug-dependent specific cell lysis by various B7H3CAR-3Gly-EBD mutants in the presence of the estrogen analogs 4-OHT, CMP8, or (Z)-endoxifen. Specific cell lysis was assessed using a chromium release assay. First, cells bearing B7H3CAR-3Gly-EBD (4OHT) mutants or B7H3CAR-3Gly-EBD (CMP8) mutants were cultured for 24 h in the presence of three estrogen analogs (4-OHT, CMP8, or (Z)-endoxifen) at various drug concentrations (0 nM, 1 nM, 50 nM, or 500 nM), and then co-cultured with antigen-expressing tumor lines previously labeled with radioactive Cr-51 for 4 h. After 4 h of incubation, the amount of Cr51 released in each well along with tumor lysis was quantified using a PerkinElmer TopCount. The results showed that no specific cell lysis was observed for any of the CAR-EBD mutants in the absence of estrogen analogs. The activity of B7H3CAR-3Gly-EBD(4OHT) was found to be modulated by 4OHT or (Z)-endoxifen, but not by CMP8. This was shown by 0% specific lysis in the presence of 500 nM CMP8. The activity of B7H3CAR-3Gly-EBD(CMP8) was modulated by all three estrogen analogs. Usually, 1 nM of estrogen analog is insufficient to induce maximal activity. Antigen-specific cell lysis was most frequently observed with 50 nM or 500 nM of estrogen analog, regardless of the type of EBD mutant. Incubation with increasing concentrations of various estrogen analogs showed that the specific cell lysis due to the activity of B7H3CAR-EBD was dose-dependent.

[0036] [Figure 19A-19B](Figure 19A) Drug-dependent cytokine release by various B7H3CAR-3Gly-EBD mutants in the presence of estrogen analogs 4-OHT, CMP8, or (Z)-endoxifen. (Figure 19B) Drug-dependent cytokine release by various B7H3CAR-3Gly-EBD mutants in the presence of estrogen analogs 4-OHT, CMP8, or (Z)-endoxifen. (Figures 20A and 20B) Cytokine release was assessed using Meso Scale Diagnostic assays. First, cells bearing the B7H3CAR-3Gly-EBD(4OHT) mutant or cells bearing the B7H3CAR-3Gly-EBD(CMP8) mutant were cultured for 24 hours in the presence of three estrogen analogs (4-OHT, CMP8 or (Z)-endoxifen) at various drug concentrations (0 nM, 1 nM, 50 nM or 500 nM), and then co-cultured with various tumor lines expressing B7H3 (the antigen of interest) or knocking out (KO) B7H3 for 24 hours. After 24 hours of incubation, the supernatants were collected from each co-culture and measured with a MESO QuickPlex SQ 120 instrument to confirm the amount of cytokines released. The results showed that no cytokines were released by any of the B7H3CAR-EBD mutants, regardless of drug conditions, when co-cultured with a tumor line that does not express the antigen (K562 B7H3 KO). The activity of B7H3CAR-3Gly-EBD(4OHT) was found to be modulated by 4OHT or (Z)-endoxifen, but not by CMP8. The activity of B7H3CAR-3Gly-EBD(CMP8) was modulated by all three estrogen analogs. Typically, 1 nM of estrogen analog was insufficient to induce maximal activity. The highest antigen-specific cytokine release was observed at 50 nM to 500 nM of estrogen analog, regardless of the type of CAR-EBD mutant. When cultured with increasing concentrations of various estrogen analogs, cytokine release by all CAR-EBD mutants was shown to be dose-dependent.

[0037] [Figure 20A-20B] (Figure 20A) shows the difference in target cell lysis dependent on the glycine linker when combined with huCD19CAR-EBD(4-OHT) targeting K562+CD19 tumor cells. (Figure 20B) shows the difference in target cell lysis dependent on the glycine linker when combined with huCD19CAR-EBD(4-OHT) targeting Raji tumor cells. (Figures 20A and 20B) Specific cell lysis was assessed using a chromium release assay. First, various huCD19CAR-EBD(4OHT) effector lines with one, two or three glycines between the CAR and EBD linker were cultured for 24 hours in the presence of two estrogen analogues (4-OHT or (Z)-endoxifen) at various drug concentrations (0 nM, 1 nM, 50 nM or 500 nM) and then co-cultured for 4 hours with antigen-expressing tumor lines (K562+CD19 or Raji parental cells) previously labeled with radioactive Cr-51. After 4 hours of incubation, the amount of Cr51 released into each well along with tumor lysis was quantified using a PerkinElmer TopCount. In the absence of added estrogen analogues, the least cell lysis was observed for all CAR-EBD(4OHT) lines with linkers consisting of one, two or three glycines. In the absence of inducing drugs, the highest activity was observed with the CAR-2Gly-EBD(4OHT) construct. For huCD19CAR-EBD(4OHT), the single glycine linker induced the least specific cell lysis in the absence of estrogen analogs, while the two and three glycine linkers induced similarly the most specific cell lysis in the presence of 50 nM or 500 nM estrogen analogs. Incubation with increasing concentrations of various estrogen analogs demonstrated that the specific cell lysis due to CAR-EBD activity remained dose-dependent.

[0038] [Figure 21]Drug-dependent specific cell lysis was reproducible across multiple T cell donors. Specific cell lysis was assessed by chromium release assays using huCD19CAR-EBD(4OHT) effector lines with one glycine, two glycines or three glycines linker from two T cell donors. Effector cell lines from two donors were cultured for 4 h in the presence of two estrogen analogs (4-OHT or (Z)-endoxifen) at various drug concentrations (0 nM, 1 nM, 50 nM or 500 nM, only the lowest and highest drug conditions are shown) and then co-cultured for 4 h with an antigen-expressing tumor line (K562 CD19) previously labeled with radioactive Cr-51. After 4 h of incubation, the amount of Cr51 released in each well along with tumor lysis was quantified using a PerkinElmer TopCount. The measurements showed that drug-dependent specific cell lysis by CAR-EBD was reproducible across different T cell donors.

[0039] [Figure 22]We show the differential cytokine release dependent on the glycine linker when combined with huCD19CAR-EBD(4-OHT) targeting K562+CD19 tumor cells. Cytokine release was assessed using a Meso Scale Diagnostic assay. First, various huCD19CAR-EBD(4OHT) effector lines with one, two or three glycine linkers between the CAR and EBD were cultured for 24 hours in the presence of two estrogen analogs (4-OHT or (Z)-endoxifen) at various drug concentrations (0 nM, 1 nM, 50 nM or 500 nM) and then co-cultured with an antigen-expressing tumor line (K562 CD19) for 24 hours. After 24 hours of incubation, supernatants were collected from each co-culture and measured on a MESO QuickPlex SQ 120 instrument to confirm the amount of cytokine release. In the absence of estrogen analogs, the amount of cytokines released was very low. When cultured with increasing concentrations of various estrogen analogs, cytokine release from CAR-EBD lines with one, two, or three glycine linkers was shown to be dose-dependent. Typically, 1 nM of estrogen analog is insufficient to induce maximal activation. Estrogen analogs at 50 nM to 500 nM induced the greatest antigen-specific cytokine release, regardless of the type of CAR-EBD line with one, two, or three glycine linkers.

[0040] [Figure 23]We show the differential cytokine release dependent on the glycine linker when combined with huCD19CAR-EBD(4-OHT) targeting Raji tumor cells. Cytokine release was assessed using a Meso Scale Diagnostic assay. First, various huCD19CAR-EBD(4OHT) effector lines with one, two or three glycine linkers between CAR and EBD were cultured for 24 hours in the presence of two estrogen analogs (4-OHT or (Z)-endoxifen) at various drug concentrations (0 nM, 1 nM, 50 nM or 500 nM) and then co-cultured with an antigen-expressing tumor line (Raji parental cells) for 24 hours. After 24 hours of incubation, supernatants were collected from each co-culture and measured on a MESO QuickPlex SQ 120 instrument to confirm the amount of cytokine released. In the absence of estrogen analogs, the amount of cytokine released was very low. When cultured in increasing concentrations of various estrogen analogs, the cytokine release of CAR-EBD lines with one, two or three glycine linkers was shown to be dose-dependent. Typically, 1 nM of estrogen analog was insufficient to induce maximal activity. Estrogen analogs at 50 nM to 500 nM induced the greatest antigen-specific cytokine release, regardless of the type of CAR-EBD line with one, two or three glycine linkers.

[0041] [Figure 24]Drug-dependent cytokine release was reproducible across multiple T cell donors. Cytokine release was assessed using Meso Scale Diagnostic assays with huCD19CAR-EBD(4OHT) effector lines with one glycine, two glycines, or three glycines linker between the CAR and EBD from two T cell donors. Effector lines from each donor were co-cultured with various tumor lines expressing CD19 (the antigen of interest) for 24 hours. After 24 hours of incubation, supernatants were collected from each co-culture and measured on a MESO QuickPlex SQ 120 instrument to confirm cytokine release. For donor 1, huCD19CAR was tested in all drug conditions, whereas for donor 2, huCD19CAR was evaluated only in the absence of drug and in the presence of 500 nM drug. All donors showed a trend toward increased release of IFN-γ, IL-2, and TNFα with increasing concentrations of estrogen analogue, indicating that drug-dependent cytokine release by CAR-EBD is reproducible across different T cell donors.

[0042] [Diagram 25]Drug-dependent specific cell lysis by two huCD19CAR-EBD(4OHT) variants with different linkers between CAR and EBD(4OHT) (no glycine linker or with a linker consisting of one glycine) in the presence of the estrogen analogues 4-OHT or (Z)-endoxifen. Specific cell lysis was assessed in donor 3 using a chromium release assay. First, huCD19CAR-EBD(4OHT) linker variants (without a glycine linker (NoGly) or with a linker consisting of one glycine (1Gly)) were cultured for 24 h in the presence of two estrogen analogues (4-OHT or (Z)-endoxifen) at various drug concentrations (0 nM, 1 nM, 50 nM, or 500 nM). Then, each variant was co-cultured for 4 h with an antigen-expressing tumor line previously labeled with radioactive Cr-51. After 4 hours of incubation, the amount of Cr51 released into each well with tumor lysis was quantified using a PerkinElmer TopCount. Measurements showed that this huCD19CAR-EBD(4OHT) system was more stringent than initially observed with huCD19CAR-EBD(4OHT) from donor 1 or donor 2 in the absence of inducing drug (Figure 22: Direct comparison with huCD19CAR-EBD(4OHT) with a single glycine linker). Small amounts of killing were still observed with variants without and with a single glycine linker. Both variants with and without a glycine linker could be modulated with 4OHT or (Z)-endoxifen, with complete killing (similar to constitutive huCD19CAR) achieved at 50 and 500 nM with each drug. Neither drug performed better than the other in this assay, and variants with no and single glycine linkers appeared to confer comparable modulation of the CAR-EBD system with respect to antigen-specific cell lysis, as measured by chromium release assays.

[0043] [Figure 26] Anti-ζ chain western blots evaluating various huCD19CAR-EBD(4OHT) with different glycine linkers in the presence of 0 nM or 500 nM 4OHT or (Z)-endoxifen. Anti-CD3ζ western blot analysis was used to evaluate protein processing and expression of huCD19CAR-EBD with different linkers between CAR and EBD. Whole cell lysates were prepared from huCD19CAR-EBD(4OHT) T cell lines with one, two or three glycine linkers between the CAR and EBD domains after 24 hours of incubation in drug conditions of 0 nM or 500 nM 4OHT or (Z)-endoxifen. Whole cell lysates from mock T cell lines and T cell lines containing only CAR were also prepared under the same conditions. Western blots were imaged and analyzed using a LiCOR Odyssey. Furthermore, protein bands were quantified using a LiCOR Odyssey. CAR-EBD lines with one, two, or three glycine linkers all showed expression of the CAR ζ chain in both the absence and maximum drug conditions. Quantification of the CAR-EBD ζ chain bands showed that addition of 500 nM 4OHT or 500 nM (Z)-endoxifen increased CAR-EBD protein expression approximately three-fold. No such change was observed in the constitutive CAR ζ chain band.

[0044] [Figure 27]Changes in cell surface expression of huCD19CAR-1Gly-EBD(4OHT) upon drug induction / withdrawal. CD4 and CD8 T cells (1:1 ratio mixture) expressing the huCD19CAR-1Gly-EBD(4OHT) construct were cultured in medium containing 1 μM 4-OHT for various periods of time, or cultured without 1 μM 4-OHT for various periods of time. Each culture was then stained with a fluorochrome-conjugated antibody specific for the scFv portion of the huCD19CAR-1Gly-EBD(4OHT) construct. The MFI (median fluorescence intensity) of each cell population was then measured at each time point shown on the graph. Addition or removal of the drug altered cell surface expression of the CAR-EBD construct. The kinetics of this change was very slow in both directions (day after day), not dissimilar to known degrons, indicating a separate regulatory mechanism.

[0045] [Fig. 28A-28B](Figure 28A) CD107a activation assay of various huCD19CAR-EBD(4OHT) linker variants acted on K562+CD19t. (Figure 28B) CD107a activation assay of various B7H3CAR-3Gly-EBD variants acted on K562+CD19t. (Figure 29A and Figure 29B) CD4 and CD8 T cells (mixtures in 1:1 ratio) expressing various huCD19CAR-EBD or B7H3CAR-EBD constructs with different linkers between the CAR and EBD domains (single glycine linker (1Gly), two glycine linkers (2Gly), or three glycine linkers (3Gly)) and different types of EBD domains (EBD(4OHT), EBD(CMP8), or EBD(ES8)) were analyzed to examine a) the regulatory ability of these cells (e.g., switching on / off with various drugs) and b) the dose response of activity to drug induction. To perform this experiment, T cells (including mock and constitutively active CAR control) were pretreated for 24 hours in the presence of various concentrations of the targeting drug and then exposed to tumor lines expressing the target antigen (CD19 or B7H3). After 4 hours of co-culture of T cells with target tumors at various drug concentrations, a) CD107a staining and flow cytometry were performed to assess T cell activation and degranulation, and b) human scFv (capable of detecting both B7H3CAR and huCD19CAR) staining and flow cytometry were performed to assess CAR-EBD on the surface of T cells. The MFI (median fluorescence intensity) of each staining for each sample under each condition was measured and plotted on a logarithmic scale against drug concentration. The EC10 (10% effective concentration) and EC90 (90% effective concentration) for CD107a expression were calculated in Graphpad Prism and used as an estimate of the drug concentration required to switch each construct "on" and "off". Positive and negative controls for each CAR are shown in the bar graphs.A slight shift in MFA of CD107a was observed between mock and uninduced CAR-EBD T cells, suggesting the presence of minimal uncontrolled degranulation in all EBD constructs, but this degranulation was more pronounced in the huCD19CAR-EBD construct than in the B7H3CAR-EBD construct. EBD(4OHT) was responsive to 4-OHT and (Z)-endoxifen, but not to CMP8 or ES8. Degranulation responsiveness to target antigen in the presence of inducers was roughly comparable among the various glycine linker variants. EBD(4OHT) showed highly sensitive to the intended inducers, initiating activation at single-digit nanomolar concentrations (EC10) and being fully activated at double-digit nanomolar concentrations (EC90). EBD(4OHT) can be used to modulate either huCD19CAR or B7H3CAR, with similar dose response characteristics (similar EC10 / EC90) between the two CARs, despite the higher background of the huCD19CAR construct. EBD(CMP8) is less sensitive to drug induction than EBD(4OHT). EBD(ES8) was highly sensitive to induction by ES8, but the degree of induction by ES8 was at most lower than that by 4OHT. Both EBD(CMP8) and EBD(ES8) are responsive to 4OHT. However, EBD(CMP8) is not responsive to ES8, and EBD(ES8) is not responsive to CMP8, indicating that multiple EBD domains can be regulated independently.

[0046] [Figure 29A-29B](Figure 29A and Figure 29B) CAR expression by different huCD19CAR-EBD linker variants on K562+CD19t. CD4 and CD8 T cells (1:1 ratio mixtures) expressing different huCD19CAR-EBD or B7H3CAR-EBD constructs with different linkers between the CAR and EBD domains (single glycine linker (1Gly), two glycine linker (2Gly), or three glycine linker (3Gly)) and different types of EBD domains (EBD(4OHT), EBD(CMP8), or EBD(ES8)) were analyzed to determine a) the regulatory ability of these cells (e.g., switching on / off with different drugs) and b) the dose response of activity to drug induction. To perform this experiment, T cells (including mock and constitutively active CAR control) were pretreated for 24 hours in the presence of various concentrations of the target drug and then exposed to tumor lines expressing the target antigen (CD19 or B7H3). After 4 hours of co-culture of T cells and tumor with various drug concentrations, a) CD107a staining and flow cytometry were performed to evaluate T cell activation and degranulation, and b) human scFv (capable of detecting both B7H3CAR and huCD19CAR) staining and flow cytometry were performed to evaluate CAR-EBD on the surface of T cells. The MFI (median fluorescence intensity) of each staining for each sample under each condition was measured and plotted against the drug concentration on a logarithmic scale. It was observed that the changes in cell surface CAR-EBD observed in the previous experiment (Figure 25) were not only time-dependent but also drug concentration-dependent, as shown in Figure 30A. Changes in cell surface CAR-EBD directly correlate with the ability to induce EBD, independent of the presence of non-specific estrogen analogs (e.g., CMP8) (i.e., EBD increases when cells are able to be activated, but EBD does not necessarily require cell activation). (Figure 29B) Relative activation and cell surface expression of CAR-EBD are shown using the exact same cells (each point in the stacked line graph represents the two fluorescent dyes for the same cell population).The drug-induced changes in cell surface expression of CAR-EBD were independent of drug-induced activation of CAR-EBD (increased CAR expression required higher drug concentrations than were required to enhance activation). The results of this experiment suggest that although CAR-EBD activation correlates with its cell surface expression, the mechanism of CAR-EBD activation is distinct from mere cell surface expression.

[0047] [Figures 30A-30C] Incucyte cytotoxicity assay of huCD19CAR-1Gly-EBD(4OHT). CD4 and CD8 T cells (1:1 ratio mixture) expressing huCD19CAR-EBD-1Gly-EBD(4OHT) were pre-incubated with 500 nM of inducing drug for 24 hours or were not pre-incubated before assay. These T cells were then plated with CAR target expressing tumor lines ((Figure 30A) K562+CD19, (Figure 30B) Raji and / or (Figure 30C) Be2+CD19) constitutively expressing mCherry (for detection and quantification). Tumor lines were seeded at a density of 10,000 cells / well and co-cultured at a ratio of 8:1 or 4:1 (T cells:tumor cells) in the presence or absence of 500 nM 4-OHT (ratios are noted in annotations where appropriate). Each sample was placed in an Incucyte device and imaged every 3-4 hours for several days. The mCherry signal was then quantified and plotted as a function of time. Constitutively active appropriate CAR T cells were used as positive controls, and mock T cells were used as negative controls. No difference in functionality was observed between CAR-EBD T cells that were preincubated with drug ("24-hour pretreatment") and those that were not ("0-hour pretreatment"). This result further demonstrated that CAR-EBD is mechanistically distinct from degrons. Degron technology takes time to accumulate in sufficient amounts to be active (typically 3-6 hours). In contrast, the "on" kinetics of CAR-EBD killing in the Incucyte cytotoxicity assay can be observed almost instantly.

[0048] [Fig. 31A-31B](FIG. 31A) Incucyte cytotoxicity assay of various B7H3CAR-3Gly-EBD variants. CD4 and CD8 T cells (mixtures at 1:1 ratio) expressing B7H3CAR-3Gly-EBD(4OHT), B7H3CAR-3Gly-EBD(CMP8) or B7H3CAR-3Gly-EBD(ES8) were pre-incubated with 500 nM of inducing drug for 24 hours or were not pre-incubated before assay. These T cells were then plated with CAR target-expressing tumor lines (K562 and / or Be2) that constitutively express mCherry (for detection and quantification). Tumor lines were seeded at a density of 10,000 cells / well and co-cultured at a 4:1 ratio (T cells:tumor cells) in the presence or absence of 500 nM of each drug (B7H3CAR-3Gly-EBD(4OHT) was supplemented with 500 nM 4OHT, B7H3CAR-3Gly-EBD(CMP8) was supplemented with 500 nM CMP8, and B7H3CAR-3Gly-EBD(ES8) was supplemented with 500 nM ES8). Each plate was placed in an Incucyte instrument and images were taken every 3-4 hours over several days. The mCherry signal was then quantified and plotted as a function of time. As positive controls, constitutively active appropriate CAR T cells were used, and as negative controls, mock T cells were used. All three B7H3CAR-3Gly-EBD variants had a potent antitumor "on" state and a well-regulated "off" state, indicating that each B7H3CAR-3Gly-EBD variant had a highly modulated activity. (Figures 31A and 31B) Shown are B7H3CAR-EBD(CMP8) constructs exposed to K562+mCherry. Figure 31A shows a comparison of drug-free conditions with drug, and Figure 31B shows a comparison of drug-free conditions with drug added after 24 hours (black vertical line). By 24 hours, the amount of tumor that had grown was beyond the capacity of the donor itself or the B7H3CAR-3Gly-EBD(CMP8) T cells at this ratio to overcome, but activation of B7H3CAR-3Gly-EBD(CMP8) T cells with CMP8 immediately altered the kinetics of tumor growth.

[0049] [Fig. 32A-32C](Figure 32A-C) CD4 and CD8 T cells (1:1 ratio mixture) expressing B7H3CAR-3Gly-EBD(4OHT), B7H3CAR-3Gly-EBD(CMP8) or B7H3CAR-3Gly-EBD(ES8) were pre-incubated with 500 nM of inducing drug for 24 hours or were not pre-incubated before assay (500 nM 4OHT for B7H3CAR-3Gly-EBD(4OHT), 500 nM CMP8 for B7H3CAR-3Gly-EBD(CMP8), and 500 nM ES8 for B7H3CAR-3Gly-EBD(ES8). These T cells were then plated with CAR target-expressing tumor lines (K562 and / or Be2) that constitutively express mCherry (for detection and quantification). Tumor lines were seeded at a density of 10,000 cells / well and co-cultured at a 4:1 ratio (T cells:tumor cells) in the presence or absence of 500 nM of the appropriate drug. Each sample was placed in an Incucyte device and imaged every 3-4 hours for several days. The mCherry signal was then quantified and plotted as a function of time. As a positive control, constitutively active appropriate CAR T cells were used, and as a negative control, mock T cells were used. In one experiment, T cells were repeatedly challenged with tumor cells to assess differences in the impact on functional lifespan between the various constructs. (Figure 32A) Shown is an Incucyte cytotoxicity assay evaluating the impact of estrogen analogs on B7H3CAR. This control assay showed that the addition of estrogen analogs (e.g., 4-OHT) did not reduce tumor growth (note: mock condition) or CAR T cell functionality (note: B7H3CAR condition). (Figure 32B) Incucyte cytotoxicity assay of B7H3CAR-3Gly-EBD variants. No difference in killing kinetics of CAR-EBD T cells was observed when pretreated with inducing drug for 24 hours versus when inducing drug was added at the beginning of the experiment. (Figure 32C) Incucyte cytotoxicity assay of various B7H3CAR-3Gly-EBD variants.Both CAR-EBD(4OHT) and CAR-EBD(ES8) cells demonstrated highly regulated killing of Be2+mCherry tumor cells. Be2 cells were allowed to attach to the plate 24 hours before the addition of T cells.

[0050] [Diagram 33]Representative sequences supporting the present disclosure are set forth below: estrogen receptor (SEQ ID NO:1), wild-type estrogen binding domain (EBD) (SEQ ID NO:2) with possible N-terminal and C-terminal cleavage sites underlined, estrogen receptor with a G521R mutation (SEQ ID NO:3), EBD with a G521R mutation (SEQ ID NO:4), estrogen receptor with an E353A mutation (SEQ ID NO:5), EBD(E353A) (SEQ ID NO:6), coding sequence for EBD(E353A) (SEQ ID NO:7), estrogen receptor with an L384M and M421G mutation (SEQ ID NO:8), EBD with an L384M and M421G mutation (SEQ ID NO:9), L384M mutation, M421G mutation and G521R mutation. Estrogen receptor with mutations (SEQ ID NO:10), EBD (L384M, M421G, G521R) used in combination with the estrogen analog CMP8 (SEQ ID NO:11, also referred to herein as EBD(CMP8)), EBD with G400V, M543A and L544A mutations (SEQ ID NO:12), EBD with G400V, M543A and L544A mutations (referred to herein as ERT2 or EBD(4-OHT)) (SEQ ID NO:13), coding sequence for ERT2 (SEQ ID NO:14), coding sequence for EBD(CMP8)(L384M, M421G, G521R) (SEQ ID NO:15), EGFRVIII(806). scFv (SEQ ID NO: 16), EGFRVIII(806) scFv coding sequence (SEQ ID NO: 17), EGFR scFv (SEQ ID NO: 18), huCD19(G01S) scFv (SEQ ID NO: 19), muCD19(FMC63) scFv (SEQ ID NO: 20), CD19 scFv (SEQ ID NO: 21), CD19 scFv coding sequence (SEQ ID NO: 22), CD20(Leu 16) scFv (SEQ ID NO: 23), CD20 scFv coding sequence (SEQ ID NO: 24), CD22(m971) scFv (SEQ ID NO: 25), B7H3(hBRCA84D) scFv (SEQ ID NO: 26), L1CAM(CE7) scFv (SEQ ID NO: 27), EphA2(2A4) scFv (SEQ ID NO: 28), EpHA2(4H5) scFv (SEQ ID NO: 29), FITC(E2) scFv (SEQ ID NO: 30), GD2 (hu3F8)scFv (SEQ ID NO: 31), Her2 (Herceptin) scFv (SEQ ID NO: 32), IL13Ra2 (hu08) VlVh scFv (SEQ ID NO: 33), IL13Ra2 hu08 VhV1 scFv (SEQ ID NO: 34), IL13Ra2 (hu07) VhV1 scFv (SEQ ID NO: 35), IL13Ra2 (hu07) VhVl scFv (SEQ ID NO: 36), oaGD2 (8B6) VlVh scFv (SEQ ID NO: 37), ROR1 (R12) scFv (SEQ ID NO: 38), CD33 (h2H12) VhVl scFv (SEQ ID NO: 39), CD33 (h2H12) VlVh scFv (SEQ ID NO: 40), mesothelin (P4) scFv (SEQ ID NO: 41), VAR2CSA (ID1-DBL2Xb) scFv (SEQ ID NO: 42), amino acid sequence of anti-IL13Ra2 (IL13 zetakine) (SEQ ID NO: 43), FITCE2 scFv (SEQ ID NO: 44), FITCE2 TyrH133Ala scFv (SEQ ID NO: 45), FITCE2 HisH131Ala scFv (SEQ ID NO: 46), FL (4M5.3) scFv (SEQ ID NO: 47), FL (4D5Flu) scFv (SEQ ID NO: 48), FL (4420) scFv (SEQ ID NO: 49), DNP scFv (SEQ ID NO: 50), DNP scFv (SEQ ID NO: 51), DNP scFv (SEQ ID NO: 52), DNP scFv (SEQ ID NO: 53), DNP VH of scFv (Ab-1; 1BAF) (SEQ ID NO: 54), VL of DNP scFv (Ab-1; 1BAF) (SEQ ID NO: 55), VH of DNP scFv (Ab-2; XC) (SEQ ID NO: 56), VL of DNP scFv (Ab-2; XC) (SEQ ID NO: 57), VH of DNP scFv (Ab-3) (SEQ ID NO: 58), VL of DNP scFv (Ab-3) (SEQ ID NO: 59), VL-linker-VH of DNP scFv (Ab-3) (SEQ ID NO: 60), VH-linker-VL of DNP scFv (Ab-3) (SEQ ID NO: 61), VH-linker-VL of DNP scFv (1BAF) (SEQ ID NO: 62), VL-linker-VH of DNP scFv (1BAF) (SEQ ID NO: 63), DNP VH-linker-VL of scFv(XC) (SEQ ID NO: 64), VL-linker-VH of DNP scFv(XC) (SEQ ID NO: 65), VH-linker-VL of DNP scFv(1BAF) (SEQ ID NO: 66), DNPVL-linker-VH of scFv(1BAF) (SEQ ID NO: 67), VH-linker-VL of DNP scFv(XC) (SEQ ID NO: 68), VL-linker-VH of DNP scFv(XC) (SEQ ID NO: 69), VH-linker-VL of DNP scFv(Ab-3) (SEQ ID NO: 70), DNP scFv; DNPVH-linker-VL of scFv (Ab-3) (SEQ ID NO: 71), glycine serine linker (SEQ ID NO: 72), glycine serine linker (SEQ ID NO: 73), glycine serine linker (SEQ ID NO: 74), glycine serine linker (SEQ ID NO: 75), coding sequence of (G4S)3 linker (SEQ ID NO: 76), coding sequence of (G4S)4 linker (SEQ ID NO: 77), short spacer (IgG4 (short hinge)) (SEQ ID NO: 78), coding sequence of short spacer (IgG4 (short hinge)) (SEQ ID NO: No. 79), short spacer 2 (another IgG4 linker) (SEQ ID NO: 80), medium length spacer (SEQ ID NO: 81), long spacer with a single mutation (L235D) (SEQ ID NO: 82), long spacer with a double mutation (L235D, N297Q) (SEQ ID NO: 83), glycine linker, coding sequence for glycine linker (SEQ ID NO: 84), P2A (SEQ ID NO: 85), coding sequence for P2A (SEQ ID NO: 86), T2A (SEQ ID NO: 87), coding sequence for T2A (SEQ ID NO: 88), E2 A (SEQ ID NO: 89), F2A (SEQ ID NO: 90), DHFRdm (SEQ ID NO: 91), DHFRdm coding sequence (SEQ ID NO: 92), CD19t (SEQ ID NO: 93), CD19t coding sequence (SEQ ID NO: 94), truncated Her2 (Her2) (SEQ ID NO: 95), Her2tG (SEQ ID NO: 96), EGFRt (SEQ ID NO: 97), GM-CSF signal peptide (SEQ ID NO: 98), CD8 signal peptide (SEQ ID NO: 99), His tag (SEQ ID NO: 100), Flag tag (SEQ ID NO: 101), Flag tag (SEQ ID NO: 102), Flag tag (SEQ ID NO: 103), Xpress tag (SEQ ID NO: 104), Avi tag (SEQ ID NO: 105), calmodulin binding peptide (CBP) tag (SEQ ID NO: 106), polyglutamic acid tag (SEQ ID NO: 107), HA tag (SEQ ID NO: 108), HA tag (SEQ ID NO: 109), HA tag (SEQ ID NO: 110), Myc tag (SEQ ID NO: 111), Strep tag (SEQ ID NO: 112), STREP® tag II (SEQ ID NO: 113), Softag 1 (SEQ ID NO: 114), Softag3 (SEQ ID NO: 115), V5 tag (SEQ ID NO: 116), MND promoter (SEQ ID NO: 117), EGFR806CAR-ERT2 (SEQ ID NO: 118), EGFR806CAR-ERT2-P2A-DHFRdm-T2A-CD19t protein sequence shown in the table (sequences shown in the table combined to form SEQ ID NO: 1306), coding sequence for EGFR806CAR-ERT2-P2A-DHFRdm-T2A-CD19t shown in the table (sequences shown in the table combined to form SEQ ID NO: 125). a CD8α hinge / transmembrane domain (SEQ ID NO:126), an alternative CD8 hinge / transmembrane domain (SEQ ID NO:127), an alternative CD8 hinge / transmembrane domain (SEQ ID NO:128), an alternative human CD8α chain hinge (SEQ ID NO:129), an intracellular signaling domain of 4-1BB / CD3ζ (SEQ ID NO:130), a coding sequence for the intracellular signaling domain of 4-1BB / CD3ζ (SEQ ID NO:131), a coding sequence for the intracellular signaling domain of 4-1BB / CD3ζ (SEQ ID NO:132), a CD3ζ ICD (SEQ ID NO: 121), coding sequence of CD3ζ ICD (SEQ ID NO: 124), CD3ε chain of surface glycoprotein of T cells (CD3ε) (SEQ ID NO: 133), CD3ε ECD (SEQ ID NO: 134), CD3ε ICD (SEQ ID NO: 135), CD3ε TM (SEQ ID NO: 136), CD3δ chain of surface glycoprotein of T cells (CD3δ) (SEQ ID NO: 137), CD3δ ECD (SEQ ID NO: 138), CD3δ ICD (SEQ ID NO: 139), CD3δ TM (SEQ ID NO: 140), CD3ζ chain of surface glycoprotein of T cells (CD3ζ) (SEQ ID NO: 141), CD3ζ ECD (SEQ ID NO: 142), CD3ζ ICD (SEQ ID NO: 143), CD3ζ TM (SEQ ID NO: 144), CD70 [Homo sapiens] (SEQ ID NO: 145), TL1A i.e. tumor necrosis factor (ligand) superfamily member 15 (SEQ ID NO: 146), OX40 (SEQ ID NO: 147), OX40 ECD (SEQ ID NO: 148), OX40 ICD (SEQ ID NO: 149), OX40 TM (SEQ ID NO: 150), CD27 antigen precursor (SEQ ID NO: 151), CD27 ECD (SEQ ID NO: 152), CD27 ICD (SEQ ID NO: 153), CD27 TM (SEQ ID NO: 154), cytokine receptor CD30 (SEQ ID NO: 155), CD30ECD (SEQ ID NO:156), CD30 ICD (SEQ ID NO:157), CD30 TM (SEQ ID NO:158), CD40 i.e. human tumor necrosis factor receptor superfamily member 5 (SEQ ID NO:159), CD40 ECD (SEQ ID NO:160), CD40 ICD (SEQ ID NO:161), CD40 TM (SEQ ID NO:162), HVEM (SEQ ID NO:163), HVEM ECD (SEQ ID NO:164), HVEM ICD (SEQ ID NO:165), HVEM TM (SEQ ID NO:166), DR3 (SEQ ID NO:167), DR3 ECD (SEQ ID NO:168), DR3 ICD (SEQ ID NO:169), DR3 TM (SEQ ID NO:170), 4-1BB i.e. human tumor necrosis factor receptor superfamily member 9 precursor (SEQ ID NO:171), 4-1BB ECD (SEQ ID NO:172), 41BB ICD (SEQ ID NO:120), coding sequence of 4-1BB ICD (SEQ ID NO:123), 4-1BB TM (SEQ ID NO: 173), interleukin 2 receptor subunit alpha (CD25) (SEQ ID NO: 174), CD25 ECD (SEQ ID NO: 175), CD25 ICD (SEQ ID NO: 176), CD25 TM (SEQ ID NO: 177), T cell specific surface glycoprotein CD28 (SEQ ID NO: 178), CD28 ECD (SEQ ID NO: 179), CD28 ICD (SEQ ID NO: 180), CD28 ICD (SEQ ID NO: 181), coding sequence of ICD of human CD28 (SEQ ID NO: 182), CD28 TM (SEQ ID NO: 183), transmembrane domain of CD28 (SEQ ID NO: 119), coding sequence of CD28tm (SEQ ID NO: 122), CD79a (SEQ ID NO: 184), CD79a ECD (SEQ ID NO: 185), CD79a ICD (SEQ ID NO: 186), CD79a TM (SEQ ID NO: 187), CD79b (SEQ ID NO: 188), CD79b ECD (SEQ ID NO: 189), CD79b ICD (SEQ ID NO: 190), CD79b TM (SEQ ID NO: 191), signaling lymphocyte activation molecule family member 1 (SLAMF1, CD150) (SEQ ID NO: 192), SLAMF1 ECD (SEQ ID NO: 193), SLAMF1 ICD (SEQ ID NO: 194), SLAMF1 TM (SEQ ID NO: 195), inducible T cell costimulator (ICOS, CD278) (SEQ ID NO: 196), ICOS ECD (SEQ ID NO: 197), ICOS ICD (SEQ ID NO: 198), ICOSTM (SEQ ID NO: 199), TNFRSF18 protein (CD357, GITR) (SEQ ID NO: 200), GITR ECD (SEQ ID NO: 201), GITR ICD (SEQ ID NO: 202), GITR TM:LGWLTVVLLAVAACVLLLTSA (SEQ ID NO: 203), caspase recruitment domain containing protein 11 (CARD11) (SEQ ID NO: 204), DAP10 (SEQ ID NO: 205), DAP10 ECD (SEQ ID NO: 206), DAP10 ICD (SEQ ID NO: 207), DAP10 TM (SEQ ID NO: 208), DAP12 (SEQ ID NO: 209), DAP12 ECD (SEQ ID NO: 210), DAP12 ICD (SEQ ID NO: 211), DAP12 TM (SEQ ID NO: 212), high affinity immunoglobulin epsilon receptor subunit alpha (SEQ ID NO: 213), high affinity immunoglobulin epsilon receptor subunit beta (SEQ ID NO: 214), FcR-gamma, i.e., high affinity immunoglobulin epsilon receptor subunit gamma precursor (SEQ ID NO: 215), tyrosine protein kinase Fyn (SEQ ID NO: 216), tyrosine protein kinase Lck (SEQ ID NO: 217), LAT (SEQ ID NO: 218), LRP (SEQ ID NO: 219), NKG2D (SEQ ID NO: 220), NOTCH1 (SEQ ID NO: 221), NOTCH2 (SEQ ID NO: 222), NOTCH3 (SEQ ID NO: 223), NOTCH4 (SEQ ID NO: 224), transmembrane receptor tyrosine protein kinase ROR2 (SEQ ID NO: 225), tyrosine protein kinase RYK (SEQ ID NO: 226), lymphocyte cytoplasmic protein 2 (Slp76) (SEQ ID NO: 227), pre-T cell receptor alpha chain precursor (pTα) (SEQ ID NO: 228), T cell receptor alpha chain (TCRα) (SEQ ID NO: 229), T cell receptor beta chain (TCRβ) (SEQ ID NO: 230), T cell receptor interacting molecule (TRIM) protein (SEQ ID NO: 231), ZAP70 protein (SEQ ID NO: 232), Patched 2 (PTCH2) (SEQ ID NO: 233), programmed cell death protein 1 (PD1) (SEQ ID NO: 234), PD1 ECD (SEQ ID NO: 235), PD1 ICD (SEQ ID NO: 236), PD1 TM (SEQ ID NO: 237), programmed cell death 1 ligand 1 (PD-L1) (SEQ ID NO: 238), PD-L1 ECD (SEQ ID NO: 239), PD-L1 ICD (SEQ ID NO: 240), PD-L1TM (SEQ ID NO:241), programmed cell death 1 ligand 2 (PD-L2) (SEQ ID NO:242), PD-L2 ECD (SEQ ID NO:243), PD-L2 ICD (SEQ ID NO:244), PD-L2 TM (SEQ ID NO:245), CTLA4 (SEQ ID NO:246), CTLA4 ECD (SEQ ID NO:247), CTLA4 ICD (SEQ ID NO:248), CTLA4 TM (SEQ ID NO:249), T cell immunoglobulin mucin 3 (TIM3) (SEQ ID NO:250), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) (SEQ ID NO:251), CEACAM-1 ECD (SEQ ID NO:252), CEACAM-1 ICD (SEQ ID NO:253), CEACAM-1 TM (SEQ ID NO:254), carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM-3) (SEQ ID NO:255), CEACAM-3 ECD (SEQ ID NO:256), CEACAM-3 ICD (SEQ ID NO: 257), CEACAM-3 TM (SEQ ID NO: 258), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) (SEQ ID NO: 259), lymphocyte activation gene 3 protein (LAG3) (SEQ ID NO: 260), LAG3 ECD (SEQ ID NO: 261), LAG3 ICD (SEQ ID NO: 262), LAG3 TM (SEQ ID NO: 263), V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) (SEQ ID NO: 264), VISTA ECD (SEQ ID NO: 265), VISTA ICD (SEQ ID NO: 266), VISTA TM (SEQ ID NO: 267), B-lymphocyte and T-lymphocyte attenuator (BTLA) (SEQ ID NO: 268), BTLA ECD (SEQ ID NO: 269), BTLA ICD (SEQ ID NO: 270), BTLA TM (SEQ ID NO: 271), T-cell immunoreceptor with Ig and ITIM domains (TIGIT) (SEQ ID NO: 272), TIGIT ECD (SEQ ID NO: 273), TIGIT ICD (SEQ ID NO: 274), TIGIT TM (SEQ ID NO: 275), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) (SEQ ID NO: 276), LAIR1 ECD (SEQ ID NO: 277), LAIR1 ICD (SEQ ID NO: 278), LAIR1 TM (SEQ ID NO: 279), T-lymphocyte activation antigen CD80 (SEQ ID NO: 280), CD80 ECD (SEQ ID NO: 281), CD80 ICD (SEQ ID NO: 282), CD80TM (SEQ ID NO: 283), T-lymphocyte activation antigen CD86 (SEQ ID NO: 284), CD86 ECD (SEQ ID NO: 285), CD86 ICD (SEQ ID NO: 286), CD86 TM (SEQ ID NO: 287), CD160 antigen (SEQ ID NO: 288), natural killer cell receptor 2B4 (SEQ ID NO: 289), 2B4 ECD (SEQ ID NO: 290), 2B4 ICD (SEQ ID NO: 291), 2B4 TM (SEQ ID NO: 292), CD276 antigen (B7-H3) (SEQ ID NO: 293), B7-H3 ECD (SEQ ID NO: 294), B7-H3 ICD (SEQ ID NO: 295), B7-H3 TM (SEQ ID NO: 296), V-set domain-containing T-cell activation inhibitor 1 (VTCN1) (SEQ ID NO: 297), VTCN1 ECD (SEQ ID NO: 298), VTCN1 ICD, VTCN1 TM (sequence number 299), killer cell immunoglobulin-like receptor (sequence number 300), adenosine receptor A2a (sequence number 301), galectin 9 (GAL9) (sequence number 302), transforming growth factor beta receptor I (TGFRβ) (sequence number 303), MHC class I (sequence number 304) and part of MHC class II (sequence number 305). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Genetic engineering has made great advances in activating immune system cells to kill cancer cells or infected cells. For example, T cells have been engineered to express molecules that have an extracellular portion that binds to a specific target antigen and an intracellular portion that induces the action of the T cell when the extracellular portion binds to the target antigen. In one example, the extracellular portion can be designed to bind to a target antigen found on cancer cells or infected cells, and when the target antigen of the cancer cell or infected cell binds to the extracellular portion, the intracellular portion activates the T cell and the cell that binds to the extracellular portion is destroyed by the T cell. An example of such a molecule is the chimeric antigen receptor (CAR).

[0052] As described herein, CARs comprise synthetically engineered proteins that contain a ligand binding domain that binds to an antigen associated with a disease or disorder. The ligand binding domain is linked to one or more intracellular signaling domains of an immune cell. The ligand binding domain may be derived, for example, from an antibody, a receptor (e.g., a T cell receptor), or a ligand of a receptor (e.g., a cytokine or chemokine).

[0053] Although CAR-expressing T cells can exhibit potent antitumor activity, they can also exhibit significant toxicity, including cytokine release syndrome (cytokine release syndrome), tumor lysis syndrome (TLS), and chronic B cell depletion induced by transplanted cells, all of which are due to the unregulated function of constitutively expressed and active CARs. Such toxicity may limit the application of CAR-based therapy.

[0054] Although methods to eliminate CAR-T cells, such as the elimination of CAR-T cells using suicide genes, can reduce such toxicity, this method may reduce the antitumor activity before it is fully exerted, and it is thought that this will have a significant impact on the therapeutic efficacy. As an alternative method to control the activity of a protein, a degron sequence can be used to control the degradation or stability of the protein. However, the effect of the degron sequence is delayed because it depends on the degradation of the protein in the cell. Therefore, there is a need to find a rapid and controllable method that can control the activity of CAR and other proteins expressed in vivo.

[0055] The present disclosure provides a CAR that can be activated by antigen binding after being expressed in vivo, and that can be controlled by administering a drug molecule. If activation can be controlled without depending on protein degradation, safety, which is important for immune cell therapy using CAR, can be improved.

[0056] The present disclosure achieves such an advance by incorporating a heat shock protein 90 (hsp90) binding domain into the intracellular portion of the CAR, which in the absence of drug molecules can bind to hsp90 and prevent other important intracellular molecules required for CAR activation following antigen binding from interacting with the CAR.

[0057] When the drug molecule is present, it displaces the bound hsp90 from the hsp90-binding domain site and / or induces a conformational change such that upon antigen binding, intracellular signaling occurs.

[0058] This mechanism is illustrated in Figures 1A-1C. In these figures, the estrogen binding domain (EBD) is shown as the hsp90 binding domain. The EBD may be derived from the natural estrogen receptor, but must contain at least one mutation such that the EBD binds to the drug molecule without binding to estrogen. In the embodiment illustrated in Figures 1A-1C, the drug molecule is 4-OHT. In the absence of the drug molecule, the hsp90 binds to the EBD and the CAR is in the "off" state (Figure 1A). When the drug molecule enters the cell, it actively acts to outcompete the hsp90 in binding to the EBD and / or causes a conformational change, and when the CAR binds to the antigen, an activation signal is transmitted by the CAR (Figures 1B, 1C).

[0059] Figures 2A and 2B show representative schematics of CARs that can be used to implement various aspects of the present disclosure. As shown in Figure 2A, the CAR includes a ligand binding domain, a spacer, a transmembrane domain, a signaling domain, and an estrogen binding domain (EBD). The CAR may further include a selection / transduction marker. Figure 2B shows a specific CAR, designated 806-ERT2 CAR. This CAR incorporates an IgG4 hinge between the EGFR806 Vh / Vl scFv and the transmembrane region. In this CAR, a Gly3 linker consisting of three glycines links the signaling domain to the ERT2 EBD. The ERT2 domain is followed by P2A-DHFRdm as a selection marker and T2A-CD19t as a transduction marker. Other CARs disclosed herein include a Gly2 linker or a Gly1 linker (such linkers are also referred to herein as linking amino acids) instead of a Gly3 linker, and still other CARs do not include a linker between the signaling domain and the EBD. Furthermore, the EBD may be included within the signaling domain (e.g., between 4-1BB and CD3ζ), between the 5' end of the signaling domain and the 3' end of the transmembrane domain, or in a portion located 5' of the transmembrane domain within the intracellular space. In a particular example, the ligand binding domain is an scFv that binds to B7H3.

[0060] The hsp90 binding domain may be derived from a binding domain that binds cortisol, androgen, progesterone, or aldosterone. Additionally, the hsp90 binding domain may be derived from a variety of other proteins that bind to hsp90, such proteins being commonly referred to as clients of hsp90. Clients of hsp90 generally include hormone receptors, transcription factors and kinases, as well as other types of molecules.

[0061] One advantage of the present disclosure is that the activity of a single-chain protein can be controlled without relying on dimerization or multimerization with another protein, and without relying on stabilization / destabilization of the protein, such as by incorporating a degron sequence. As used herein, a "degron sequence" refers to an amino acid sequence that is linked to a fusion protein by recombinant techniques to control protein stability / degradation. The degron sequence is usually linked to the C-terminus of the fusion protein. Examples of degron sequences are described, for example, in US Patent Publication No. 2014 / 0255361, and include RRRG and RRRGN (SEQ ID NO: 306).

[0062] Various aspects of the present disclosure are described in more detail below with further options. Various aspects of the present disclosure are described under the following headings: (i) drug molecules and hsp90 binding domains; (ii) ligand binding domains; (iii) intracellular signaling domains; (iv) transmembrane domains; (v) linkers; (vi) tags and selection markers; (vii) alternative transmembrane receptors; (viii) cells genetically engineered to express activity-inducible fusion proteins; (ix) ex vivo and in vivo cell recombination methods; (x) production of activity-inducible fusion proteins; (xi) recombinant formulations, recombinant-derived formulations and drug compositions; (xii) methods of use; (xiii) kits; (xiv) clients of hsp90; (xv) variants; (xvi) representative embodiments; (xvii) experimental examples shown in Figures 9-17; and (xviii) conclusion. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the present disclosure.

[0063] (i) a drug molecule and an hsp90-binding domain The drug molecule used with the activity-inducing fusion protein disclosed herein can overcome hsp90 in binding to the hsp90-binding domain present on the activity-inducing fusion protein and / or undergo a conformational change that results in intracellular signaling. In certain examples, the binding domain present on the activity-inducing fusion protein is present on the intracellular segment of the CAR. Overcoming hsp90 in binding to the hsp90-binding domain and / or undergoing a conformational change allows the CAR to be activated after antigen binding (see, for example, Figures 8A-9C). In certain examples, the hsp90-binding domain is a hormone-binding domain or a modified version thereof.

[0064] In some embodiments, the drug molecule is a small molecule estrogen analogue, including tamoxifen and its salts and metabolites, as well as compounds having similar structures as described herein.

[0065] Tamoxifen is an estrogen antagonist / partial agonist and is commercially available as an FDA-approved drug. Tamoxifen has a proven safety profile, favorable pharmacokinetic profile, good tissue distribution, and a low partition coefficient between the extracellular space and the cytoplasm. Tamoxifen is often administered orally as a pharmaceutical acceptable salt. For example, tamoxifen citrate (RN 54965-24-1, MW563.643) is indicated for the treatment of metastatic breast cancer and is used as an adjuvant in the treatment of breast cancer in women who have undergone mastectomy and axillary lymph node dissection and breast radiation. Tamoxifen citrate is also indicated for the reduction of breast cancer incidence in women at high risk of developing breast cancer.

[0066] Tamoxifen (CAS RN: 10540-29-1) is also known as 2-(4-((1Z)-1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl-ethanamine or (Z)-2-(p-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethylamine (IUPAC) and has the molecular formula C 26 H 29 It is designated NO and its molecular weight (MW) is 371.52 g / mol.

[0067] Tamoxifen metabolites that may be useful in some of the approaches described herein include the major metabolites N-desmethyltamoxifen (RN 31750-48-8, MW 357.494), 4-hydroxytamoxifen (4-OHT) (RN 68392-35-8, MW 387.52, afimoxifen), and endoxifen. These compounds are known and have been reported by Robinson et al. (Metabolites, pharmacodynamics, and pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer patient. Drug Metab Dispos January 1991 19:36-43). Additional metabolic products useful in some of the approaches described herein include cis-4-hydroxytamoxifen (RN 174592, MW 387.52; afimoxifen, E isomer), described in Crewe et al. (Metabolism of Tamoxifen by recombinant human cytochrome P-450 enzymes: Formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen, Drug Metab Dispos, 30(8): 869-874, 2002, FIG. 1), and 4'-hydroxytamoxifen ((Z)-4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-phenylbut-1-en-2-yl)phenol).

[0068] Compounds having a structure similar to tamoxifen that are useful in some of the approaches described herein include cis-tamoxifen (RN 13002-65-8, MW 371.521), 4-methyl tamoxifen (RN 73717-95-5, MW 385.548), N-desmethyl tamoxifen (RN 31750-48-8, MW 357.494), (Z)-desethyl methyl tamoxifen (RN 15917-50-7, MW 357.494), (E)-desethyl methyl tamoxifen (RN 31750-45-5, MW 357.494), trans-4-hydroxy tamoxifen (RN 68047-06-3, MW 387.52), afimoxifen (RN 68392-35-8, MW 387.52), and tamoxifen-containing cis- or cis-(4-hydroxy) tamoxifen (RN 68392-35-8, MW 387.52). 387.52, 4-hydroxytamoxifen), afimoxifen, E isomer (RN 174592-47-3, MW 387.52), 4-chlorotamoxifen (RN 77588-46-6, MW 405.966), 4-fluorotamoxifen (RN 73617-96-6, MW 389.511), toremifene (RN 89778-26-7, MW 405.966), desethyl tamoxifen (RN 19957-51-8, MW 343.47), (E)-desethyl tamoxifen (RN 97151-10-5, MW 343.47), (Z)-desethyl tamoxifen (RN 97151-11-6, MW 343.47), miproxifen (RN 129612-87-9, MW 429.6), 2-(p-(β-ethyl-α-phenylstyryl)phenoxy)triethylamine (RN 749-86-0, MW 399.575), droloxifene (RN 82413-20-5, MW 387.52), 4-iodo-tamoxifen (RN 116057-68-2, MW 497.413), dihydrotamoxifen (RN 109640-20-2, MW 373.537), (E)-N,N-dimethyl-2-(4-(1-(2-methylphenyl)-2-phenyl-1-butenyl)phenoxy)ethanamine (RN 97150-96-4, MW 385.548), and 4-hydroxytoremifene (RN 110503-62-3, MW421.965); and pharma- ceutically acceptable salts, hydrates or solvates thereof.

[0069] Citrate salts of tamoxifen or compounds having a structure similar to tamoxifen that are useful in some of the approaches described herein include tamoxifen citrate (RN 54965-24-1, MW 563.64), 2-(p-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethylethylamine citrate (RN 7244-97-5, 563.64), (E)-tamoxifen citrate (RN 76487-65-5, MW 563.64), toremifene citrate (RN 89778-27-8, MW 598.088), droloxifene citrate (RN 97752-20-0, MW 563.64), and the like. 579.64), 2-(p-(1,2-bis(p-methoxyphenyl)-1-butenyl)phenoxy)triethylamine citrate (RN 42920-39-8, MW 651.748), 2-(4-(1,2-diphenylethenyl)phenoxy)-N,N-diethyl-ethanamine 2-hydroxy-1,2,3-propanetricarboxylate (RN 40297-42-5, MW 563.643), 2-(p-(α-phenylstyryl)phenoxy)triethylamine citrate (RN 102433-95-4, MW 563.64), 2-(p-(2-(p-methoxyphenyl)-1-phenyl-1-butenyl)phenoxy)triethylamine citrate (1:1) (RN 42824-34-0, MW 637.72), 2-(p-(1-(p-methoxyphenyl)-2-phenylpropenyl)phenoxy)triethylamine citrate (RN 13554-24-0, MW 607.696), 2-(p-(α-(p-methoxyphenyl)styryl)phenoxy)triethylamine citrate monohydrate (RN 13542-71-7, MW 593.669), 2-(p-(p-methoxy-α-phenylphenethyl)phenoxy)triethylamine citrate (RN 16421-72-0, MW 595.685), α-(p-(2-(diethylamino)ethoxy)phenyl)-β-ethyl-p-methoxy-α-phenylphenethyl alcohol citrate (1:1) (RN 35263-93-5, MW 639.737), 1-(p-(2-(diethylamino)ethoxy)phenyl)-2-(p-methoxyphenyl)-1-phenylethanol citrate (MW 611.68), α-p-(2-(diethylamino)ethoxy)phenyl)-β-ethyl-α-(p-hydroxyphenyl)-p-methoxyphenethyl alcohol citrate (RN 35263-96-8, MW 655.737), and 2-(p-(p-methoxy-α-methylphenethyl)phenoxy)-triethylamine citrate (RN 15624-34-7, MW 533.614).

[0070] In certain embodiments, the drug molecule is tamoxifen, 4-OHT, ES8 or CMP8. In certain embodiments, the drug molecule is fulvestrant or raloxifene.

[0071] Representative hormone binding domains include estrogen receptor with at least one mutation that inhibits or blocks binding to endogenous estrogen / estradiol. The protein sequence of this estrogen receptor is shown in FIG. 33 as SEQ ID NO: 1. The point mutation of this estrogen receptor (G521R (SEQ ID NO: 3)) inhibits binding to endogenous estrogen but confers nano-level specificity for tamoxifen metabolite 4-OHT, fulvestrant and other estrogen analogs. This particular embodiment utilizes the G521R mutant binding domain (EBD) of estrogen receptor shown in SEQ ID NO: 4. Also, a particular embodiment utilizes EBD with E353A mutation (SEQ ID NO: 6) and drug molecule ES8 as described in Shi & Koh, Chemistry & Biology 8 (2001) 501-510. In another embodiment, an EBD with two point mutations (L384M and M421G (SEQ ID NO: 9)) or three point mutations (L384M, M421G, and G521R (SEQ ID NO: 11)) described in Gallinari et al., Chemistry & Biology, Vol. 12, 883-893 (2005) can be used with the drug molecule CMP8. Another mutation (G400V, M543A, and L544A (SEQ ID NO: 7)) also blocks binding to estradiol but allows binding to tamoxifen metabolites and other estrogen analogs. Thus, in some embodiments, an EBD with the sequence shown in SEQ ID NO: 13 is used. For additional information regarding estrogen receptor-based drug systems, see Indra et al., Nucleic Acids Research, 1999, Vol. 27, No. 22 (4324-4327) and Giacomello et al., Int. J. Dev. Biol. 45: 833-838 (2001).

[0072] In some embodiments, the effective amount of a drug that enables the activity of a fusion protein in the presence of a physiological event involved is an amount that increases the activity of the fusion protein compared to the uninduced and / or basal activity. For example, in some embodiments, the effective amount of a drug that enables the activity of a CAR in the presence of antigen binding is an amount that increases the activity of the CAR compared to the uninduced and / or basal activity. In another embodiment, an effective amount of a drug enables stimulatory, co-stimulatory, or inhibitory immune signaling activity in the presence of ligand binding compared to the uninduced and / or basal activity. Such an effective amount can be determined using known drug doses and pharmacokinetic profiles.

[0073] Furthermore, in addition to the drugs exemplified herein, other drugs may be selected based on proven safety, favorable pharmacokinetic profile, tissue distribution, low partition coefficient between the extracellular space and the cytoplasm, and / or low toxicity.

[0074] (ii) Ligand-binding domain In certain embodiments, the extracellular ligand-binding domain is a molecule capable of specifically binding to a target antigen. Exemplary ligand-binding domains include antibodies or binding fragments thereof, receptors (e.g., T cell receptors), and ligands for receptors (e.g., cytokines and chemokines).

[0075] As understood by those skilled in the art, a full-length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a variable region, a first constant region, a second constant region, and a third constant region, and each light chain comprises a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, or μ chains, and mammalian light chains are classified as λ or κ chains. Immunoglobulins containing α, δ, ε, γ, or μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, or IgM, respectively. Full-length antibodies are "Y" shaped. The stem of a Y-shaped full-length antibody is composed of the second and third constant regions (and a fourth constant region in IgE and IgM) of each of the two heavy chains linked together, and a disulfide bond (interchain bond) is formed at the hinge. The gamma, alpha and delta heavy chains have a constant region consisting of three immunoglobulin domains linked in tandem (in series) and a hinge region that provides flexibility; the mu and epsilon heavy chains have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain", respectively. Each arm of a full-length Y-shaped antibody is composed of the variable and first constant regions of one heavy chain linked to one light chain consisting of a variable and constant region. The light and heavy chain variable regions are responsible for binding to antigens.

[0076] Light and heavy chain variable regions contain a "framework" region flanked by three hypervariable regions, also called "complementarity determining regions" or "CDRs."CDR combinations may be numbered as described, for example, by Kabat numbering (Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) ("Kabat" numbering scheme); Chothia (Al-Lazikani et al. (1997) JMB 273:927-948 ("Chothia" numbering scheme)); Martin (Abinandan et al. (2008) Mol Immunol. 45:3832-3839 ("Martin" numbering scheme)); Gelfand (Gelfand and Kister (1995) Proc Natl Acad Sci USA. 92:10884-10888; Gelfand et al. (1998) Protein Eng. 11:1015-1025; Gelfand et al. (1996) Proc Natl Acad Sci USA. 93:3675-3678; Gelfand et al. (1998) J Comput Biol. 5:467-477 ("Gelfand" numbering scheme);Contact (MacCallum et al. (1996) J. Mol. Biol. 262:732-745 (Contact numbering scheme);IMGT (Lefranc et al. (2003) Dev Comp Immunol 27(1):55-77 ("IMGT" numbering scheme);AHo (Honegger and Pluckthun (2001) J Mol Biol 309(3):657-670 ("AHo" numbering scheme);North (North et al. (2011) J Mol Biol. 406(2):228-256 (the "North" numbering scheme); or other numbering schemes.

[0077] Various software programs and bioinformatics tools can be used to determine the CDR sequences. Examples of such software programs and bioinformatics tools include ABodyBuilder (Leem et al. (2016) MAbs 8(7):1259-1268), PIGSPro (Lepore et al. (2017) Nucleic Acids Res 45(W1):W17-W23), Kotai Antibody Builder (Yamashita et al. (2014) Bioinformatics 30(22):3279-3280), Rosetta Antibody (Weitzner et al. (2017) Nature Protocols 12:401-416), Paratome (Kunik et al. (2012) Nucleic Acids Res 40: W521-W524), Antibody i-Patch (Krawczyk et al. (2013) Protein Eng Des Sel 26(10):621-629), and proABC-2 (Ambrosetti et al. (2020) Bioinformatics 36(20):5107-5108).

[0078] The sequences of the framework regions of various light or heavy chains are relatively conserved among species, including humans. The framework region of an antibody is a combination of multiple framework regions in the light and heavy chains that make up the antibody, and is responsible for positioning and aligning the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to the epitope of an antigen. The CDRs of each chain are generally numbered CDR1, CDR2, and CDR3, starting from the N-terminus, and are generally identified by the chain in which the CDR is located. Thus, the CDRs present in the variable domain of the heavy chain of an antibody are called CDRH1, CDRH2, and CDRH3, and the CDRs present in the variable domain of the light chain of an antibody are called CDRL1, CDRL2, and CDRL3. If the specificity of an antibody differs (i.e., if the binding sites for various antigens are different), the CDRs that the antibody has will also differ. Although it is the CDRs that differ from antibody to antibody, only a few amino acid positions within each CDR are directly involved in binding to the antigen. These amino acid positions within the CDRs are called specificity determining residues (SDRs).

[0079] "V H " or "VH" refers to the variable region of an immunoglobulin heavy chain. L " or "VL" refers to the variable region of an immunoglobulin light chain.

[0080] Antibodies that specifically bind to cell surface molecules can be produced using methods to obtain monoclonal antibodies, phage display methods, methods to make human or humanized antibodies, or methods using transgenic animals or plants genetically engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available, which can be screened for antibodies or fragments thereof that can bind to target molecules. Phage display libraries of human antibodies are also available. Once the amino acid or polynucleotide sequences encoding the antibodies have been identified, these sequences can be isolated and / or sequenced. Many related antibodies are known and commercially available.

[0081] In some embodiments, the antibodies specifically bind to a surface molecule of a cancer cell or a virally infected cell and do not cross-react with non-specific components such as bovine serum albumin or other unrelated antigens.

[0082] An "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically bind to an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments, single chain variable (scFv) antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments containing a VH domain and a CH1 constant domain, linear antibodies, single domain antibodies (e.g., sdAb (VL or VH)), camelid heavy chain variable domains only (VHH), multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment containing two Fab fragments linked at the hinge region by a disulfide bridge), and isolated CDR or other epitope-binding fragments of antibodies (Harlow et al., 1999 in Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989 in Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). The antigen-binding fragment can also be incorporated into a single domain antibody, maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR or bis-scFv (see, e.g., Hollinger and Hudson (2005) Nature Biotechnology 23:1126-1136).

[0083] In certain embodiments, the binding domain may comprise a humanized form of a non-human (e.g., murine) antibody or an antigen-binding fragment thereof. Humanized antibodies include antibodies in which one or more human immunoglobulin constant framework and variable regions are fused to the binding region (e.g., CDRs) of an animal (non-human) immunoglobulin. Such humanized antibodies are designed to avoid immune responses against the non-human antibody from which the binding region is derived while retaining the binding specificity of the non-human antibody. In certain embodiments, the binding domain may comprise a fully human antibody or antibody fragment thereof, where the entire molecule is of human origin or contains amino acid sequences identical to the human form of the antibody or immunoglobulin.

[0084] "scFv" refers to a recombinant fusion protein that comprises an antibody-derived VH and VL linked by a linker and can be expressed as a single-chain polypeptide. An scFv retains the specificity of the original intact antibody. In certain embodiments, the linker linking the variable regions may comprise a glycine serine linker, such as the glycine serine linkers shown in SEQ ID NOs: 72-75 or the glycine serine linkers described elsewhere herein. In certain embodiments, an scFv may comprise a VL variable region and a VH variable region in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, and an scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0085] There are various methods for identifying and selecting a particular TCR for use in a CAR, for example, many TCR sequences that bind to specific antigen fragments are known and publicly available.

[0086] A TCR for use with a particular antigen can be identified, for example, by isolating T cells that bind to a particular antigen / MHC complex and determining the sequence of the TCR chain that binds to the antigen / MHC complex. The TCR gene encoding the TCR can be readily cloned, for example, by 5' RACE using primers corresponding to sequences specific to the TCR alpha and beta chain genes.

[0087] In certain embodiments, it may be necessary to combine the TCR α and TCR β chains after determining the sequence (i.e., to perform the analysis of the combined TCR chains). If necessary, the TCR chains can be combined using various methods. For example, the TCR chains can be combined in silico using computer-based methods such as immunological gene alignment software available from IMGT, JOINSOLVER, VDJSolver, SoDA or iHMMune-align, or other similar tools for annotating VDJ gene segments. Assays such as PairSEQ® (Adaptive Biotechnologies Corp., Seattle, WA) have also been developed.

[0088] In certain embodiments, the recombinant TCR includes a single chain T cell receptor (scTCR) that includes a Vα / β chain and a Cα / β chain (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ) or a Vα-Cα pair, a Vβ-Cβ pair, or a Vα-Vβ pair, specific for a target of interest (e.g., a peptide-MHC complex).

[0089] Cancer antigens are proteins produced by cancer cells, and viral antigens are proteins produced by virus-infected cells. The ligand-binding domain of the CAR disclosed herein can be selected to bind to cancer antigens or viral antigens. In some embodiments, the cancer antigens or viral antigens are preferentially expressed or overexpressed on cancer cells or infected cells compared to other cells of the same type of tissue. In some embodiments, the cancer antigens or viral antigens are cell surface molecules present on cancer cells or virus-infected cells, and are substantially absent on normal tissues or have their expression restricted to non-essential normal tissues.

[0090] Representative cancer antigens include carcinoembryonic antigen (CEA), prostate-specific antigen, prostate stem cell antigen (PSCA), PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrinB2, CD19, CD20, CD22, CD23, CD123, CS-1, CE7, hB7H3, ROR1, mesothelin, c-Met, GD-2, MAGE A3 TCR, EGFR, EGFRvIII, EphA2, IL13Ra2, L1CAM, oaGD2, GD2, B7H3, CD33, FITC, VAR2CSA, MUC16, PD-L1, ERBB2, folate receptor (FOLR), CD56; glypican 2, disialoganglioside, EpCam, L1-CAM, Lewis Y, WT-1, tyrosinase-related protein 1 (TYRP1 / gp75); GD2, B-cell maturation antigen (BCMA), CD24, SV40 T, carbonic anhydrase IX (CAIX); and CD133. Other examples are known to those of skill in the art.

[0091] Certain embodiments utilize a ligand binding domain that specifically binds to HER2, CE7, hB7H3, EGFR, EGFRvIII, CD19, CD20, CD22, EphA2, IL13Ra2, L1CAM, oaGD2, B7H3, CD33, mesothelin, ROR1, FITC, or VAR2CSA.

[0092] In certain embodiments, the scFvs utilized in accordance with the teachings of the present disclosure include huCD19(G01S) scFv, muCD19(FMC63) scFv, CD20(Leu 16) scFv, CD22(m971) scFv, B7H3(hBRCA84D) scFv, L1CAM(CE7) scFv, EGFR scFv, EGFRVIII(806) scFv, EphA2(2A4) scFv, EpHA2(4H5) scFv, FITC(E2) scFv, GD2(hu3F8) scFv, Her2 (Herceptin) scFv, IL13Ra2(hu08)VlVh scFv, IL13Ra2 hu08 VhV1 scFv, IL13Ra2(hu07)VhV1 scFv, IL13Ra2(hu07)VhVl Representative scFv sequences include oaGD2(8B6) VlVh, ROR1(R12) scFv, CD33(h2H12) VhVl scFv, CD33(h2H12) VlVh scFv, mesothelin (P4) scFv, VAR2CSA(ID1-DBL2Xb) scFv, or IL13Ra2 (IL13 zetakine). See FIG. 33 for sequences of these representative scFvs.

[0093] Binding domains that bind to representative viral antigens can also be used, including coronavirus antigens: spike (S) protein; cytomegalovirus antigens: envelope glycoprotein B and CMV pp65; Epstein-Barr virus antigens: EBV EBNAI, EBV P18, and EBV P23; hepatitis virus antigens: Hepatitis B virus S protein, M protein, and L protein, Hepatitis B virus pre-S antigen, HBCAG DELTA, HBV HBE, Hepatitis C virus RNA, HCV NS3, and HCV NS4; herpes simplex virus antigens: immediate early protein and glycoprotein D; HIV antigens: gene products of the gag gene, pol gene, or env gene, such as HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, Nef protein and reverse transcriptase; influenza virus antigen: hemagglutinin and neuraminidase; Japanese encephalitis virus antigen: E protein, ME protein, ME-NS1 protein, NS1 protein, NS1-NS2A protein and 80% E protein; measles virus antigen: measles virus fusion protein; rabies virus antigen: rabies virus glycoprotein and rabies virus nucleoprotein; respiratory syncytial (RS) virus antigen: RSV fusion protein and M2 protein; retrovirus antigen: VP7sc; rubella virus antigen: E1 protein and E2 protein; and varicella zoster virus antigen: gpI and gpII. For more examples of viral antigens, see Fundamental Virology, Second Edition, eds. Fields, BN and Knipe, DM (Raven Press, New York, 1991).

[0094] In certain embodiments, the binding domain is specific for a ligand on a B cell, and in particular the ligand on a B cell for which the binding domain is specific is CD1d, CD5, CD19, CD20, CD21, CD22, CD23 / FcεRII, CD24, CD25 / IL-2 Rα, CD27 / TNFRSF7, CD32, CD34, CD35, CD38, CD40(TNFRSF5), CD44, CD45, CD45.1, CD45.2, CD54(ICAM-1), CD69, CD72, CD79, CD80, CD84 / SLAMF5, LFA-1, CALLA, BCMA, B cell receptor (BCR), IgM, IgD, B220 / CD45R, C1q R1 / CD93, CD84 / SLAMF5, BAFF R / TNFRSF13C, B220 / CD45R, B7-1 / CD80, B7-2 / CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM / TNFRSF14, BLIMP1 / PRDM1, CXCR4, DEP-1 / CD148 or EMMPRIN / CD147.

[0095] The binding domain of the CAR can also bind to other immune cell antigens found, for example, on natural killer T (NKT) cells, natural killer cells (also known as K cells or killer cells), tumor infiltrating lymphocytes (TIL), bone marrow infiltrating lymphocytes (MIL), MAIT cells, macrophages, monocytes, and / or dendritic cells. These cells and representative cell surface antigens are described elsewhere herein.

[0096] The binding domains described herein can also bind to haptens. A hapten can be any small molecule that, when combined with a larger carrier, such as a protein, induces the production of antibodies that specifically bind to the hapten (either free or bound to the carrier). Haptens include peptides, other larger chemical entities, and aptamers. In some embodiments, the hapten can be any of the haptens provided in the database of haptens available on the World Wide Web at the following URL: crdd.osdd.net / raghava / haptendb / .

[0097] In some embodiments, the hapten is fluorescein, urushiol, quinone, biotin, or dinitrophenol and / or derivatives thereof. In certain embodiments, the hapten is 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);nicotine (3-(1-methyl-2-pyrrolidyl)pyridine;black leaf;morphine (morphine;morphine sulfate);2,4-dinitrochlorobenzene (1-chloro-2,4-dinitrobenzene;DNCB;dinitrochlorobenzene);4-chloro-6-(ethylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid);structurally related s-triazines (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));Ametryne (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine);Deethylatrazine (DEA) (structurally related s-triazines);Deisopropylatrazine (DIA) (structurally related s-triazines);Deethyldeisopropylatrazine (DEDIA) (structurally related s-triazines);Deethyldeisopropylatrazine (DEDIA) (structurally related s-triazines);Hydroxyatrazine (HA) (structurally related s-triazines);Deisopropylhydroxyatrazine 2-Hydroxyatrazine (atrazine derivative);2-Hydroxypropazine (structurally related s-triazines);2-Hydroxysimazine;N-(4-Amino-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (modified: R 1 = NH2, R 2 = NH(CH2)3COOH, R 3= OH);Sulcofuron;5-Chloro-2-{4-chloro-2-[3-(3,4-dichlorophenyl)ureido]phenoxy}benzenesulfonic acid;Flucofuron (1,3-bis(4-chloro-α,α,α-trifluoro-m-tolyl)urea);Agatharesinol;Secuirin C;Sugiresinol;Hydroxysugiresinol;Hinokiresinol;Coniferyl alcohol;Cinnamyl alcohol;p-Coumaric acid;Cinnamic acid;p-Coumaric acid;Cinnamic acid;Hinokinin;Guaiacylglycerol-β-guaiacyl ether;Morphine-3-glucuronide (M3 G);Codeine;Norcodeine;6-Monoacetylmorphine;(+)-Methamphetamine;Ceftazidime;Phenobarbital;p-Hydroxyphenobarbital;p-Aminophenobarbital;Cyclobarbital;3'-Ketocyclobarbital;3'-Hydroxycyclobarbital;Secobarbital;Barbital;Methalbital;Barbituric acid;Thiopental;Thiobarbituric acid;Primidone;Gluthethimide;Pentobarbital;Heroin;Diacetylmorphine;Levalorphan;L-11-Allyl-1,2,3,9,10 ,10a-Hexahydro-4H-10,4a-iminoethanophenanthren-6-ol;Pethidine (Demerol;Drantin;Meperidine;1-methyl-4-phenylpiperidine-4-carboxylate ethyl;Isonipecaine);Methamphetamine;d-Desoxyephedrine;Methedrine;Tolupropamine;Pratalgin;Pragman;Benzoylecgonine;3-Carboxymethylmorphine;Cocaine;5-Benzimidazole carboxylic 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;desoxymetasone;triamcinolone;9α-fluoro-11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20-dione;fluocortolone;6α-fluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione;cortisol;11β,17,21-Trihydroxypregn-4-ene-3,20-dione;Prednisone;17,21-Dihydroxypregn-4-ene-3,11,20-trione;Methylprednisolone;11β,17,21-Trihydroxy-6α-methylpregna-1,4-diene-3,20-dione;Triamcinolone hexacetonide;21-(3,3-Dimethyl-1-oxobutoxy)-9α-fluoro-11-hydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione;Carbofuran;Me 2,3-Dihydro-2,2-dimethyl-7-benzofuranyl ethylcarbamate;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;Propoxur;Aldicarb;Methomyl;Benalaxyl;Methyl N-(phenylacetyl)-N-(2,6-xylyl)-DL-alaninate;Bn-Ba(4-[2-(N-phenylacetyl-N-2,6-xylyl)-DL-alaninate) 4-[2-(N-phenylacetyl-N-2,6-xylyl-DL-alanine;Benalaxyl derivatives;Furalaxyl;Metalaxyl;Acetochlor;Dimetachlor;Metolachlor;2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide;Diethoxyethyl;Benzoylpropethyl;Benzoylpropethyl;2,4,5-Trichlorophenoxyacetic acid;2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide;Diethoxyethyl 2,4,5-Trichlorophenoxyacetic acid;2,4,5-T;Weedone;2,4-Dichlorophenoxybutyric acid (2,4-DB);2,4-DB;Butanoic acid;4-(2,4-dichlorophenoxy)-;Butoxone;Embutone;MCPA;2-Methyl-4-chlorophenoxyacetic acid;Metaxon;Dichlorprop (2,4-DP);1-[(2-chloro)phenylsulfonyl]monoamidosuccinic acid;Chlorsulfuron;Chlorbromuron;Amidosulfuron;Chlorotoluron;Isoproturon;Diuron;Linuron;O-Methyl-O-(4-nitrophenyl)-N-(4-carboxybutyl)-phosphoramidothioate;Parathion methyl;O,O-Dimethyl O-4-nitrophenyl phosphorothioate;Metaphos;Wolfatox;Dimethyl parathion;Metacide;Parathion ethyl;Diethyl p-nitrophenyl thiophosphate;O,O-Diethyl O-(p-nitrophenyl) phosphorothioate;Fenitrothion;O,O-Dimethyl O-4-nitro-m-tolyl phosphorothioate;Fenthion, O,O-Dimethyl O-4-methylthio-m-tolyl phosphorothioate;Bromophos, O-4-bromo-2,5-dichlorophenyl O,O-Dimethyl phosphorothioate;Chlorpyrifos methyl, O,O-Dimethyl O-3,5,6-trichloro-2-pyridyl Phosphorothioates; Parathion methyl oxide; Paraoxon; Phosphoric acid; O,O-Diethyl O-(4-nitrophenyl) ester; Diazinon; O,O-Diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate; Azinphos methyl; Pirimiphos methyl; O-2-Diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate; Methidathion; S-2,3-Dihydro-5-methoxy-2-oxo-1,3,4-thiadiazol-3-ylmethyl O,O-dimethyl phosphorodithioate; Dimethyl chlorothiophosphate; 4-Nitrophenol; p-Nitrophenol; Phenol derivatives (modifications on the benzene ring: R; 1 = OH, R 2 = NO2, R 3 = H, R 4 = CH2COOH, R 5 = H, R 6= H);2-nitrophenol;o-nitrophenol;3-nitrophenol;m-nitrophenol;2,4-dinitrophenol;3,4-dinitrophenol;2,5-dinitrophenol;2,4-dinitro-6-methylphenol;2,3,6-trinitrophenol;2-chlorophenol;4-chloro-3-methylphenol;fenitroxone;3-methyl-4-nitrophenol;nonylphenol;HOM(3-[2-hydroxy-5-nitrobenzylthio]propionic acid;phenol;Delor 103;polychlorinated biphenyls;Delor 104;polychlorinated biphenyls;Delor 105,polychlorinated biphenyls,Delor 106;4,4'-Dichlorobiphenyl, PCBs;2,4,4'-Trichlorobiphenyl;PCBs, 2,4'-;PCBs;2,2'-Dichlorobiphenyl, PCBs;2,4,5-Trichlorobiphenyl, PCBs;3,3',4,4'-Tetrachlorobiphenyl, PCBs;PCBs;2,2',4,4',5,5'-Hexachlorobiphenyl;2-(5-Carboxypentanoylamino)-4,4'-dichlorobiphenyl;Biphenyl derivatives;4-Chlorophenoxyacetic acid;2-Chlorophenoxyacetic acid;DDT, 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane;DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene;p-Chlorophenol;4-Chlorophenol;m-Chlorophenol;3,4-Dichlorophenoxy 3,5-Dichlorophenol;2,3,4-Trichlorophenol;2,3,5-Trichlorophenol;3-Methylindole;3-Methylindole derivatives;4-(3-Methylindol-5-yloxy)butanoic acid;4-(3-Methylindol-5-yloxy)butanoic acid;3-Methylindole derivatives;6-[n-3-Methylindol-5-yloxycarbonyl)amino]hexanoic acid;6-[n-3-Methylindol-5-yloxycarbonyl)amino]hexanoic acid;3-Methylindole derivatives;2-[4-(3-Methylindol-6-yl)but-1-ylsulo]acetic acid;2-[4-(3-Methylindol-6-yl)but-1-ylsulo]acetic acid;3-Methylindole derivatives;4-(3-Methylindol-6-yl-4-oxo)butanoic acid;4-(3-Methylindol-6-yl-4-oxo)butanoic acid;3-Methylindole derivatives;6-(3-Methylindol-7-yloxy)hexanoic acid;6-(3-Methylindol-7-yloxy)hexanoic acid;Indole;Indole-3-carboxylic acid;Indole derivatives: Indole-3-acetic acid;Indole-3-acetic acid;Indole derivatives: Indole-3-propionic acid;Indole-3-propionic acid;Indole derivatives: Indole-3-carbinol, Indole-3-carbinol;Tryptophan;Tryptamine;5-Methoxyindole-3-carboxaldehyde, 5-methoxytryptamine;5-Methoxyindole;6-Methoxyindole;7-Methoxyindole, EB1089 (seocalcitol);EB1 089 (seocalcitol) derivatives;(22E,24E)-des-A,B-24-homo-26,27-dimethyl-8-[(E)-N-(2-carboxyethyl)-carbamoylmethylidene]-cholesta-22,24-dien-25-ol;1α-25-dihydroxyvitamin D3;25(OH)D3, 25-hydroxyvitamin D3, 24R,25(OH)2D3;24R,25-dihydroxyvitamin D3;Vitamin D2, ergocalciferol;Vitamin D3;Cholecalciferol;EB1446;EB1436;EB1445;EB1470;Deethylhydroxyatrazine (DEHA) (structurally related s-triazines);Irgarol 1051;Fluorescein isothiocyanate;FITC, metanephrine, normetanephrine;Propazine; Terbuthylazine; 6-chloro-N-(1,1-dimethylethyl)-N'-ethyl-1,3,5-triazine-2,4-diamine; (structurally related s-triazines); Ametryne (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine (modifications: iPr / SCH3 / Et, R 1 = (CH3)2-CH-NH-, R 2 = -SCH3, R 3 = -NH-CH2-CH3; Irgarol; Cyanazine (Modification: R 1 = Cl, R 2 = NHCH2CH3, R3 = NHCCN(CH3)2);OH-Terbuthylazine;Terbuthylazine-2OH;Hydroxytriazine (EQ-0027);Deisopropylatrazine (structurally related s-triazines);Desethylterbuthylazine (structurally related s-triazines);Desethyl-deisopropylatrazine (structurally related s-triazines);Atratone;Terbutryn (structurally related s-triazines);Atrazine derivatives (modified: R 1 = -NHCH(CH3)2, R 2 = -S(CH2)2COOH, R 3 = -NHC2H5); cyanuric chloride; trifluralin; (structurally related s-triazines) tBu / C4 / SCH3 (modification: R 1 = -NH-C-(CH3)3, R 2 = -NH(CH2)3COOH, R 3 = -SCH3; sulfamethazine; (structurally related s-triazines) 6-[[[4-chloro-6-(methylamino)]-1,3,5-triazin-2-yl]amino]hexanoic acid (modifications: Me / Cl / C6, R 1 = -NHCH3, R 2 = -Cl, R 3 = -NH(CH2)5COOH); (Structurally related s-triazines) Procyadin (Modification: R 1 = -Cl, R 2 = -NHcyclopropyl, R 3 = -NHCCN(CH3)2); (structurally related s-triazines); Prometon (modification: R 1 = -OCH3, R 2 = -NHCH(CH3)2, R 3 = -NHCH(CH3)2); (Structurally related s-triazines) Atrazine mercapturic acid (AM) (Modification: R 1 = -SCH2CH(NHAc)COOH, R 2 = -NHCH2CH3, R 3 = -NHCH(CH3)2); (structurally related s-triazines), desethylatrazine mercapturic acid (desethyl AM) (modification: R 1= -NAcCys, R 2 = -NH2, R 3 = -NHCH(CH3)2); (structurally related s-triazines); Deisopropylatrazinemercapturic acid (Deisopropyl AM) (modification: R 1 = -NAcCys, R 2 = -NHCH2CH3, R 3 = -NH2); (structurally related s-triazines); didealkylated atrazine mercapturic acid (didealkylated AM) (modification: R 1 = -NAcCys, R 2 = -NH2, R 3 = -NH2); (structurally related s-triazines); simazine mercapturate (modification: R 1 = -NAcCys, R 2 = -NHCH2CH3, R 3 = -NHCH2CH3); (Structurally related s-triazines) (Modification: R 1 = -S(CH2)2COOH, R 2 = -NHCH2CH3, R 3 = -NHCH2CH3); (Structurally related s-triazines) (Modification: R 1 = -Cl, R 2 = -NHCH(CH3)2, R 3 = -NH(CH2)2COOH); (structurally related s-triazines) (modification: R 1 = -Cl, R 2 = -NHCH2CH3, R 3 = -NH(CH2)2COOH); (structurally related s-triazines); atrazine mercapturic acid methyl ester (AM methyl ester) (modification: R 1 = -NAcCysME, R 2 = -NHCH2CH3, R 3 = -NHCH(CH3)2); N-acetylcysteine; S-benzylmercapturic acid; (structurally related s-triazines); Simetryne (modification: R 1 = -SCH3, R 2 = -NHCH2CH3, R 3= -NHCH2CH3);Metribuzin;4-Amino-6-tert-butyl-4,5-dihydro-3-methylthio-1,2,4-triazin-5-one;Sulfa drugs;N 4 -Acetylsulfamethazine (modification: N 4-Acetylsulfamethazine;Sulfa drugs;Sulfathiazole;Sulfathiazole;Sulfamerazine;Sulfamerazine;Sulfaquinoxaline;Sulfaquinoxaline;Sulfachlorpyridazine;Sulfachlorpyridazine;Sulfapyridine;Sulfadimethoxine;Sulfadimethoxine;Sulfamethoxazole;Sulfamethoxazole;Sulfisoxazole;Sulfisoxazole;Sulfamethizole;Sulfamethizole;Sulfanilamide;Sulfanilamide;Sulfaguanidine;Sulfag anidine;sulfadiazine;sulfadiazine;sulfamethoxypyridiazine;sulfamethoxypyridiazine;pentachlorophenoxypropionic acid;pentachlorophenol;PCP;2,3,5,6-tetrachlorophenol;1,2,4,5-tetrachlorobenzene;2,4,6-trichlorophenol;2-methoxy-3,5,6-trichloropyridine;1,3,5-trichlorobenzene;1,3-dichlorobenzene;2,4,5-trichlorophenol;2,6-dichlorophenol;3,5,6-trichloro-2 -Pyridinoxyacetic acid;3,5,6-trichloro-2-pyridinol;TCP;2,4-Dichlorophenol;2,5-Dichlorophenol;DNC;4,4'-Dinitrocarbanilide;(Structurally related s-triazines);Dichloroatrazine;(Structurally related s-triazines);Dichlorosimazine;1-((6-chloropyridin-3-yl)methyl)imidazolidine-2-imine;Pyridine derivatives;6-Chloropyridine-3-carboxylic acid;Nicotinic acid;Pyridine derivatives;N-((6-chloropyridin-3-yl)methyl )-N-Methylacetamide;(6-Chloropyridin-3-yl)-N-methylmethanamine;(6-Chloropyridin-3-yl)methanol;Imidacloprid;1-(6-Chloropyridinmethyl)-N-nitroimidazolidin-2-ylideneamine;Acetamiprid;(E)-N1-[(6-Chloropyridinmethyl)-N2-cyano-N1-methylacetamidine;Nitenpyram;Deltamethrin;1(R)-cis-α(S)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylic acid cyano(3-phenoxyphenyl)methyl ester;DON;Deoxynivalenol;DON derivatives;15-AcDON (15-acetyldeoxynivalenol);DON derivatives;-AcDON (3-acetyldeoxynivalenol);DON derivatives;3,15-DiacDON (3,15-diacetyldeoxynivalenol);DON derivatives;3,7,15-TriacDON (3,7,15-triacetyldeoxynivalenol);NIV (nivalenol);Nivalenol;NIV derivatives;4-AcNIV (fusarenon X);Flutanil;α,α,α-trifluoro-3'-isopropoxy-o-toluanilide;Mepronil;Mebenil; Benodani;24,25(OH)2D3;(24R)-24,25-dihydroxyvitamin D3;24S,25(OH)2D3;24S,25-dihydroxyvitamin D3;25R,26(OH)2D3;25R,26-dihydroxyvitamin D3;25S,26(OH)2D3;25S,26-dihydroxyvitamin D3;1,24,25(OH)3D3;1,24,25-trihydroxyvitamin D3;1,25-lactone;(23S,25R)-1,25(OH)2D3-26,23-lactone;24,25(OH)2-7-DHC;24,25(O H)2-7-dehydrocholesterol;25(OH)D3-3S;25(OH)D3 3-sulfate;24,25(OH)2D3-hemiglutarate derivatives;11α-hemiglutaryloxy-(24R)-24,25-dihydroxyvitamin D3;24,25(OH)2D3-hemiglutarate derivatives;(24R)-24,25-dihydroxyvitamin D3-3-hemiglutarate;24R,25(OH)2D2;24S,25(OH)2D2;25(OH)D2;1,24(OH)2D3;2,3,6-trichlorophenol;Tetrachlorohydroquinone;Pe Pentachloroaniline;Pentachlorobenzene;2,3-Dinitrotoluene;,4-Dinitrotoluene;2,4,5-Trichloronitrobenzene;3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid;2,3,4,6-Tetrachlorophenol;2,4,6-Trichloroanisole;2,4,6-TCA;Pentabromophenol;PBP;2,4,6-Tribromophenol;2,4,6-TBP;2-Bromo-4-chlorophenol;2-B-4-CP, 2,4-Dibromophenol;2,4-DBP;2,6-Dibromophenol;2,6-DBP;4-bromophenol;4-BP;Furosemide;Ampicillin;Amoxicillin;6-aminopenicillanic acid (6-APA);Azlocillin;Bacampicillin;Carbenicillin;Epicillin;Cloxacillin;Dicloxacillin;Methampicillin;Methicillin;Moxalactam;Oxacillin;Penicillin G;Benzylpenicillin;Penicillin V;Phenoxymethylpenicillin;Phenethicillin;Piperacillin;Ticarcillin;Ampicillin hydrolysate; Penicillin G hydrolysate;3-phenoxybenzoic acid (3-PBAc);Chlorpyrifos;Chlorpyrifos derivatives;HClo1;Derivatives synthesized directly from the study chlorpyrifos by replacing the chlorine at position 6 with a 3-mercaptopropanoic acid spacer arm;Chlorpyrifos derivatives;HTCP (modified: the TCP metabolite HTCP was prepared by hydrolysis of the thiophosphate ester of HClo1);Zeatin riboside (trans isomer);Zeatin (trans isomer);N; 6 -(2-Isopentenyl)-adenosine;IPA;N 6-(2-Isopentenyl)-adenine;2-iP;benzyladenine;kinetin;monuron;monolinuron;fenuron;nebulon;propanil;propham;chloropropham;4-chloroaniline;methylurea derivatives;1-(3-carboxypropyl)-3-(4-chlorophenyl)-1-methylurea;methylurea derivatives;1-(5-carboxypentyl)-3-(4-chlorophenyl)-1-methylurea;metobromuron;sennoside B;SB;sennoside B with erythro configuration between C-10 and C-10';sennoside A ( Modified forms: Sennoside A in the threo configuration between C-10 and C-10; Rhein; Emodin; Aloe-emodin; Barbaloin; 1,4-Dihydroxyanthraquinone; Rhaponticin; Gallic acid; Vanillic acid; Caffeic acid; Homogentisic acid; Esculin; Cinnamtannin B1; Baicalin; Naringin hydrate; Wogonin; Wogonin 7-O-β-glucuronide; Curcumin; δ1-Tetrahydrocannabinolic acid; δ1-Tetrahydrocannabinol; (±)-cis-4-aminopermethrin; 3-(4-aminophenoxy)benzyl (±)-cis-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate;Permethrin;trans-permethrin;cis-permethrin;Cypermethrin;Fenothrin;Resmethrin;Cyfluthrin;trans-permethrinic acid esfenvalerate;Fulvalinate;Fenpropathrin;cis-permethrinic acid;4-phenoxybenzoyl alcohol; Diuron derivatives;1-(3-carboxypropyl)-3-(3,4-dichlorophenyl)-1-methylurea;Siduron;Terbuthiuron;Burban;Trifluralin;2,6-Dinitro-N-propyl-N-(2-carboxyethyl)-4-(trifluoromethyl)benzenamine;TR-13;2-Ethyl-7-nitro-1-propyl-5-(trifluoromethyl)-1H-benzimidazole;Benefin;2,6-Dinitro-N-butyl-N-ethyl-4-(trifluoromethyl)benzenamine;TR-2;2,6-Dinitro-N-propyl -4-(Trifluoromethyl)benzenamine;Ethalfluralin;2,6-Dinitro-N-ethyl-N-(2-methyl-2-propenyl)-4-(trifluoromethyl)benzenamine;TR-40;N-(2,6-Dinitro-4-(trifluoromethyl)phenyl)-N-propylpropanamide;TR-15;2-Ethyl-4-nitro-6-(trifluoromethyl)-1H-benzimidazole;TR-3;2,6-Dinitro-4-(trifluoromethyl)benzenamine;TR-6;3-Nitro-5-(trifluoromethyl)-1,2-benzene Benzenediamine;TR-9;5-(trifluoromethyl)-1,2,3-benzenetriamine;TR-21;4-(dipropylamino)-3,5-dinitrobenzoic acid;TR-36M;3-Methoxy-2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine;Oryzalin;3,5-Dinitro-4-(dipropylamino)benzenesulfonamide;Pendimethalin;2,6-Dinitro-N-(1-ethylpropyl)-3,4-dimethylbenzenamine;Pentagalloylglucose;Pyrene;Pyrene-1-carboxaldehyde;Fur Phenanthrene;Benzo(a)pyrene;3,4-benzopyrene;Anthracene;3,4-benzopyrene;Acenaphthene;Fluorene;Chrysene;1,2-benzphenanthrene;Benzo[g,h,i]perylene;Benzo[e]pyrene;Acenaphthylene;Fluoranthene;Benzo(j,k)fluorene;Indeno-1,2,3-cd-pyrene;1,10-(1,2-phenylene)pyrene;Benzo[a]anthracene;1,2-benzanthracene;Benzo(k)fluoranthene;Naphthalene;Benzo[a]fluoranthene;Dibenzo[ah]anthracene;1,2,5,6-Dibenzanthracene;2,3-Diaminonaphthalene;2,6-Dinitroaniline;17-β-Estradiol (ED);Estra-1,3,5(10)-triene-3,17-β-diol;Trifluralin derivatives;2,6-Dinitro-4-trifluoromethylaniline;Trifluralin derivatives;N-(2,6-Dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid;Trifluralin derivatives;N-(2,6-Dinitro-4-trifluoromethylphenyl)-N-methyl-6-aminohexanoic acid;Trifluralin derivatives N-(2,6-dinitro-4-trifluoromethylphenyl)-N-propyl-6-aminohexanoic acid;Trifluralin derivatives;N-(2,6-dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid methyl ester;Trifluralin derivatives;N-(2,6-dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid tert-butyl ester;Benfluralin;Ethalfluralin;Trifluralin derivatives;2,6-Dinitro-4-trifluoromethylphenol;Isopropanol;Aniline;2-Hydroxybenzotri Fluoride; N-propyl-6-aminohexanoic acid; N-methyl-6-aminohexanoic acid; MHPG derivatives; D-MHPG (D-3-methoxy-4-hydroxyphenyl glycol); MHPG derivatives; L-MHPG (L-3-methoxy-4-hydroxyphenyl glycol); MHPG derivatives; DL-MHPG (DL-3-methoxy-4-hydroxyphenyl glycol); mixture of isomers of D-MHPG and L-MHPG; MHPG derivatives; DL-MHPG-SO4 (DL-3-methoxy-4-hydroxyphenyl glycol sulfate), modified form is D-MHP May contain isomeric mixtures of G-SO4 and L-MHPG-SO4;Serotonin;5-HT;5-hydroxydopamine (5-4HDA);3,4-dihydroxyphenylglycol (DOPEG);Dopamine;4-(2-aminoethyl)pyrocatechol;3-hydroxytyramine;3,4-dihydroxyphenethylamine;L-3,4-dihydroxyphenylalanine;L-DOPA;Vanillomandelic acid;DL-VMA;Homovanillic acid;Norepinephrine;DL-NE;D-epinephrine;DE;3-methoxytyramine;MTA;3-methoxytyrosine;MTyr;3,4-Dihydroxymandelic acid;DL-DOMA;3,4-Dihydroxyphenylacetic acid;DOPAC;L-Phenylalanine;Tyramine;p-Tyramine;4-(2-Aminoethyl)phenol;D-Mandelic acid;Homocatechol;Octopamine;DL-Octopamine;Azinphosethyl;S-(3,4-Dihydro-4-oxobenzo[d]-[1,2,3]-triazin-3-ylmethyl)O,O-diethylphosphorodithioate;Phosmet;O,O-Dimethyl S-Phthalimidomethyl phosphorodithioate;Folpet;N-[(trichloromethyl)thio]phthalimide;Tetramethrin;(1-Cyclohexene-1,2-dicarboximido)methyl-2,2-dimethyl-3-(2-methylpropenyl)-cyclopropanecarboxylate;N-(Bromomethyl)phthalimide;N-(Chloromethyl)benzazimide;6-(N-phthalimidoylmethylthio)hexanoic acid (MFH);Br Bromacil derivatives;5-Bromo-3-sec-butyl-6-methyluracil;Bromacil derivatives;5-Bromo-6-(hydroxymethyl)-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinone;Bromacil derivatives;5-Bromo-3-(2-methylpropyl-6-methyl-2,4(1H,3H)-pyrimidinone;Metabolites of Bromacil;Bromacil derivatives;3-Hydroxy-1-methylpropyl-6-methyl-2,4(1H ,3H)-Pyrimidinedione (modified: Bromacil metabolites);Bromacil derivatives;6-Methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified: Bromacil metabolites);Terbacil derivatives;[5-Chloro-3-(1,1-dimethylethyl)-6-(hydroxymethyl)-2,4(1H,3H)-pyrimidinedione;Terbacil;3-tert-butyl-5-chloro-6-methyluracil;Bromacil derivatives Derivatives;Ethyl 5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-1-yl)hexanoate;Bromacil derivatives alkylated at N-1;Bromacil derivatives 5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-1-yl)hexanoic acid (modified:Bromacil derivatives alkylated at N-1);Bromacil derivatives;Bromo-6-(bromomethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified: a bromacil derivative with a methyl group at the 6th position); [5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]-2-carboxylpropanoic acid (modified: a bromacil derivative with a methyl group at the 6th position); 3-[5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]propanoic acid (modified: a bromacil derivative with a methyl group at the 6th position) Bromacil derivatives;5-bromo-1,6-dimethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione, a derivative of bromacil;5-bromo-1-butyl-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione, a derivative of bromacil;Butachlor;N-Butoxymethyl-2-chloro-2',6'-diethylacetanilide;Amidoclor;N-[(acetylamino)methyl]-2-chloro-N-(2,6-diethylphenyl)acetamide;Nicarbazin;4,6 -Dimethyl-2(1H)-pyrimidinone-containing N,N'-bis(4-nitrophenyl) compounds (modified: DNC+HDP);2-Hydroxy-4,6-dimethylpyrimidine;HDP;Imazalil;[1-(β-allyloxy-2,4-dichlorophenethyl)imidazole];Imazalil derivatives;EIT-0073 (modified imazalil with -O(CH2)5-COOH group instead of the original -OCH2CH=CH2 group);Penconazole;(RS)-1-(2,4-dichloro-β-propylphenethyl)-1H-1,2,4-trimethylpyrimidinone; azole;hexaconazole;(RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)hexan-2-ol;propiconazole;cis-trans-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole;diclobutazole;(2RS,3RS)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol;triflumizole;(E)-4-Chloro-α,α,α-trifluoro-N-(1-imidazol-1-yl-2-propoxyethylidene)-o-toluidine;Imazalil derivatives;EIT-0183;Imazalil derivatives;EIT-0180;Imazalil derivatives;EIT-0111;Imazalil derivatives;EIT-0158;Imazalil derivatives;K-240;Chlorothalonil;Tetrachloroisophthalonitrile (Modifications on the benzene ring: R; 1 = CN, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); chlorothalonil derivative 2,4,5,6-tetrachloro-3-cyanobenzamide (modifications on the benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); chlorothalonil derivative 2,5,6-trichloro-4-hydroxyisophthalonitrile (modifications on the benzene ring: R 1 = CN, R 2 = Cl, R 3 = CN, R 4 = OH, R 5 = Cl, R 6 = Cl); 3-carbamyl-2,4,5-trichlorobenzoic acid (modification on the benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = COOR, R 4 = H, R 5 = Cl, R 6 = Cl); pentachloronitrobenzene (modification on the benzene ring: R 1 = NO2, R 2 = Cl, R 3 = Cl, R 4 = Cl, R 5 = Cl, R 6 = Cl); benzene hexachloride; hexachlorobenzene; BHC; lindane (modifications on the benzene ring: R1 = Cl, R 2 = Cl, R 3 = Cl, R 4 = Cl, R 5 = Cl, R 6 = Cl); 2,4,5,6-tetrachlorophenol (modification on the benzene ring: R 1 = OH, R 2 = Cl, R 3 = H, R 4 = Cl, R 5 = Cl, R 6 = Cl); carbaryl derivatives; ethyl carbamate (modification: R 1 = OCONHCH2CH3, R 3 = H);1-Naphthol;1-Naphthaleneacetamide;-(1-Naphthyl)acetamide;Carbaryl derivatives;1-Methylcarbonate (modified: R 1 = OCOOCH3, R 2 = H); carbaryl derivatives; 1-ethyl carbonate (modification: R 1 = OCOOCH2CH3, R 2 = H); the carbaryl derivative 2-ethyl carbonate (modification: R 1 = H, R 2 = OCOOCH2CH3); carbaryl derivatives; 1-ethylthiocarbonate (modification: R 1 = OCOSCH2CH3, R 2 = H); carbaryl derivatives; 2-ethylthiocarbonate (modification: R 1 = H, R 2 = OCOSCH2CH3); naptalam; N-1-naphthylphthalamic acid; Carbaryl derivatives; 3-hydroxycarbaryl (modification: R 1 = OCONHCH3, R 2 = H, R 3 = OH, R 4 = H, R 5 = H); the carbaryl derivative 4-hydroxycarbaryl (modification: R 1 = OCONHCH3, R 2 = H, R 3 = H, R 4 = OH, R5 = H); The carbaryl derivative 5-hydroxycarbaryl (modification: R 1 = OCONHCH3, R 2 = H, R 3 = H, R 4 = H, R 5 = OH); carbaryl derivatives; 1-(5-carboxypentyl)-3-(1-naphthyl)urea (modification: R 1 = NHCONH(CH2)5COOH, R 2 = H); (Structurally related s-triazines adiprothrin; 4-azido-N-isopropyl-6-methylthio-1,3,5-triazin-2-ylamine (modification: R 1 = -SCH3, R 2 = -N3, R 3 = -CH(CH3)2); (structurally related s-triazines); 2-(ethylamino)-4-(methylthio)-6-aminotriazine (modification: R 1 = -SCH3, R 2 = -NH-C2H5, R 3 = -NH2); (Structurally related s-triazines, 2-amino-4-(methylthio)-6-(isopropylamino)triazine (modification: R 1 = -SCH3, R 2 = -NH2, R 3 = -NH-CH(CH3)2); (Structurally related s-triazine, 2-amino-4-methoxy-6-(isopropylamino)triazine (modification: R 1 = -OCH3, R 2 = -NH2, R 3= -NH-CH(CH3)2;TCP derivatives (3,5,6-trichloro-2-pyridinol derivatives);3-(3,5-dichloro-6-hydroxy-2-pyridyl)thiopropanoic acid;p-nitrosuccinilidic acid (PNA-S);PNA-S;PNA-C;p-nitro-cis-1,2-cyclohexanedicarboxylic acid;Nitroaniline derivatives;2-Nitroaniline;o-Nitroaniline;3-Nitroaniline, a nitroaniline derivative;m-Nitroaniline;4-Nitroaniline, a nitroaniline derivative;p-Nitroaniline;Aromatic alcohols;4-Nitrobenzyl alcohol;4-Nitrophenethyl alcohol, an aromatic alcohol;2-Nitrobenzyl alcohol, an aromatic alcohol;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-Carbofuranphenol;Hydroxycarbofuran;Ketocarbofuran;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-anilinate;Furathiocarb;2,3-Dihydro-2,2-dimethyl-7-benzofuranyl 2,4-Dimethyl-5-oxo-6-oxa-3-thia-2,4-diazadecanoate;Carbofuran derivatives;4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]butanoic acid (BFNB) (modification: n=3, X=CH2);Endrin;Nendrin;(1R,4S,4aS,5S,6S,7R,8R,8aR)-1,2,3,4,10,10-Hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7-epoxy-1,4,5,8-dimethanonaphthalene;Heptachlor;1,4,5,6,7,8,8-Heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene;Chlordane;1,2,4,5,6,7,8,8-Octachloro-2,3,3a,4,7,7a-hexahydro-4,7-methanoindene;Endosulfan (modified: α and β forms) isomeric mixture of;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 isomers (modified: 1S,2R isomer, R:Ph); fenvalerate isomers (modified: 1R,2S isomer, R:Ph); fenvalerate isomers (modified: 1R,2R isomer, R:Ph); fenvalerate isomers (modified: 1S,2R / S isomer, R:Ph); fenvalerate isomers (modified: 1R,2R / S isomer, R:Ph); fenvalerate isomers; fenvalerate (modified: 1R / S,2R / S isomer, R:Ph); Thiabendazole;2-(thiazol-4-yl)benzimidazole;Thiabendazole derivatives;5-Hydroxythiabendazole (modified: 5-OH-TBZ);Thiabendazole derivatives;5-NH2-TBZ;Thiabendazole derivatives;Methylbenzimidazole carbamate;Albendazole;Mebendazole;Fenbendazole;Thiabendazole derivatives;2-Succinamidothiabendazole;Thiabendazole derivatives;2-Succinamidothiabendazole;Cambendazole;Fenvalerate haptens;(S)-4-Chloro-α-(1-methylethyl)benzeneacetic acid 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; Benzyl 3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetate;Fenvalerate haptens;3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetic acid;Fenvalerate haptens;Benzyl 6-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]hexanoate; Fenvalerate haptens;6-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]hexanoic acid (fenvalerate haptens);4-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]benzenepropanoic acid;(S)-Fenvalerate acid;(structurally related s-triazines);Atrazine mercapturate (modified: R; 1 = -SCH2CH(NHCOCH3)COOH, R 2 = -NHCH2CH3, R 3= -NHCH(CH3)2;Fenthion hapten;Methyl O-[3-methyl-4-(methylthio)phenyl] N-(3-carboxypropyl) phosphoramidothioate (modified form called hapten B);Fenthion derivatives;Fenthion oxide;Fenthion derivatives;Fenthion oxide;Pirimiphos-ethyl;4-(methylthio)-m-cresol;Chlorpyrifos derivatives;Chlorpyrifos-oxon;Fenchlorphos;O,O-Dimethyl O-2,4,5-Trichlorophenyl phosphorothioate;Trichloronate;O-Ethyl O-2,4,5-Trichlorophenyl ethylphosphonothioate;Diclofenthion;O-2,4-Dichlorophenyl O,O-Diethyl phosphorothioate;Parathion;O,O-Diethyl O-4-nitrophenyl phosphorothioate;Thiophos;Chlorpyrifos derivatives (modified form: synthesis of AR1 has been reported);Chlorpyrifos derivatives;O-Ethyl O-(3,5,6-trichloro-2-pyridyl) O-(3-carboxypropyl) phosphorothioate;(PO);Chlorpyrifos derivative O-Ethyl O-(3,5,6-trichloro-2-pyridyl) N-(5-carboxyethyl) phosphoramidothioate;(PN1)(Modification: Amide bond of thiophosphate reagent);Chlorpyrifos derivative;O-Ethyl O-(3,5,6-trichloro-2-pyridyl) N-(2-Carboxyethyl) phosphoramidothioate;(PN1) (modification: amide bond of appropriate thiophosphate reagent);Triadimefon;(RS)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one;GR151004;(4-[[5-[3-[2-(dimethylamino)ethyl]]-5-benzofuranyl]-3-pyridinyl]acetyl]morpholine dihydrochloride;Diflubenzuron; 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea; (Structurally related s-triazine, SprAAT (modification: R 1 = SCH2CH2COOH, R 2 = NH2, R 3 = NH2); (structurally related s-triazines); SBeAAT (modification: R 1= S(C6H4)COOH, R 2 = NH2, R 3 = NH2); (structurally related s-triazines); SAAT (modification: R 1 = SH, R 2 = NH2, R 3 = NH2); (structurally related s-triazines); CDAT (modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH2); (Structurally related s-triazines, CDET (modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH(CH2CH3); (Structurally related s-triazines CDIT (modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH(CH(CH3)2)); (Structurally related s-triazines); CDDT (Modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH[C(O)CH3]; (Structurally related s-triazines ammeline; OAAT (modification: R 1 = OH, R 2 = NH2, R 3 = NH2); (Structurally related s-triazines, ammelides; OOAT (modification: R 1 = OH, R 2 = OH, R 3 = NH2); (Structurally related s-triazines cyanuric acid; OOOT (modification: R 1 = OH, R 2 = OH, R 3 = OH); (structurally related s-triazines); melamine; AAAT (modification: R 1 = NH2, R 2 = NH2, R 3 = NH2); the structurally related s-triazine N-isopropylammeline; OIAT (modification: R 1 = OH, R2 = NH[CH(CH3)2], R 3 = NH2); the structurally related s-triazine N-ethylammeline; OEAT (modification: R 1 = OH, R 2 = NHCH2CH3, R 3 = NH2); structurally related s-triazines; N-ethyl ammelide; OOET (modification: R 1 = OH, R 2 = OH, R 3 = NHCH2CH3); the structurally related s-triazines cyromazine, CyPAAT (modification: R 1 = NH(C3H5), R 2 = NH2, R 3 = NH2); the structurally related s-triazines diamino-s-triazine; HAAT (modification: R 1 = H, R 2 = NH2, R 3= NH2);PCBs;2,5,3',4'-Tetrachlorobiphenyl (modification: IUPAC no.: 70);PCB 2,4,5,3',4'-Pentachlorobiphenyl (modification: IUPAC no.: 118);PCB 2,2',5,5'-Tetrachlorobiphenyl (modification: IUPAC no.: 52);PCBs;6-[3,3',4'-Trichlorobiphenyl-4-yl)oxy]hexanoic acid;Metolazone;Trade names: Mykrox, Zaroxolyn;Furfuryl benzoate;DDT metabolites;DDA;Paraquat;1,1'-Dimethyl-4,4'-bipyridinium ion;Diethylcarbamazine;THP;2,4,6-Triphenyl-N-(4-hydroxyphenyl)-pyridinium;o-DNCP;Dinitrocarboxyphenol;PCBs;3-Chlorobiphenylol (modification: IUPAC no. 2);PCBs;3,4'-dichlorobiphenyl (modified: IUPAC No. 13);PCBs;3,5-dichlorobiphenyl (modified: IUPAC No. 14);PCBs;3,4,5,3',4'-pentachlorobiphenyl (modified: IUPAC No. 126);2,3,3',4'-tetrachlorobiphenyl (modified: IUPAC No. 56);2',3,4,5-tetrachlorobiphenyl (modified: IUPAC No. 76);3,3',5,5'-tetrachlorobiphenyl (modified: IUPAC No. 80);2,4,5,2',5'-pentachlorobiphenyl (modified: IUPAC No. 101);2,3,3',4,4'-pentachlorobiphenyl (modified: IUPAC No. 105);2,3,6,3',4'-Pentachlorobiphenyl (modified: IUPAC No. 110);3,3',4,5,5'-Pentachlorobiphenyl (modified: IUPAC No. 127);3,4,5,3',4',5'-Hexachlorobiphenyl (modified: IUPAC No. 169);2,3,3',4,4',5-Hexachlorobiphenyl (modified: IUPAC No.156);3,4,3',4'-Tetrabromobiphenyl;3,4,5,3',4',5'-Hexabromobiphenyl;2,4,5,2',4',5'-Hexabromobiphenyl;Dibenzofurans and dioxins;2,3,7,8-Tetrachlorobenzofuran;2,3,7,8-Tetrachlorodibenzo-p-dioxin;3,4',5-Trichloro-4-biphenylol;3,3',5,5'-Tetrachloro-4,4'-biphenyldiol;3,4,3',4'-Tetrachlorodiphenylether;1,2-Dichlorobenzene;1,4-Dichlorobenzene;1,2,4-Trichlorobenzene;3,4-Dichloroaniline;DDT metabolites;4,4'-DDT;4,4'-DDD retronecine;3,4-Dichlorobiphenyl (modified: IUPAC No. 12);3,4,3'-trichlorobiphenyl (modified: IUPAC No. 35);PCBs;3,4,4'-trichlorobiphenyl (modified: IUPAC No. 37);3,4,3',5-tetrachlorobiphenyl (modified: IUPAC No. 78);3,4,3',5'-tetrachlorobiphenyl (modified: IUPAC No. 79);3,4,4',5-tetrachlorobiphenyl (modified: IUPAC No.81);DDT metabolites;p,p'-DDT (modified: p,p'-dichlorodiphenyltrichloroethane);o,p'-DDT (modified: o,p'-dichlorodiphenyltrichloroethane);p,p'-DDE (modified: p,p'-DDE);o,p'-DDE (modified: o,p'-);p,p'-DDD (modified: p,p'-DDD);o,p'-DDD (modified: o,p'-DDD);dichophosphatase 4,4-Dichloro-α-(trichloromethyl)benzhydrol;Ciprazine;6-Chloro-N-cyclopropyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine;Structurally related s-triazines;Dipropetrine;6-(Ethylthio)-N,N'-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine;Triethazine;6-Chloro-N,N,N'-triethyl-1,3,5-triazine-2,4-diamine;6-Hydroxyatrazine;Hexazinone;3-Cyclohexyl-6-dimethylamino-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione;TNT;2,4,6-Trinitrotoluene;Tetraconazole (M14360);1-[2-(2,4-dichlorophenyl)-3- (1,1,2,2-Tetrafluoroethoxy)propyl]-1H-1,2,4-triazole;DTP;2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propanol;Imazaril;Fenarimol;(RS)-2,4'-Dichloro-α-(pyrimidin-5-yl)benzhydryl alcohol;Lupanine metabolites;(+)-Lupanine (Modification: R = H);Lupanine metabolites;(+)-13-Hydroxylupanine (Modification: R = OH);Lupanine metabolites;Hemisuccinate ester of (+)-13-hydroxylupanine (Modification: R = OCO-(CH2)2COOH);Lupanine metabolites;cis-Hexahydrophthalate ester of (+)-13-hydroxylupanine (Modification: R = OCOC6H. 10 COOH); lupanine metabolites; α-isolupanine; lupanine metabolites; hydroxylupanine; sparteine; cysteine; multiflorin; epirupinine; (structurally related s-triazines); cyanazine acid (modified: R 1 = Cl, R 2= NHCH2CH3, R 3 = NHCCOOH(CH3)2); structurally related s-triazines (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)3COOH); structurally related s-triazines (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NHCH2COOH); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)4COOH); norflurazon; 4-chloro-5-(methylamino)-2-[3-(trifluoromethyl)phenyl]-3(2H)-pyridazinone; norflurazon derivatives; desmethylnorflurazon; metofluran; chloro-5-(dimethylamino)-2-[(3-trifluoromethyl)phenyl]-3(2H)-pyridazinone; pyrazone; chloridazone; 5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone (active ingredient); dichlorophenyl-pyridazon; (a structurally related s-triazine, azidoatrazine (modification: R 1 = N3, R 2 = NHCH(CH3)2, R 3 = NHCH2CH3); alachlor, 2-chloro-2',6'-diethyl-N-methoxymethylacetanilide; trichothecolumn (modification: R 1 = H, R 2 = OH, R 3 = H, R 4 = O, R 5= H);DON derivatives;Acetyl-T-2;DON derivatives;T-2 tetrol tetraacetate;Chlorpyrifos derivatives;Monodechloro-CP;Bromophos derivatives;Bromophos methyl;Bromophos derivatives;Bromophos ethyl dicapton;2-Chloro-4-nitrophenyl O,O-dimethyl phosphorothioate;Tetrachlorvinphos;(Z)-2-chloro-1-(2,4,5-trichlorophenyl)vinyl dimethyl phosphate;Triclopyr;3,5,6-Trichloro-2-pyridyloxyacetic acid;Picloram;4-Amino-3,5,6-trichloropyridine-2-carboxylic acid;Formononetin;Biochanin A;5,7-Dihydroxy-4'-methoxyisoflavone (modified: 4'-methyl ether of genistein);Equol;(7-Hydroxy 3-(4'-hydroxyphenyl)-xy-chroman;2'-Methoxyformononetin;Daidzein;7-Hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one;Genistein;Quercetin;3,3',4',5,7-Pentahydroxyflavone;3,5,7,3',4'-Pentahydroxyflavone;matheucinol;Coumestrol;(Structurally related s-triazines);Hydroxysimazine (modified:R 1 = OH, R 2 = NHCH2CH3, R 3 = NHCH2CH3);angustifoline;Alodan;1-Methyl-4-phenyl-4-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;Amorolfine;Fenpropidin;(RS)-1-[3-(4-tert-butylphenyl)-2-methylpropyl]piperidine;(Structurally related s-triazines)(Modification:R 1 = Cl, R 2 = Cl, R 3 = NHCH2CH3); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = Cl, R3 = NHCH(CH3)2); (Structurally related s-triazines) (Modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)5COOH); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = NHCH(CH3)2, R 3 = NHCH2COOH); (structurally related s-triazines) (modification: R 1 = Cl, R 2 = NHCH(CH3)2, R 3 = NH(CH2)5COOH); structurally related s-triazines; cyanazine amide (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NHCCONH2(CH3)2); hydroxycyanadic acid (modification: R 1 = OH, R 2 = NHCH2CH3, R 3 = NHCCOOH(CH3)2); deethylsimazine (modification: R 1 = Cl, R 2 = NH2, R 3= NHCH2CH3);Albendazole sulfoxide;[5-(propylthionyl)-1H-benzimidazol-2-yl]-, methyl ester;Albendazole sulfone;5(6)-Alkylbenzimidazoles;2-Amino-5-(propylthio)benzimidazole;5(6)-Alkylbenzimidazoles;2-Amino-5-(propylsulfonyl)benzimidazole;Oxibendazole;5-Propoxybenzimidazole-2-methylcarbamate;5(6)-Arylbenzimidazoles;Fenbendazole sulfone (modified: sulfone metabolite of fenbendazole);5(6)-Arylbenzimidazoles;4'-Hydroxy Droxifenbendazole;5(6)-Arylbenzimidazoles;Oxfendazole (modification: Oxfendazole is the sulfoxide metabolite of fenbendazole);5(6)-Arylbenzimidazoles;Flubendazole;Benzimidazole metabolites;2-Aminobenzimidazole;Benzimidazole metabolites;5-Aminobenzimidazole;Benzimidazole metabolites;2-Acetylbenzimidazole;Benzophenone;Diphenylmethanone;Phenyl ketone;Diphenyl ketone;Benzoylbenzene;Benzaldehyde;Benzaldehyde;4-Bromo-2,5-dichlorophenol;Acephate;O,S-Dimethyl Acetyl phosphoramidothioate;Methamidophos;O,S-Dimethyl phosphoramidothioate;Dichlorvos;2,2-Dichlorovinyl dimethyl phosphate;Phenthoate;S-α-Ethoxycarbonylbenzyl O,O-Dimethyl phosphorodithioate;EPN;Ethyl p-nitrophenyl thionobenzenephosphonate;Bioresmethrin;(1R,3R)-2,2-Dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate 5-benzyl-3-furylmethyl (modification: the ISO common name for the unseparated isomeric mixture of this substance is resmethrin);Flufenoxuron;1-[4-(2-chloro-α,α,α-trifluoro-p-tolyloxy)-2-fluorophenyl]-3-(2,6-difluorobenzoyl)urea;Amitrole;1H-1,2,4-Triazol-3-ylamine;Molinate;S-Ethyl Azepane-1-carbothioate;Molinate derivatives (modified: S-2-carboxyethyl hexahydroazepine-1-carbothioate); Molinate derivatives (modified: S-5-carboxypentyl hexahydroazepine-1-carbothioate); Molinate derivatives (modified: Molinate sulfone); Molinate derivatives (modified: S-(p-aminobenzyl) hexahydroazepine-1-carbothioate); Molinate derivatives (modified: S-2-(p-aminophenyl)ethyl hexahydroazepine-1-carbothioate); Hexamethyleneimine; Thiobencarb (Bolero); Butyrate (Sutan); EPTC (Eptam); Cycloate (Roneet); Pebulate (Tillam); Vernolate (Vernam); Aflatoxin M1; AFM1 (modified: AFM1); Aflatoxin B1; AFB1 (modified: AFB1); Aflatoxin G1; AFG1 (modified: AFG1; aflatoxin M2; AFM2 (modified: AFM2); aflatoxin B2; AFB2 (modified: AFB2); aflatoxin G2; AFG2 (modified: AFG2); aflatoxin B2α; AFB2α (modified: AFB2α); aflatoxin G2α; AFG2α (modified: AFG2α); KB-6806; 6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl) (modified: R; 1 = NH2, R 2 = CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivatives) (modification: R 1 = NH2, R 2 = CH2CH(CH3)2, R 3 = CH3); Hapten name: KB-6806 (benzimidazole derivative) (modification: R 1 = NH2, R 2 = CH(CH2CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivatives) (modification: R 1 = NHCOCH3, R 2 = CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivatives) (modification: R 1 = H, R 2= CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivatives) (modification: R 1 = NH2, R 2 = CH(CH3)2, R 3 = CH3); KB-6806 (benzimidazole derivatives) (modification: R 1 = NH2, R 2 = CH(CH3)2, R 3 = =N(→O)CH3(N-oxide);KB-6806 (benzimidazole) derivative (modification: R 1 = NH2, R 2 = CH(CH3)2, R 3 = H); KB-6806 (benzimidazole derivatives) (modification: R 1 = NH2, R 2 = CH2CH3, R 3= CH3);Aminopraoxon;Phosphoric acid;O,O-Diethyl O-(4-aminophenyl) ester;Methyl parathion;Phosphorothioic acid;O,O-Diethyl O-(4-nitrophenyl) ester;Diethylphenyl phosphate;Phenylphosphonic acid;O,O-Diethyl ester;Diethyl phosphate;Ethylphosphonic acid;O,O-Diethyl ester;p-Nitrophenyl phosphate;Phosphoric acid;O-(4-nitrophenyl) ester;Folate;Phosphorodithioic acid;O,O-Diethyl S-[(ethylthio)methyl] ester;Ethion;Bis(phosphorodithioic acid);S,S'-Methylene O,O,O',O'-Tetraethyl ester;Carbophenothione;Phosphorodithioic acid;O,O-Diethyl S-[[(4-chlorophenyl)thio]methyl] ester;Disulfoton;Phosphorodithioic acid;O,O-Diethyl S-[(2-ethylthio)ethyl] ester;TS;N-[4-(carboxymethyl)-2-thiazolyl)sulfanilamide;NS;N-(4-nitrophenyl)sulfanilamide;Sulfamoxole;Sulfacetamide;DNP-SL;Spin-labeled dinitrophenyl (modifications: the synthesis of DNP-SL was reported by Balakrishnan et al. (1982); the formula is given in Anglister et al. (1984));β-ecdysone;Benzimidazole derivatives;5(6)-[carboxypentyl)thio]-2-(methoxycarbonyl)amino]-benzimidazole;2-hydroxybiphenyl;H BP;Atrazine caproic acid;Lysophosphatidic acid (LPA);1-Acyl-2-hydroxy-sn-glycero-3-phosphate;Berberine;Palmatine;9-Acetylberberine;Corydalin;Coptisine;Berberubine;8-Oxoverbaine;Papaverine;Berberine derivatives;9-O-Carboxymethylberberine;Phencyclidine;1-(1-Phenylcyclohexyl)piperidine;Methoxychlor;Endosulfan derivatives;4-Oxobutanoic acid, 4-(4,5,6,7,8,8-hexachloro-3a,4,7,7a-tetrahydro-4,7-methano-1H-indenyl-1-oxy);Endosulfan derivatives;4-Oxybutanoic acid, 4-(1,3,4,5,6,7,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindanyl-2-oxy;Endosulfan derivatives (modification: hemisuccinate of endosulfandiol);Triazole derivatives;5-(3-hydroxypropyl)-3-amino-2H-1,2,4-triazole;Triazole derivatives;5-(3-hydroxypropyl)-3-(2-nitrophenylsulfenyl)amino-2H-1,2,4-triazole;Triazole derivatives;3-amino-5-[(3-succinyloxy) propyl]-2H-1,2,4-triazole;Triazole derivatives;3-amino-1,2,4-triazole-5-thiol;Triazole derivatives;3-[(2-nitrophenylsulfenyl)amino-2H-1,2,4-triazole-5-thiol;Triazole derivatives;2H-1,2,4-triazole-5-thiol;Triazole derivatives;4-Methyl-1,2,4-triazole-3-thiol;Triazole derivatives;(1,2,4-triazol-2-yl)acetic acid;1,2,4-triazole;4-Nitrophenyl 4'-carboxymethylphenyl phosphates;triazole derivatives;4-amino-1,2,4-triazole;triazole derivatives;3-acetamido-1H-1,2,4-triazole;triazole derivatives;3-amino-1,2,4-triazole-5-carboxylic acid hemihydrate;triazole derivatives;2-(4-chlorophenyl)-2-(1,2,4-triazol-1-yl)-methylhexanoic acid;succinic acid;imidazole;L-histidine;L-glutamic acid;permethrin derivatives;3-phenoxybenzyl 2,2-Dimethylcyclopropane-1,3-dicarboxylate;3-Phenoxybenzaldehyde;Flucythrinate;Chrysanthemic acid;2,4-Dinitrophenyl;DNP;Thiram haptens;4-[carbodithioate(methyl)-amino]butanoic acid disodium;Thiram haptens;5,11-Dimethyl-6,10-dithioxo-7,9-dithia-5,11-diazadodecanoic acid;Thiram haptens;2-{[(dimethylamino)carbothioyl]sulfanyl}ethanoic acid;Thiram haptens;4-{[(dimethylamino)carbothioyl]sulfanyl}butanoic acid;Thiram haptens;6-{[(dimethylamino)carbothioyl]sulfanyl}hexanoic acid;Thiram haptens;11-{[(dimethylamino)carbothioyl]sulfanyl}undecanoic acid;Thiram haptens;2-{[(dimethylamino)carbothioyl]sulfanyl}ethanoic acid;Thiram;Tetramethylthiuram monosulfide;Tetraethylthiuram disulfide;Dimethyldithiocarbamic acid sodium salt;Dimethyldithiocarbamic acid zinc salt;Diethyldithiocarbamic acid sodium salt;N,N,N',N'-Tetramethylthiourea;Nabam;Zineb;Maneb;Ethylenethiourea;Chlorpyrifos hapten;O,O-Diethyl O-[3,5-dichloro-6-[(2-carboxyethyl)thio]-2-pyridyl] Phosphorothioates;2-Succinamidobenzimidazole;2-Benzimidazolecarbamic acid methyl ester;MBC;Benzimidazole;2-Benzimidazolylurea;Succinamide;Ethyl carbamate;Urea;N-Methylurea;N,N'-Dimethylurea;Brevetoxin PbTx-3;Organophosphorus haptens;O,O-Diethyl O-(5-carboxy-2-fluorophenyl) Phosphorothioates;Chlorpyrifos-ethyl;Anandamide hapten;N-Arachidonyl-7-amino-6-hydroxyheptanoic acid;Anandamide;Arachidonic acid;Docosatetraenoylethanolamide;Dihomo-gamma-linolenylethanolamide;2-Arachidonylglycerol;2-Arachidonylglycerol ether;Stearoylethanolamide;Heptadecanoylethanolamide;Prostaglandin E1;3-Hydroxy-2-(3-hydroxy-1-octenyl)-5-oxocyclopentaneheptanoic acid;Alprostadil;PGE1;Prostaglandin D2;PGD2;Prostaglandin A2;PGA2;Prostaglandin B2;PGB2;Prostaglandin F; 2α ;7-[3,5-dihydroxy-2-(3-hydroxy-1-octenyl)cyclopentyl]-5-heptenoic acid;Dinoprost;PGF 2α ; Prostaglandin F 1α ;PGF 1α ;6-Keto-prostaglandin F 1α ;6-keto-PGF 1α;13,14-dihydro-15-keto-prostaglandin E2;13,14-dihydro-15-keto-PGE2;13,14-dihydro-15-keto-prostaglandin F 2α ;14-dihydro-15-keto-PGF 2α ;5α,7α-dihydroxy-11-ketotetranorpostane-1,16-dioic acid;15-keto-PGF 2α;TXB2;Prostaglandin E2;7-[3-hydroxy-2-(3-hydroxy-1-octenyl)-5-oxocyclopentyl]-5-heptenoic acid;Dinoprostone;PGE2;hCG-α-(59-92)-peptide (34 residues);Paraquat derivatives;Paraquat hexanoate (PQ-h);Monoquat;Diquat;9,10-Dihydro-8a,10a-diazoniaphenanthrene;MPTP;1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine;1,2-Naphthoquinone;N-Acetyl-S-(1,2-dihydroxy- 4-naphthyl)cysteine;N-acetyl-S-(1,4-dihydroxy-2-naphthyl)cysteine;N-acetyl-S-(1,2-dihydroxy-1-hydroxy-1-naphthyl)cysteine;2-chloro-2',6'-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;Alachlor ethanesulfonic acid;Isoproturon hapten;3-(4-isopropylphenyl)-1-carboxypropyl-1-methylurea;Chlorotoluron;3-(3-chloro-p-tolyl)-1,1-dimethylurea;Metoxuron;3-(3-chloro N-(3-chloro-4-methylphenyl)-1,1-dimethylurea;Metamitron;4-Amino-4,5-dihydro-3-methyl-6-phenyl-1,2,4-triazin-5-one;Mecoprop;(RS)-2-(4-chloro-o-tolyloxy)propionic acid;Propyzamide;3,5-Dichloro-N-(1,1-dimethylpropynyl)benzamide;Paraquat dichloride;MCPB;4-(4-chloro-o-tolyloxy)butyric acid;Chlorotoluron hapten;N-(3-chloro-4-methylphenyl)-N-methyl-N-carboxypropylurea;Metsulfuron;Methyl 2-[3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)ureidosulfonyl]benzoate;Captopril haptens;Captopril-4-(maleimidomethyl)-cyclohexane carboxylic acid (MCC);Captopril haptens;Captopril disulfide modified;Mercaptoethanol-MCC;Mercaptoethanol-4-(maleimidomethyl)-cyclohexane carboxylic acid, modified;Captopril haptens;Captopril without MCC;Acreaticid A;Acreaticid B;Solamargine;Solasonine;Solanine -S;Praplin;Solasodine;Cassianin;Tomatine;Lycopersicin;Tomatidine;3-O-β-D-glucopyranosyl-solasodine;O-α-L-rhamnosyl-1(1→2)-3-O-β-D-glucopyranosyl-solasodine;3-O-β-D-galactopyranosyl-solacidine;O-β-D-glucopyranosyl-1(1→3)-3-O-β-D-galactopyranosyl-solasodine;12-hydroxysolamardine;12-hydroxysolasonine;Isoanguivine;Solaberin I;Solaberin II;Xylosyl-β-solamardine;α-solanine ;α-Chaconine;Dioscin;Indole derivatives;β-Indoleacetic acid;2-Bromo-4,6-dinitroaniline;2-Chloro-4,6-dinitroaniline;Tetryl;2,4,6-Trinitrophenyl-n-methylnitramine;Nitramine;Tetralite;Tetryl;2-Amino-4,6-dinitrotoluene;2,4-Dinitroaniline;3,5-Dinitroaniline;2-Amino-4,6-dinitrobenzoic acid;Disperse Blue 79;N-[5-[bis[2-(acetyloxy)ethyl]amino]-2-[(2-bromo-4,6-dinitrophenyl) Azo]-4-ethoxyphenyl]acetamide;1,3-dinitrobenzene;2,6-dinitrotoluene;4-amino-2,6-dinitrotoluene;1,3,5-trinitrobenzene;Nisergoline;Ethylmorphine;7,8-didehydro-4,5-epoxy-3-ethoxy-17-methylmorphinan-6-ol;Dihydromorphine;Dihydrocodeine;Dihydromorphinone;Hydromorphone;Dihydrocodeinone;Hydrocodone;Naltrexone;N-cyclopropylmethyl-14-hydroxydihydromorphinone;Dextromethorphan;(±)-3-Methoxy-17-methylmorphinan;Homatropine;Endorphins and their modified derivatives (type: β-endorphin);Met-enkephalin;DALEA;D-Ala(2)-D-Leu(5)-enkephalinamide;Vincristine;22-Oxovincaleukoblastine;Leulocristine;VCR;LCR;OCT;22-Oxacalcitriol;OCT-3-HG;22-Oxacalcitriol-3-hemiglutarate;24(OH)O CT;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;Benzeneethanamine;PEA;m-Tyramine;o-Tyramine;Dimethoxyphenethylamine;Thymidine glycol monophosphate acid;5,6-dihydroxythymidine monophosphate;thymidine monophosphate;thymidine glycol;thymine glycol;5,6-dihydrothymidine;thymidine;thymine;5-methyluracil;2,4-dihydroxy-5-methylpyrimidine;AMP;Adenosine monophosphate;CMP;Cytidine monophosphate;Carbamazepine;5-carbamoyl-5H-dibenz[b,f]azepine;Neopterin isomers;D-erythroneopterin;Neopterin isomers;L-erythroneopterin;Neopterin Pterin isomers;D-Threonopterin;Biopterin isomers;L-Erythrobiopterin;Biopterin isomers;D-Erythrobiopterin;Biopterin isomers;L-Threonobiopterin;Biopterin isomers;D-Threonobiopterin;Pterin-6-carboxylic acid;C7H5NiO3;Pterin;Thromboxane B2;(5Z,9α,13E,15S)-9,11,15-trihydroxythromboxane-5,13-dien-1-oic acid;15-keto prostaglandin F; 2α;Fumonisin B1;Macrofusin;FB1;Thyroliberin;TRH;Thyrotropin-releasing factor;Thyrotropin-releasing hormone;TRF;Protirelin;Rhopremone;Thyroliberin-OH;TRH-OH;Diketopiperazine;Cyclo(HP);TRH analogs;Methylated TRH;TRH analogs;TRH extended peptide;TRH-Gly;TRH extended peptide;TRH-Gly-Lys-Arg;TRH extended peptide;TRH-Gly-Lys-Arg-Ala;TRH extended peptide;P7(modified:QHPGLRF);TRH extended peptide;P10(modified :SLRQHPGLRF);TRH extension peptide;Ps5(modified:proTRH[178-199]);TRH extension peptide;TRH-Ps5(modified:proTRH[172-199];hypothalamic peptide;LHRH;cyanoginosine LA;cyanoginosine LB;cyanoginosine LR;cyanoginosine LY;cyanoginosine AY;cyanoginosine FR;cyanoginosine YR;Ne-acetyl-lysine-containing peptide;Gly-Lys(Ac)-e-aminocaproic acid(Aca)-Cys;benzoic acid;benzenecarboxylic acid;phenylformic acid;drasilic acid;m -Hydroxybenzoic acid;3-Hydroxybenzoic acid;o-Methoxybenzoic acid;2-Methoxybenzoic acid;o-Toluic acid;2-Methylbenzoic acid;o-Chlorobenzoic acid;2-Chlorobenzoic acid;o-Aminobenzoic acid;2-Aminobenzoic acid;Thiosalicylic acid;2-Mercaptobenzoic acid;o-Sulfhydrylbenzoic acid;Salicylamide;2-Hydroxybenzamide;Saligenin;Saligenol;o-Hydroxybenzyl alcohol;Salicyl alcohol;2-Cyanophenol;2-Hydroxyphenylacetic acid;p-Hydroxybenzoic acid;p-Aminobenzoin acid;4-aminobenzoic acid;vitamin Bx;bacterial vitamin H1;p-toluic acid;p-methylaminobenzoic acid;p-chlorosalicylic acid;4-chloro-2-hydroxybenzoic acid;2,4-dihydroxybenzoic acid;β-resorcylic acid;2,4-dihydroxybenzenecarboxylic acid;BRA;4-aminosalicylic acid;4-amino-2-hydroxybenzoic acid;p-aminosalicylic acid;gentisic acid;2,5-dihydroxybenzoic acid;5-hydroxysalicylic acid;picolinic acid;o-pyridinecarboxylic acid;2-pyridinecarboxylic acid;picolinic acid N-oxide;3-Hydroxypicolinic acid;2-Hydroxynicotinic acid;7-Methylguanine;N; 2 -Carboxymethyl-N 7 -Methylguanine;2-(7-methyl-6-oxo-6,7-dihydro-1H-purin-2-ylamino)acetic acid;7-Methylxanthine;7-Methyluric acid;7-Methyladenine;Guanine;2-Amino-1,7-dihydro-6H-purin-6-one;2-Aminohypoxanthine;Adenine;6-Aminopurine;6-Amino-1H-purine;6-Amino-3H-purine;6-Amino-9H-purine;7-(2-carboxyethyl)guanine;7-CEGua;7-Ethylguanine;2-Amino-7-ethyl-1H-purin-6(7H)-one;7-(2,3-dihydroxypropyl)guanine;2-Amino-7-(2,3-dihydro 2-amino-7-(2-hydroxyethyl)-1H-purin-6(7H)-one; 7-(2-[(2-hydroxyethyl)amino]ethyl)-guanine; 2-amino-7-(2-(2-hydroxyethylamino)ethyl)-1H-purin-6(7H)-one; 7-carboxymethylguanine; 2-(2-amino-6-oxo-1,6-dihydropurin-7-yl)acetic acid; fluorescein; urushiol; quinone; biotin; His tag; FLAG tag; Strep tag; Myc tag; HA tag; Spot tag; or NE tag.

[0098] Representative antibodies having a binding domain that binds to haptens include 3-methylindole antibody; 3F12; 3-methylindole antibody; 4A1G; 3-methylindole antibody; 8F2; 3-methylindole antibody; 8H1; 3-methylindole antibody; fumonisin B1 antibody; 1,2-naphthoquinone antibody; 15-acetyldeoxynivalenol antibody; (2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propanol) antibody (DTP antibody); 22-oxacalci antibody; Triol antibodies (As-1, As-2 and As-3); (24,25(OH)2D3) antibody (Ab11); (24,25(OH)2D3) antibody (Ab3); (24,25(OH)2D3) antibody (Ab3-4); 2,4,5-trichlorophenoxyacetic acid antibody; (2,4,5-trichlorophenoxyacetic acid) antibody; (2,4,6-trichlorophenol) antibody; (2,4,6-trichlorophenol) antibody; 2,4,6-trinitrotoluene (TNT) antibody; 2,4-dichlorophenoxyacetic acid (MAb B5 / C3; E2 / B5; E2 / G2; F6 / C10; and F6 / E5; (2,4-dichlorophenoxyacetic acid) antibody; 2-hydroxybiphenyl antibody; (3,5,6-trichloro-2-pyridinol) antibody (LIB-MC2; LIB-MC3); (3,5,6-trichloro-2-pyridinol) antibody (LIB-MC2 MAb); 3-acetyldeoxynivalenol (3-AcDON) antibody; 3-phenoxybenzoic acid (3-PBAc) antibody; 4-nitrophenol antibody; 4-nitrophenyl 4'-carboxymethylphenylphosphate antibody; 7-(carboxyethyl)guanine (7-CEGua) antibody (antibody group specific for 7-meGua); 7-methylguanine (7-MEGua) antibody; ABA antibody; acephate antibody (antiserum 8377); acetyllysine antibody (mAb AL3D5;AL11;AKL3H6;AKL5C1;Aculaetiside-A antibody;Aflatoxin M1 (AFM1) antibody (mAb A1;N12;R16;FF32);Agatharesinol antibody;Agatharesinol antibody;Amidochlore antibody;Amitrol antibody (1a-BSA antibody);Ampicillin antibody (AMPI I 1D1 and AMPI II 3B5);Anandamide antibody (9C11.C9C;30G8.E6C;7D2.E2b;13C2 MAb;Atrazine antibody;Atrazine antibody;Atrazine antibody;Atrazine antibody;Atrazine antibody;Atrazine antibody;Atrazine antibody;Atrazine antibody (4063-21-1MAb cell line mAb and scAb);Atrazine antibody (4D8 scAb and 6C8 scAb);Atrazine antibody (C193);Atrazine antibody (rabbit / sheep);Atrazine antibody (K4E7);Atrazine antibody (MAb: AM7B2.1);Atrazine antibody (scAb);Atrazine mercapturic acid antibody;(azinphosmethyl) antibody (MAb:LIB-MFH14;LIB-MFH110);Benalaxyl antibody;Benzimidazole carboxylic acid;Benzimidazole antibody (Ab 587);Benzo[a]pyrene antibody;Benzo(a)pyrene antibody (10C10 MAb and 4D5 MAb; (benzoylphenylurea) antibodies (mainly diflubenzuron antibodies); berberine antibodies; β-indoleacetic acid antibodies; biopterin (L-erythro form) antibodies; brevetoxin PbTx-3 antibodies; bromacil antibodies; bromophos antibodies; bromophosethyl antibodies; butachlor antibodies; captopril-MCC antibodies; carbamazepine (CBZ) antibodies; carbaryl antibodies; carbaryl antibodies (LIB-CNH32; LIB-CNH33, LIB-CNH36; LIB-CNH37; LIB-CNH45; LIB-CNA38); carbaryl antibodies (LIB / CNH-3.6 MAb;Carbofuran antibody (LIB-BFNB-52;LIB-BFNB-62;LIB-BFNB-67);Carbofuran antibody (LIB-BFNP21);CDA antibody;CDA antibody (2-[2-chloro-(2,6'-diethyl)acetanilide]butanoic acid);CDA antibody (2-[2-chloro-(2',6'-diethyl)acetanilide]ethanoic acid);CDA antibody (5-(4-chloroacetamido-3,5-diethyl)phenoxypentanoic acid);Ceftazidime antibody;(Chlorodiamino-s-triazine) antibody (CAAT) (PAb1-8);Chlorothalonil antibody;Chlorpyrifos antibody;Chlorpyrifos antibody;Chlorpyrifos antibody (LIB-AR1.1 MAb;LIB-AR1.4 MAb);Chlorpyrifos antibody (LIB-C4);(Chlorpyrifos) antibody (LIB-C4 MAb);Chlorpyrifos antibody (LIB-PN1 MAb); chlorpyrifos antibody (LIB-PN2 MAb);Chlorpyrifos antibody (LIB-PO MAb); Chlorsulfuron antibody; Chlorsulfuron antibody; Chlorotoluron antibody (antiserum); Cyanoginosin LA antibody (mAb 2B2-2; 2B2-7; 2B2-8; 2B2-9; 2B2-10; 2B5-5; 2B5-8; 2B5-14; 2B5-15; 2B5-23); (D-3-Methoxy-4-hydroxyphenylglycol) antibody; DDA antibody; DDT antibody (PAb and MAb); DDT MAb (LIB1-11; LIB5-21; LIB5-25; LIB5-28; LIB5-212; LIB5-51; LIB5-52; LIB5-53); DEC antibody (Diethylcarbamazine antibody); DEHA antibody; (Delor 103) antibody; Deltamethrin antibody; Deltamethrin antibody (Del 01~Del 12 MAb and PAb); deoxynivalenol (DON) antibody; deoxynivalenol (DON) antibody; dexamethasone antibody; dexamethasone antibody; dinitrophenyl (DNP) antibody; spin-labeled dinitrophenyl antibody (AN01-AN12); diuron antibody (MAb: 21; 60; 195; 202; 275; 481; 488; 520); D-MHPG antibody; DNC antibody; EB1089 antibody; ecdysone antibody; endosulfan antibody; esfenvalerate antibody (Ab7588); estradiol antibody; fenitrothion antibody (pAb and mAb); fenpropimorph antibody; fenthion antibody; fenthion antibody; FITC antibody (B13-DEI); flucofuron antibody (F2A8 / 1 / A4B3); flufenoxuron antibody; and (benzoylphenylurea) antibody; formononetin antibody; furosemide antibody (Furo-26 MAb;Furo37 MAb;furo-72 MAb;Furo 73 MAb);GR151004 antibody;hCG-α-peptide antibody (FA36);Hydroxyatrazine antibody (HYB-283-2);Hydroxysimazine antibody;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);Irgarol antibody;Isopentenyladenosine antibody;Isoproturon antibody;Anti-KB-6806 antiserum;(+)lupanin antibody;Lysophosphatidic acid (LPA) antibody;M3G Ab1 and Ab2;M3G Ab1 and Ab2;MBC antibody (anti-2-succinamidobenzimidazole antiserum);metanephrine antibody;(+)methamphetamine antibody;methiocarb antibody (LIB-MXNB31; LIB-MXNB-33; LIB-MXNH14 and LIB-MXNH-15 MAb);metolachlor antibody;metolachlor antibody;metolachlor antibody (MAb 4082-25-4);molinate antibody;monuron antibody;morphine-3-glucuronide (E3 scFv antibody);morphine antibody;morphine antibody;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;Neopterin (D-erythro) antibodies;Nicarbazin antibodies (Nic 6;Nic 7;Nic 8;and Nic 9);Nisergoline antibodies (Nic-1;Nic-2;Nic-3 & BNA-1; BNA-3; norflurazon antibody; normetanephrine antibody; (o-DNCP) antibody; P10 antibody (TRH extended peptide); paraoxon antibody (BD1 and CE3); paraquat antibody; paraquat antibody; parathion-methyl antibody; PCB antibody (antibody to 3,3',4,4'-tetrachlorobiphenyl) MAb S2B1; pentachlorophenol antibody; pentachlorophenol antibody; pentachlorophenol antibody; permethrin antibody (MAb Py-1; Py-3 and Py-4); phencyclidine antibody (Mab 6B5 Fab); phenobarbital antibody; phenobarbital antibody; (p,p'-DDT) antibody (LIB-DDT-35 and LIB-DDT5-52); premethrin antibody (Ab549); propoxur antibody (LIB-PRNP15; LIB-PRNP21; LIB-PRNB21; LIB-PRNB33); prostaglandin E2 antibody; p-tyramine antibody; pyrene antibody; retronecine antibody; retronecine antibody; salicylic acid antibody; sennoside A antibody (MAb 6G8); sennoside B antibody (MAb: 7H12; 5G6; 5C7); simazine antibody; sulfonamide antibody (anti-TS); sulcofuron antibody (S2B5 / 1 / C3); sulfamethazine antibody (21C7);Synephrine antibody;Thiabendazole antibody (antibody 300);Thiabendazole antibody (antibody 430 and antibody 448);Thiram antibody;THHP antibody (7S and 19S);Thromboxane B2 antibody;Thymidine glycol monophosphate antibody (mAb 2.6F.6B.6C);Thyroliberin (TRH) antibody;TNT antibody (AB1 antiserum and AB2 antiserum);Triadimefon antibody;Triazine antibody (AM1B5.1);Triazine antibody (AM5C5.3);Triazine antibody (AM5D1.2);Triazine antibody (AM7B2.1);Triazine antibody (SA5A1.1);Triazine serum (ametrine);Triazine serum (atrazine);Triazine serum (anti-simazine);Triazine serum (anti-simetrine);Triazine antibody;Triazine antibody;Vincristine antibody;Zearalenone antibody;Zeatin riboside antibody;E2 G2 and E4 C2;MAb Fab fragment K411B from K4E7 (isotype IgG2b with kappa light chain); LIB-BFNP23 Mab; MAbs H-7 and H-9 (MAbs against O,O-diethyl OP peptide); MoAb 33A7-1-1; MoAb 33B8-1-1; MoAb 33C3-1-1; MoAb 3C10-1-1 and MoAb 3E17-1-1; MoAb 45D6-5-1; MoAb 45E6-1-1; MoAb 45-1-1; Mutant (GlnL89Glu) of Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain); Mutant (GlnL89Glu / ValH37Ile / GluL3Val) of Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain); MAb Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7 mutant (GlnL89Glu / ValH37Ile / GluL3Val); Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7 mutant (GlnL89Glu / ValH37Ile); Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7 mutant (GlnL89Glu / ValH37Ile); Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7 mutant (GluH50Gln);Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GluH50X); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GlyH100aAla); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (GlyH100aSer); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (HisH95Phe); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (HisH95Tyr); Fab fragment K411B from MAb K4E7 (isotype IgG2b with kappa light chain) mutant (PheL32Leu); MAb Mutant (TrpH33Phe,Tyr,Leu) of Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7; Mutant (Tryl96Phe) of Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7; Mutant (TryL96Phe) of Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7; Mutant (ValH37Ile) of Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7; Mutant (ValH37Ile) of Fab fragment K411B (isotype IgG2b with kappa light chain) from MAb K4E7; P6A7 MAb; PNAS2 6 / 3 56(1)-1-5-1; PNAS2 6 / 3 56(1)-1-5-2; PNAS2 6 / 3 56(1)-1-10-4; PNAS2 6 / 3 56(1)-1-10-5 and PNAS2 6 / 3 56(1)-3-1-5; Alexa Fluor 405 / Cascade Blue dye antibody; Alexa Fluor 488 dye antibody; BODIPY FL dye 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 nitrotyrosine antibody.

[0099] Representative scFvs that bind haptens are shown in Figure 33 and include those in the form of FITCE2 scFv, FITCE2 TyrH133Ala scFv, FITCE2 HisH131Ala scFv, FL(4M5.3) scFv, FL(4D5Flu) scFv, FL(4420) scFv and DNP scFv.

[0100] In certain embodiments, the binding domain may target a small molecule ligand linked to the targeting moiety. In certain embodiments, the small molecule ligand includes folates, DUPA, NK-1R ligand, CAIX ligand, ligand of gamma-glutamyl transpeptidase, NKG2D ligand, or CCK2R ligand, which are all small molecule ligands that specifically bind to cancer cells (i.e., the receptors for these ligands are overexpressed in cancer compared to normal tissue). In certain embodiments, the targeting moiety includes fluorescein, fluorescein isothiocyanate (FITC), NHS, and / or fluorescein. In certain embodiments, the binding domain is specific for the targeting moiety. In certain embodiments, the binding domain includes anti-fluorescein antibody E2 or an antibody fragment thereof.

[0101] (iii) Intracellular signaling domain The intracellular portion of the protein comprises one or more intracellular signaling domains. In certain embodiments, the intracellular signaling domain generates a signal that promotes the immune effector function of the CAR recombinant cell. In certain embodiments, the intracellular signaling domain generates a stimulatory, costimulatory or inhibitory signal in response to binding of a ligand to the recombinant fusion protein that induces stimulatory, costimulatory or inhibitory activity. Examples of immune effector functions include cytolytic and helper activities, including secretion of cytokines. The signal generated by the intracellular signaling domain can also induce proliferation, activation, differentiation, etc. of immune cells.

[0102] A "signaling domain" refers to a functional portion of a protein that regulates cellular activity by transmitting intracellular information through a defined signaling pathway, either by generating a second messenger or by functioning as an effector that responds to a second messenger. A "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a CAR) or a costimulatory molecule with its cognate ligand, which results in a signaling event, such as signaling through the appropriate signaling domain of a CAR or recombinant receptor protein. Stimulation can result in altered expression of a particular molecule.

[0103] The intracellular signaling domain may comprise the entire intracellular region of the signaling domain or a functional fragment thereof. In certain embodiments, the intracellular signaling domain may comprise a primary intracellular signaling domain. In certain embodiments, the primary intracellular signaling domain comprises an intracellular signaling domain derived from a molecule responsible for primary or antigen-dependent stimulation. In certain embodiments, the intracellular signaling domain may comprise an intracellular costimulatory domain.

[0104] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences that contain ITAMs include those derived from CD3zeta, common FcRgamma (FCER1G), FcgammaRIIa, FcRβ (FcεR1b), CD3gamma, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0105] In certain embodiments, the CD3ζ (CD247) stimulatory domain may comprise sufficient amino acid residues from the cytoplasmic domain of the T cell receptor ζ chain or a functional fragment thereof to functionally transmit an initial signal required for activation of the cell. In certain embodiments, the CD3ζ stimulatory domain may comprise a human CD3ζ stimulatory domain or a functional fragment thereof. In certain embodiments, the CD3ζ stimulatory domain comprises the sequence set forth in SEQ ID NO: 121. In certain embodiments, the CD3ζ stimulatory domain is encoded by SEQ ID NO: 124. In certain embodiments, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure to be able to generate a signal under appropriate conditions.

[0106] In certain embodiments, the intracellular signaling domain may comprise an intracellular costimulatory domain. In certain embodiments, the intracellular costimulatory signaling domain comprises an intracellular costimulatory signaling domain derived from a molecule responsible for costimulatory signals or antigen-independent stimulation. In certain embodiments, the intracellular costimulatory signaling domain may be the intracellular portion of a costimulatory molecule. A "costimulatory molecule" is a binding partner present on an immune cell that is recognized by and specifically binds to a costimulatory ligand, thereby mediating the induction of a costimulatory response (e.g., proliferation) by the immune cell. Costimulatory molecules include cell surface molecules that contribute to an efficient immune response, other than antigen receptors and their ligands. Costimulatory molecules may be represented in the TNF receptor protein family, the immunoglobulin-like protein family, the cytokine receptor family, the integrin family, the signaling lymphocyte activation molecule (SLAM protein) family, and the activation-induced NK cell receptor family.Examples of such costimulatory molecules include MHC class I molecules, B-lymphocyte-T-lymphocyte attenuator (BTLA, CD272), Toll ligand receptor, CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS (CD278), BAFFR, HVEM (LIGHTR), ICAM-1, lymphocyte function-associated antigen 1 (LFA-1; CD11a / CD18), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80(KLRF1), NKp30, NKp44, NKp46, CD160( BY55), B7-H3(CD276), CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49d, ITGA 6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, C These include ligands that specifically bind to RTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0107] In certain embodiments, the intracellular costimulatory signaling domain comprises 4-1BB (CD137, TNFRSF9). "4-1BB" refers to a member of the tumor necrosis factor receptor (TNFR) superfamily. In certain embodiments, the costimulatory domain of 4-1BB comprises the costimulatory domain of human 4-1BB or a functional fragment thereof. In certain embodiments, the costimulatory domain of 4-1BB comprises the sequence set forth in SEQ ID NO: 120. In certain embodiments, the costimulatory domain of 4-1BB is encoded by SEQ ID NO: 123.

[0108] In certain embodiments, the intracellular costimulatory signaling domain comprises CD28. CD28 is a T cell specific glycoprotein involved in T cell activation, induction of cell proliferation, production of cytokines and promotion of T cell survival. In certain embodiments, the costimulatory domain of CD28 comprises the costimulatory domain of human CD28 or a functional fragment thereof. In certain embodiments, the costimulatory domain of human CD28 comprises the sequence shown in SEQ ID NO: 180. In certain embodiments, the costimulatory domain of human CD28 is encoded by SEQ ID NO: 182.

[0109] In certain embodiments, the intracellular signaling domain comprises a combination of one or more stimulatory domains and one or more costimulatory domains described herein. In certain embodiments, the intracellular signaling domain comprises a costimulatory domain of 4-1BB and a stimulatory domain of CD3ζ. In certain embodiments, the intracellular signaling domain comprising a costimulatory domain of 4-1BB and a stimulatory domain of CD3ζ is set forth in SEQ ID NO: 130. In certain embodiments, the intracellular signaling domain comprising a costimulatory domain of 4-1BB and a stimulatory domain of CD3ζ is encoded by the sequence set forth in SEQ ID NO: 132 or SEQ ID NO: 131.

[0110] Inhibitory immune cell molecules that can be engineered to contain an intracellular hsp90 binding domain include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD80, CD86, CD160, 2B4, B7-H3 (CD276), B7-H4 (VTCN1), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR-β.

[0111] (iv) Transmembrane domain A fusion protein can be designed to contain a transmembrane domain that, when expressed, links the extracellular portion of the fusion protein with the intracellular portion of the fusion protein The transmembrane domain can tether the protein to the cell membrane. A transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with an extracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids from the extracellular region of the protein from which the transmembrane domain is derived) and / or one or more additional amino acids associated with an intracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids from the intracellular region of the protein from which the transmembrane domain is derived). In certain embodiments, the transmembrane domain may be derived from the same protein as the protein from which the signaling domain, costimulatory domain, or hinge domain is derived. In certain embodiments, the transmembrane domain is derived from a protein other than the protein from which the other domains of the fusion protein are derived, hi certain embodiments, the transmembrane domain can be selected or modified by amino acid substitution to prevent binding to or minimize interaction with the other domains of the fusion protein.

[0112] In certain embodiments, the transmembrane domain has a three-dimensional structure, typically 15-30 amino acids long, that is thermodynamically stable in the cell membrane. The structure of the transmembrane domain can include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof.

[0113] The transmembrane domain may be of natural or recombinant origin. If of natural origin, the transmembrane domain may be of membrane-bound or transmembrane protein origin. In certain embodiments, the transmembrane domain can transmit a signal to the intracellular domain when the target binds to the extracellular ligand-binding domain of the fusion protein. In certain embodiments, the transmembrane domain may include at least the transmembrane region of the α, β or ζ chain of the T cell receptor; CD28; CD27; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; and / or CD154. In certain embodiments, the transmembrane domain is selected from the group consisting of KIRDS2; OX40; CD2; LFA-1; ICOS; 4-1BB; GITR; CD40; BAFFR; HVEM; SLAMF7; NKp80; NKp44; NKp30; NKp46; CD160; CD19; IL2Rβ; IL2Rγ; IL7Ra; ITGA1; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6; VLA-6; CD49f; ITGAD; CD11d; ITGAE; CD103; ITGAL; CD11a; ITGAM; CD11b; ITGAX; CD11c; ITGB1; CD29; ITGB2; CD18; ITGB7; TNFR2; DNAM1; SLAMF4; CD84; CD96; CEACAM1; CRT The transmembrane domain may comprise at least the transmembrane region of AM; Ly9; CD160; PSGL1; CD100; SLAMF6 (NTB-A, Ly108); SLAM; BLAME; SELPLG; LTBR; PAG / Cbp; NKG2D; and / or NKG2C. In certain embodiments, the transmembrane domain may comprise a transmembrane domain derived from the CD28 chain or the CD8 alpha chain. In certain embodiments, the transmembrane domain of CD8 comprises SEQ ID NO: 119 or SEQ ID NO: 128 and / or is encoded by SEQ ID NO: 122.

[0114] In certain embodiments, the majority of the transmembrane domain may comprise hydrophobic residues such as leucine and valine. In certain embodiments, the transmembrane domain may comprise a triplet of phenylalanine, tryptophan and valine at each end. In certain embodiments, the CD28 or CD8 hinge is located on the extracellular side of the transmembrane domain.

[0115] (v) Linker Herein, a linker may be a part of a fusion protein that serves to connect two subcomponents or subdomains of the fusion protein. In certain embodiments, a linker can provide flexibility to various components of a fusion protein. In scFv, a linker that connects the VH and VL of an antibody-derived binding domain is also described above. The linker may further include a spacer region and a linking amino acid. In certain instances, when a more rigid linker is required, a proline-rich linker can be used.

[0116] A spacer region is a type of linker region used to provide appropriate distance from and / or flexibility relative to other linked components.

[0117] In certain embodiments, the length of the spacer region can be customized for individual purposes. For example, the spacer region can be customized according to individual cell markers on the target cell to optimize the recognition and destruction by the cell upon binding of the fusion protein. In certain examples, the length of the spacer can be such that the responsiveness of the CAR-expressing cell upon binding to the antigen is enhanced compared to the absence of the spacer. In certain embodiments, the length of the spacer region can be selected according to the location of the epitope of the cell marker, the affinity of the binding domain for the epitope, and / or the ability of the CAR recombinant cell to destroy the target cell in response to the recognition of the cell marker ex vivo and / or in vivo. The spacer region can induce high expression in the CAR recombinant cell. In certain embodiments, the extracellular spacer region of the CAR can be located between the transmembrane domain and the extracellular binding domain.

[0118] Representative spacers include spacers having 10-250 amino acids, 10-200 amino acids, 10-150 amino acids, 10-100 amino acids, 10-50 amino acids, or 10-25 amino acids. In certain embodiments, the spacer region is 12, 20, 21, 26, 27, 45, or 50 amino acids long. In certain embodiments, a long spacer is greater than 119 amino acids long, a medium length spacer is 13-119 amino acids long, and a short spacer is 10-12 amino acids long.

[0119] In certain embodiments, the spacer region comprises an immunoglobulin hinge region. The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is a human immunoglobulin hinge region. The immunoglobulin hinge region may be an IgG, IgA, IgD, IgE or IgM hinge region. The IgG hinge region may be an IgG1, IgG2, IgG3 or IgG4 hinge region. In certain embodiments, the spacer region may comprise the entire or a portion of the hinge region sequence from IgG1, IgG2, IgG3, IgG4, or IgD, or a combination of the entire or a portion of the hinge region sequence from IgG1, IgG2, IgG3, IgG4, or IgD with the entire or a portion of the CH2 region; the entire or a portion of the CH3 region; or a combination of the entire or a portion of the CH2 region with the entire or a portion of the CH3 region. As used herein, the term "wild-type immunoglobulin hinge region" refers to the amino acid sequence of the hinge located between and linking the CH1 and CH2 domains of the heavy chain in the upper and middle parts of the naturally occurring antibody (in the case of IgG, IgA, and IgD), or the amino acid sequence of the hinge located between and linking the CH1 and CH3 domains of the heavy chain (in the case of IgE and IgM).

[0120] Representative spacers include a spacer consisting of only an IgG4 hinge, a spacer consisting of an IgG4 hinge linked to a CH2 domain and a CH3 domain, or a spacer consisting of an IgG4 hinge linked to a CH3 domain. In certain embodiments, the spacer comprises an IgG4 linker as set forth in SEQ ID NO: 78 or 80. The hinge region can be engineered to prevent undesired structural interactions, such as dimerization with unintended partners. Other examples of hinge regions that can be used in the fusion proteins described herein include hinge regions present in the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, CD7, and the like, which may be wild-type or variants thereof. In certain embodiments, the hinge comprises a CD8α hinge as set forth in SEQ ID NO: 129.

[0121] In certain embodiments, the spacer region comprises the hinge region of the interdomain region (stalk region) of a type II C-type lectin or the hinge region of the stalk region of a cluster of differentiation (CD) molecule. The "stalk region" of a type II C-type lectin or CD molecule refers to a portion of the extracellular domain that is located between the C-type lectin-like domain (CTLD) (e.g., similar to the CTLD of the natural killer cell receptor) and the hydrophobic portion (transmembrane domain) in a type II C-type lectin or CD molecule. For example, the extracellular domain of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, whereas the CTLD corresponds to amino acid residues 61-176, and the stalk region of the human CD94 molecule includes amino acid residues 34-60, and is therefore located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:15, 1999; for a description of other stalk regions, see further Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11, 2002). These type II C-type lectins or CD molecules may further have linking amino acids between the stalk region and the transmembrane domain or between the stalk region and the CTLD. In another example, the 233 amino acid long human NKG2A protein (UniProt ID P26715.1) has a hydrophobic portion (transmembrane domain) consisting of amino acids 71-93 and an extracellular domain consisting of amino acids 94-233. The CTLD of the human NKG2A protein comprises amino acids 119-231, and the stalk region comprises amino acids 99-116, which may be flanked by additional linking amino acids.Other type II C-type lectins or CD molecules, or their extracellular ligand-binding domains, stalk regions and CTLDs, are also known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1; NP 001773.1; AAL65234.1; and CAA04925.1 for sequences and descriptions of human CD23, human CD69, human CD72, human NKG2A and human NKG2D).

[0122] The linking amino acid may be a linker that can be used to link fusion protein domain sequences when it is not necessary and / or desirable to use a spacer to provide distance. In certain embodiments, the linking amino acid is a short amino acid sequence that can be used to link intracellular signaling domains. In certain embodiments, the length of the linking amino acid is 9 amino acids or less.

[0123] The linking amino acid may be a short oligo- or protein linker, and the length of these linkers is preferably 2 to 9 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). In certain embodiments, a glycine-serine doublet may be used as a suitable linking amino acid linker. In certain embodiments, a single amino acid, such as, for example, alanine or glycine, may be used as a suitable linking amino acid. The CAR constructs disclosed herein may further utilize a linker consisting of three glycines (Gly3) as the linking amino acid sequence.

[0124] (vi) Tags and selectable markers In certain embodiments, the fusion protein may contain one or more tags and / or express one or more selectable markers. Representative tags include His tag, Flag tag, Xpress tag, Avi tag, calmodulin-binding peptide (CBP) tag, polyglutamic acid tag, HA tag, Myc tag, Strep tag (previously called STREP® tag or STREP tag II (IBA Institut fur Bioanalytik, Germany); see, e.g., U.S. Patent Publication No. 7,981,632), Softag 1, Softag 3 and V5. See FIG. 33 for representative sequences.

[0125] Binding molecules that specifically bind to the tag sequences disclosed herein to form complexes are commercially available. For example, His tag antibodies are commercially available from manufacturers such as Life Technologies, Pierce Antibodies, GenScript, etc. Flag tag antibodies are commercially available from manufacturers such as Pierce Antibodies, GenScript, Sigma-Aldrich, etc. Xpress tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Life Technologies, GenScript, etc. Avi tag antibodies are commercially available from manufacturers such as Pierce Antibodies, IsBio, Genecopoeia, etc. Calmodulin tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, Pierce Antibodies, etc. HA tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Cell Signal, Abcam, etc. Myc tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, Cell Signal, etc. Strep tag antibodies are commercially available from manufacturers such as Abcam, Iba, Qiagen, etc.

[0126] In certain embodiments, one or more transduction markers can be co-expressed with the fusion protein, for example, using a skipping element or IRES site that allows the transduction marker and other components of the fusion protein to be expressed as separate molecules. Exemplary self-cleaving polypeptides include the 2A peptide (P2A) from porcine teschovirus-1, the 2A peptide (T2A) from Thosea asigna virus, the 2A peptide (E2A) from equine rhinitis A virus, or the 2A peptide (F2A) from foot and mouth disease virus (see, for example, FIG. 33).

[0127] In certain embodiments, the transduction marker may include a cell surface-displayed marker detectable with an antibody capable of binding to the marker and selecting cells bearing the marker. In certain embodiments, the transduction marker may include a magnetically selectable streptavidin-binding peptide (SBP) marker displayed on the cell surface by a truncated low affinity nerve growth receptor (LNGFRF) and one-step selection with streptavidin-labeled magnetic beads (Matheson et al. (2014) PloS one 9(10): e111437); or a truncated human epidermal growth factor receptor (EGFR) (tEGFR; see Wang et al., Blood 118: 1255, 2011).

[0128] In some embodiments, the transduction marker is truncated EGFR (EGFRt), truncated Her2 (Her2), truncated Her2 (Her2tG), truncated CD19 (CD19t) or the transduction marker DHFRdm.

[0129] The transduction marker may comprise a suitable fluorescent protein, including blue fluorescent protein (e.g., BFP, eBFP, eBFP2); cyan fluorescent protein (e.g., eCFP, cerulean, CyPet); green fluorescent protein (e.g., GFP-2, tagGFP, turboGFP, eGFP); orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange); red fluorescent protein (e.g., mKate, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express); yellow fluorescent protein (e.g., YFP, eYFP, Citrine, Venus); and other suitable fluorescent proteins (e.g., firefly luciferase, etc.). In a particular embodiment, the transduction marker comprises the BFP shown in FIG. 33. See also Heim and Tsien (1996) Current Biology, 6(2): 178-182; Yang et al. (1998) Journal of Biological Chemistry, 273(14): 8212-8216; Ai et al. (2007) Biochemistry, 46(20): 5904-5910; and Constantini et al. (2015) Nature Communications, 6(1): 7670.

[0130] (vii) Another transmembrane receptor Additionally, the disclosure includes stimulatory, costimulatory and inhibitory immunoreceptors that have been engineered such that their activity is induced by the presence of an intracellular hsp90 binding domain.

[0131] Stimulatory receptors include, for example, CD3.

[0132] Costimulatory immune cell molecules that can be engineered to contain an intracellular hsp90 binding domain include 4-1BB, OX40, CD40, CD30, CD27, DR3, SLAMF1, ICOS, GITR, CD25, CD28, CD79A, CD79B, CD226, CARD11, DAP10, DAP12, DR3, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, LIGHT, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TIM1, TRIM, Zap70, and PTCH2. Inhibitory immune cell molecules that can be engineered to contain an intracellular hsp90 binding domain include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD80, CD86, CD160, 2B4, B7-H3 (CD276), B7-H4 (VTCN1), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR-β. Particular embodiments include 4-1BB, OX40, CD40, CD30, CD27, DR3, SLAMF1, ICOS, GITR, CD25, CD28, CD79A, CD79B, CD226, CARD11, DAP10, DAP12, DR3, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, LIGHT, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TIM1, TRIM, Zap70, P The EBD includes an EBD linked to an intracellular signaling domain of TCH2, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD80, CD86, CD160, 2B4, B7-H3 (CD276), B7-H4 (VTCN1), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, or TGFR-β.

[0133] (viii) A cell genetically engineered to express the activity-inducible fusion protein. The present disclosure includes cells genetically engineered to express activity-inducible fusion proteins. As used herein, "genetic engineering" or "genetic manipulation" refers to the addition of additional genetic material in the form of DNA or RNA to the complete genetic material in a cell. Also, the terms "genetically engineered cell" and "recombinant cell" are used interchangeably. In certain embodiments, cells genetically engineered to express activity-inducible fusion proteins include effector immune cells. "Effector immune cells" include immune system cells that have one or more effector functions (e.g., cytotoxic / killing activity, secretion of cytokines, induction of antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)). Effector immune cells are a subtype of immune cells.

[0134] Immune cells of the present disclosure may be autologous / autologous cells ("self" cells) or non-autologous cells ("non-self" cells, e.g., allogeneic, syngeneic or xenogeneic cells). An "autologous" cell refers to a cell obtained from the same subject. An "allogeneic" cell refers to a cell that is genetically different from a comparison cell, but originates from the same species. A "syngeneic" cell refers to a cell derived from another subject that is genetically identical to a comparison cell. A "xenogeneic" cell refers to a cell that originates from a different species than a comparison cell. In certain embodiments, recombinant cells of the present disclosure may be autologous or allogeneic cells.

[0135] In certain embodiments, the genetically modified cells comprise lymphocytes. In certain embodiments, the genetically modified cells comprise T cells, B cells, natural killer (NK) cells, monocytes / macrophages, or HSPCs.

[0136] The majority of T cells have a T cell receptor (TCR) that consists of two different peptide chains (α- and β-TCR chains). γδ T cells are a small subset of T cells that have a different type of T cell receptor (TCR) that consists of one γ chain and one δ chain.

[0137] CD3 is expressed on all mature T cells. T cells can be further classified into cytotoxic T cells (CD8+ T cells, also called CTLs) and helper T cells (CD4+ T cells).

[0138] Cytotoxic T cells can destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells recognize their targets by binding to antigens bound to MHC class I molecules, which are present on the surface of almost all cells in the body.

[0139] "Central memory T cells (TCM)" refer to CTLs that have experienced an antigen and, compared to naive cells, express CD62L or CCR7 and CD45RO, but do not express CD45RA or have reduced expression of CD45RA.

[0140] "Effector memory T cells (TEM)" refer to T cells that have experienced an antigen and do not express CD62L or have reduced expression of CD62L compared to central memory cells, and do not express CD45RA or have reduced expression of CD45RA compared to naive cells. In certain embodiments, effector memory cells are negative for expression of CD62L and CCR7, and positive or negative for expression of CD28 and CD45RA compared to naive or central memory cells. Effector T cells are positive for granzyme B and perforin compared to memory or naive T cells.

[0141] Helper T cells support other immune cells through functions such as activating cytotoxic T cells and macrophages and promoting B cell maturation. Helper T cells are activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, helper T cells are responsible for regulating or supporting an active immune response by dividing rapidly and secreting cytokines.

[0142] Natural killer T (NKT) cells are a T cell subset that co-express the αβ T cell receptor and express various molecular markers typically associated with natural killer cells, such as NK1.1 (CD161), CD16 and / or CD56.

[0143] Natural killer cells (also known as K cells and killer cells) express CD8, CD16 and CD56, but not CD3. NK cells also express activating receptors (such as NKp46) and inhibitory receptors (such as NKG2A) that regulate the cytotoxic function of NK cells against tumor cells and virus-infected cells.

[0144] Tumor-infiltrating lymphocytes (TILs) are immune cells that can migrate from the blood into tumors and recognize and kill cancer cells. Bone marrow-infiltrating lymphocytes (MILs) are antigen-experienced immune cells that migrate to the bone marrow and persist there. Mucosal-associated invariant T (MAIT) cells are found in mucosa, blood, and secondary lymphoid organs (SLOs) and are innate immune-like T cells that exhibit an effector phenotype. MAIT cells display a semi-invariant T cell receptor (TCR) and are restricted by the major histocompatibility complex-related molecule MR1.

[0145] Macrophages (and their precursor cells, monocytes) are present in all tissues of the body and phagocytose apoptotic cells, pathogens and other non-self components. Monocytes / macrophages express CD11b, F4 / 80, CD68, CD11c, IL-4Rα and / or CD163.

[0146] Immature dendritic cells (i.e., preactivated dendritic cells) phagocytose peripheral antigens and other non-self components, become activated, migrate to T cell areas of lymphoid tissues, and present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and DEC-205.

[0147] Hematopoietic stem cells (HSCs) refer to undifferentiated hematopoietic cells that are capable of self-renewal and differentiation into all other types of hematopoietic cells. HSCs are CD34+.

[0148] Hematopoietic progenitor cells (HPCs) originate from HSCs and can further differentiate into mature cell types. HPCs are capable of self-renewal or can differentiate into (i) myeloid progenitors that eventually differentiate into monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets or dendritic cells, or (ii) lymphoid progenitors that eventually differentiate into T cells, B cells and NK cells. HPCs express CD24 lo Lin - CD117 + It is.

[0149] HSPC refers to a cell population that includes HSC and HPC. The HSPC cell population may be positive for CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof.

[0150] Induced pluripotent stem cells (iPSCs) refer to a type of pluripotent stem cell that is artificially created from a non-pluripotent cell, usually an adult somatic cell or a terminally differentiated cell, such as a fibroblast, hematopoietic cell, muscle cell, neuron, or epidermal cell, by the introduction or contact of a reprogramming factor.

[0151] (ix) Ex vivo and in vivo cell recombination methods The present disclosure provides methods for harvesting, enriching, culturing, and recombining cells to express activity-inducible fusion proteins (e.g., CARs) ex vivo, and / or for genetically modifying immune cells in vivo using cell-targeted delivery methods.

[0152] In certain embodiments, lymphocytes are isolated from a sample, such as blood or a blood-derived sample, or a sample obtained by apheresis or leukapheresis. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), bone marrow, thymus, cancer tissue, lymphoid tissue, spleen, or other suitable sources.

[0153] Sources of HSPCs include, for example, peripheral blood (see U.S. Pat. No. 5,004,681; U.S. Pat. No. 7,399,633; and U.S. Pat. No. 7,147,626; as well as Craddock, et al., 1997, Blood 90(12):4779-4788; Jin, et al., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292; Papayannopoulou, et al., 1998, Blood 91(7):2231-2239; Tricot, et al., 2008, Haematologica 93(11):1739-1742; and Weaver et al., 2001, Bone Marrow Transplantation 27(2):S23-S29).

[0154] Methods for collecting, anticoagulating, and processing blood samples are described, for example, in Alsever, et al., 1941, NY St. J. Med. 41:126; De Gowin, et al., 1940, J. Am. Med. Ass. 114:850; Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573; Rous and Turner, 1916, J. Exp. Med. 23:219; and Hum, 1968, Storage of Blood, Academic Press, New York, pp. 26-160.

[0155] In certain embodiments, the recovered cells may be washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, or to lyse or remove cells sensitive to a particular reagent. Isolation may include one or more of a variety of cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to a particular reagent, and / or affinity for an antibody or other binding partner (e.g., immunoaffinity).

[0156] In certain embodiments, one or more cell populations enriched, isolated and / or selected from a sample by the methods provided herein are comprised of cells positive for one or more particular markers (e.g., cell surface markers) (e.g., markers + ) or cells that highly express one or more specific markers (markers hi ), or cells negative for one or more markers (markers - ) or cells with relatively low expression of one or more markers (markers lo ).

[0157] In certain embodiments, T cells lyse red blood cells and then bind to, for example, PERCOLL TM Monocytes can be isolated from peripheral blood mononuclear cells (PBMCs) by removing monocytes, such as by centrifugation using a density gradient. In certain embodiments, specific T cell subpopulations expressing CD3, CD28, CD4, CD8, CD45RA and CD45RO are further isolated by positive or negative selection techniques. In certain embodiments, cell sorting and / or selection is performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells to be negatively selected. For example, CD4 +When cells are enriched by negative selection, a monoclonal antibody cocktail can generally be used that includes CD14, CD20, CD11b, CD16, HLA-DR and CD8 antibodies.

[0158] After the cells are isolated and / or enriched, they can be expanded to increase their numbers. In certain embodiments, the cells can be grown using methods such as those described in, for example, U.S. Patent Publication Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; and 7,172,869. T cells can be activated and expanded before or after genetic modification to express a CAR using methods described in U.S. Patent Publication No. 7,232,566; U.S. Patent Publication No. 7,175,843; U.S. Patent Publication No. 5,883,223; U.S. Patent Publication No. 6,905,874; U.S. Patent Publication No. 6,797,514; U.S. Patent Publication No. 6,867,041; or U.S. Patent Publication No. 2006 / 0121005.

[0159] Typically, T cells are expanded by contacting them with a surface bound to an agent that stimulates signals associated with the CD3-TCR complex and a ligand that stimulates costimulatory molecules on the surface of the T cells. In certain embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, e.g., anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in culture medium containing appropriate cytokines (see Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999). In certain embodiments, T cells may be activated and stimulated to expand with feeder cells and appropriate antibodies and cytokines, such as using methods described in U.S. Patent Publication No. 6,040,177; U.S. Patent Publication No. 5,827,642; or WO2012 / 129514.

[0160] In certain embodiments, K562 cells, U937 cells, 721.221 cells, T2 cells or C1R cells can be engineered to generate artificial APCs (aAPCs) that can be used to induce stable expression and secretion of various costimulatory molecules and cytokines. aAPCs are described in WO03 / 057171 and US Patent Publication No. 2003 / 0147869.

[0161] In certain embodiments, HSPCs can be isolated from or differentiated from HSPCs as described, e.g., in U.S. Patent Publication No. 7,399,633; U.S. Patent Publication No. 5,004,681; U.S. Patent Publication No. 2010 / 0183564; WO2006 / 047569; WO2007 / 095594; WO2011 / 127470; or WO2011 / 127472; Vamum-Finney, et al., 1993, Blood 101:1784-1789; Delaney, et al., 2005, Blood 106:2693-2699; Ohishi, et al., 2002, J. Clin. Invest. 110:1165-1174; Delaney, et al., 2010, Nature Med. 16(2): or Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006, or in the references cited therein. The harvesting and processing of other types of cells described herein will be known to those of skill in the art.

[0162] In certain embodiments, the isolation, incubation, growth and / or recombination steps are performed in a sterile or sealed environment and / or in an automated manner, e.g., under the control of a computer linked to the apparatus in which each step is performed. Final formulation of recombinant cells into a recombinant preparation for administration is described elsewhere herein.

[0163] Also, immune cells can be genetically modified in vivo or ex vivo using targeting-imparted viral vectors and / or nanoparticles. Viral vectors that can be used to deliver genes encoding fusion proteins to cells are described elsewhere herein, and many targeting-imparted viral vectors (e.g., pseudotyped viral vectors) are known in the art.

[0164] Representative cell-targeting nanoparticles include nanoparticles with cell-targeting ligands (e.g., CD3, CD4, CD8, CD34) attached to their surface, which are selectively taken up by a selected cell type due to the cell-targeting ligands attached to their surface. The nanoparticles then deliver genetically engineered components to express activity-inducible fusion proteins.

[0165] Representative nanoparticles include liposomes (tiny vesicles with at least one lipid bilayer surrounding an aqueous core forming a concentric sphere), liposomal nanoparticles (liposomal structures used to encapsulate smaller nanoparticles within their cores), and lipid nanoparticles (liposome-like structures that do not have the continuous lipid bilayer characteristic of liposomes). Other polymer-based nanoparticles and porous nanoparticles composed of materials capable of forming a porous network can also be used. Representative materials include metals, transition metals and metalloids (e.g., lithium, magnesium, zinc, aluminum and silica).

[0166] Nanoparticles intended for in vivo delivery and cellular uptake may have an uncharged or negatively charged coating and may be 130 nm or smaller in size. Nanoparticle dimensions can be measured using conventional techniques such as, for example, dynamic light scattering and / or electron microscopy.

[0167] (x) Production of activity-inducing fusion protein The activity-inducible fusion protein according to the present disclosure can be produced by methods known in the art. In certain embodiments, the activity-inducible fusion protein is produced using recombinant DNA technology. Standard molecular cloning techniques can be used to prepare nucleic acids encoding several regions of the activity-inducible fusion protein and construct complete coding sequences. The resulting coding regions can be inserted into expression vectors to transform cells or cell lines.

[0168] The term "gene" (used interchangeably with the terms "polynucleotide" or "nucleotide sequence") refers to a nucleic acid sequence that encodes an activity-inducible fusion protein, a component thereof, or a molecule that is co-expressed with the activity-inducible fusion protein described herein. The definition of this term includes various sequence polymorphisms, mutations and / or variants, where such changes do not substantially affect the function of the encoded protein. The term "gene" may include not only the coding sequence, but also regulatory regions such as promoters, enhancers, and termination regions. Genetic sequences that encode molecules may be DNA or RNA that induce the expression of the activity-inducible fusion protein. These nucleic acid sequences may be DNA strand sequences that are transcribed into RNA or RNA sequences that are translated into proteins.

[0169] "Encode" refers to the property that a particular nucleotide sequence within a gene, such as complementary DNA (cDNA) or messenger RNA (mRNA), serves as a template for the synthesis of another macromolecule, such as a defined amino acid sequence. Thus, a particular gene encodes a protein when its corresponding mRNA is transcribed and translated to produce the protein in a cell or other biological system. A "protein-encoding gene" includes any degenerate nucleotide sequence that encodes the same amino acid sequence or an amino acid sequence having substantially similar form and function.

[0170] Multiple polynucleotide gene sequences encoding two or more portions of the expressed activity-inducing fusion protein can be operably linked to each other and can also be linked to relevant regulatory sequences. For example, there can be a functional association between the regulatory sequence and the exogenous nucleic acid sequence, which allows the exogenous nucleic acid sequence to be expressed. In another example, a first nucleic acid sequence and a second nucleic acid sequence can be operably linked if they are arranged in a functional association. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Usually, operably linked DNA sequences are contiguous, and the coding regions are linked in the same reading frame if the coding regions are necessary or useful.

[0171] In the representative nucleic acid constructs used in the present disclosure, the nucleic acid sequence encoding the activity-inducible fusion protein is operably linked to a promoter, i.e., the nucleic acid sequence encoding the activity-inducible fusion protein and the promoter are arranged so as to promote transcription of mRNA from the DNA encoding the activity-inducible fusion protein. The promoter may be of genomic origin or synthetically produced. The promoter may be associated with an enhancer or may not be associated with an enhancer, and the enhancer may be naturally associated with a particular promoter or may be associated with another promoter. Various promoters for use in cells (e.g., CD4 promoter) are well known in the art. The promoter may be a constitutive promoter or an inducible promoter, for example, induction is associated with a particular type of cell or a particular developmental stage. Alternatively, a number of well-known viral promoters are suitable. Promoters of interest include the Simian Virus 40 (SV40) (e.g. early or late) viral promoter; the Moloney Murine Leukemia Virus (MoMLV) long terminal repeat (LTR) promoter; the Rous Sarcoma Virus (RSV) LTR promoter; the Herpes Simplex Virus (HSV) (thymidine kinase) promoter; the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter; the heat shock protein 70 kDa (HSP70) promoter; the ubiquitin C (UBC) promoter; or the phosphoglycerate kinase 1 (PGK) promoter.

[0172] In certain embodiments, a signal sequence can be used that directs the activity-inducible fusion protein to the cell membrane surface, and this signal sequence may include the endogenous signal sequence of the N-terminal part of the activity-inducible fusion protein. In some cases, it may be desirable to replace this endogenous signal sequence with another signal sequence. Meanwhile, the selected signal sequence should be compatible with the secretory pathway of the cell expressing the activity-inducible fusion protein so that the activity-inducible fusion protein is presented on the cell surface in the cell expressing it. In certain embodiments disclosed herein, the signal peptide of GM-CSF or the signal peptide of CD8 is utilized.

[0173] Similarly, the terminal region may be constituted by the native or endogenous transcriptional terminal region of the nucleic acid sequence encoding the C-terminal portion of the activity-inducing fusion protein. Alternatively, the terminal region may be derived from another source. In most cases, the source of the terminal region will not generally have a significant effect on the expression of the recombinant protein, and a variety of terminal regions can be used without adversely affecting expression.

[0174] As is well understood by those skilled in the art, in some cases, several amino acids at both ends of the binding domain of an activity-inducible fusion protein (e.g., CAR) can be deleted, for example, typically 10 or less amino acids, more generally 5 or less amino acids, can be deleted at both ends of the binding domain of the activity-inducible fusion protein. It may also be desirable to insert several amino acids, typically 10 or less amino acids, more generally 5 or less amino acids, at the boundary of the binding domain. The deletion or insertion of amino acids may be the result of necessity in constructing an activity-inducible fusion protein, and may provide effects such as convenient restriction sites, easy manipulation, and improved expression. In addition, substitution of one or more amino acids with other amino acids may occur for the same reason.

[0175] In any of the embodiments described herein, the polynucleotide may include a sequence encoding a self-cleaving polypeptide between the polynucleotide segment encoding the activity-inducible fusion protein and the polynucleotide encoding a selection marker (e.g., a transduction marker) (e.g., EGFRt, Her2tG, CD19t, or DHFRdm). Exemplary nucleic acid sequences encoding 2A peptides are provided, for example, by Kim et al. (PLOS One 6:e18556 (2011)) and Donnelly et al. (J. Gen. Virol. 82:1027-1041 (2001)).

[0176] Introduction of the desired gene encoding the activity-inducible fusion protein into cells can be accomplished by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate transfection, infection with a viral or bacteriophage vector containing the gene sequence, cell fusion, chromosome-based gene transfer, microcell-based gene transfer, spheroplast fusion, in vivo nanoparticle-based delivery, mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359), liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63:607-618), ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83), triple-helical DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282). Various techniques for introducing foreign genes into cells are known in the art (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92), and such techniques may be used as long as they do not unduly disrupt the necessary development and physiological functions of the recipient cells. These techniques can be used to stably transfer genes into cells so that the foreign genes are expressed in the cells, and in certain cases, are preferably heritable and expressed in their progeny cells.

[0177] In certain embodiments, the gene encoding the activity-inducing fusion protein can be incorporated into a vector and introduced into a cell. A "vector" is a nucleic acid molecule that can transport another nucleic acid. A vector can be, for example, a plasmid, a cosmid, a virus, or a phage. An "expression vector" is a vector that can induce the expression of a protein encoded by one or more genes incorporated into the expression vector when placed under appropriate conditions.

[0178] Viral vectors can be derived from various kinds of viruses. "Lentivirus" refers to a virus of the retrovirus genus that can infect dividing and non-dividing cells, and usually shows high viral titers. Some examples of lentivirus include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0179] Further examples of viral vectors include those derived from foamy viruses, adenoviruses (e.g., adenovirus type 5 (Ad5), adenovirus type 35 (Ad35), adenovirus type 11 (Ad11), adenovirus type 26 (Ad26), adenovirus type 48 (Ad48) or adenovirus type 50 (Ad50)), adeno-associated viruses (AAV; see, e.g., U.S. Pat. No. 5,604,090; Kay et al., 2000; Nakai et al., z1998), alphaviruses, cytomegaloviruses (CMV), flaviviruses, herpes viruses (e.g., herpes simplex virus), influenza viruses, papilloma viruses (e.g., human papilloma virus and bovine papilloma virus; see, e.g., U.S. Pat. No. 5,719,054), pox viruses, vaccinia viruses, and the like. See Kozarsky and Wilson (1993); Rosenfeld et al. (1991); Rosenfeld et al. (1992); Mastrangeli et al. (1993); Walsh et al. (1993); and Lundstrom (1999). Additional examples include modified vaccinia Ankara (MVA), NYVAC, or virus strains derived therefrom. Other examples include avian poxvirus vectors, such as fowlpox virus vectors (e.g., FP9), and canarypox virus vectors (e.g., ALVAC and virus strains derived therefrom).For further information regarding viral vectors for gene delivery, see Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503;Rosenfeld, et al., 1991, Science 252:431-434;Rosenfeld, et al., 1992, Cell 68:143-155;Mastrangeli, et al., 1993, J. Clin. Invest. 91:225-234;Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300;Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686;Miller, et al., 1993, Meth. Enzymol. 217:581-599; Naldini et al. (1996) Science 272(5259): 263-267; Naldini et al. (1996) Proceedings of the National Academy of Sciences 93(21): 11382-11388; Zufferey et al. (1997) Nature biotechnology 15(9): 871-875; Dull et al. (1998) Journal of virology 72(11): 8463-8471; U.S. Patent Publication No. 6,013,516; and U.S. Patent Publication No. 5,994,136.

[0180] Targeted genetic engineering methods may also be used. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is a recombinant nuclease system used in bacterial genetic engineering. Information on the CRISPR-Cas system and its components can be found in, for example, U.S. Patent Publication No. 8697359, U.S. Patent Publication No. 8771945, U.S. Patent Publication No. 8795965, U.S. Patent Publication No. 8865406, U.S. Patent Publication No. 8871445, U.S. Patent Publication No. 8889356, U.S. Patent Publication No. 8889418, U.S. Patent Publication No. 8895308, U.S. Patent Publication No. 8906616, U.S. Patent Publication No. 8906617, U.S. Patent Publication No. 8906619 ... No. 8,932,814, U.S. Patent Publication No. 8,945,839, U.S. Patent Publication No. 8,993,233, and U.S. Patent Publication No. 8,999,641 and related applications thereof; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724 , WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473, WO2015 / 089486, WO2016205711, WO2017 / 106657 and WO2017 / 127807 and related applications thereof.

[0181] Certain embodiments use zinc finger nucleases (ZFNs) as gene editing reagents. ZFNs are a type of site-specific nuclease engineered to bind and cleave DNA at specific locations.For further information regarding ZFNs and ZFNs useful within the teachings of the present disclosure, see, e.g., U.S. Patent Publication Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013, No. 219; U.S. Patent Publication No. 7,030,215; U.S. Patent Publication No. 7,220,719; U.S. Patent Publication No. 7,241,573; U.S. Patent Publication No. 7,241,574; U.S. Patent Publication No. 7,585,849; U.S. Patent Publication No. 7,595,376; U.S. Patent Publication No. 6,903,185; U.S. Patent Publication No. 6,479,626; U.S. Patent Publication No. 2003 / 0232410 and U.S. Patent Publication No. 2009 / 0203140, as well as Gaj et al., Nat Methods, 2012, 9(8):805-7;Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8;Kim et al., Genome Res, 2012, 22(7): 1327-33;Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646;Miller, et al. Nature biotechnology 25, 778-785 (2007);Bibikova, et al. Science 300, 764 (2003);Bibikova, et al. Genetics 161, 1169-1175 (2002);Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000);Kim,et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0182] In certain embodiments, the gene editing reagent can be a transcription activator-like effector nuclease (TALEN), which is a fusion protein that contains a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. For further information regarding TALENs, see U.S. Patent Publication No. 8,440,431; U.S. Patent Publication No. 8,440,432; U.S. Patent Publication No. 8,450,471; U.S. Patent Publication No. 8,586,363; and U.S. Patent Publication No. 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0183] Cells that have been successfully engineered to express an activity-inducible fusion protein ex vivo can be selected, for example, based on expression of a transduction marker, and can be further processed.

[0184] (xi) Recombinant preparations, recombinant-derived preparations and pharmaceutical compositions The formulations described herein may include cells that have been genetically engineered ex vivo (i.e., recombinant formulations) or may include viral vectors or nanoparticles that can engineer cells in vivo to express the CAR (recombinant-induced formulations). As described herein, the compositions of the invention include drug molecules that bind to the hsp90 binding domain displayed on the expressed activity-induced fusion protein and / or cause a conformational change in the activity-induced fusion protein to generate intracellular signaling upon ligand binding.

[0185] A "pharmaceutical" formulation or composition comprises an active compound (eg, genetically modified cells, viral vectors, nanoparticles or drug molecules) for administration formulated in a pharma- ceutically acceptable carrier.

[0186] "Pharmaceutically acceptable" refers to compounds, materials and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without causing undue toxicity, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. In certain cases, pharma- ceutically acceptable carriers are those approved by the relevant regulatory authorities (e.g., the U.S. Food and Drug Administration (US FDA)).

[0187] Depending on the situation and the active compound to be delivered, a "pharmaceutical acceptable carrier" includes adjuvants, excipients, glidants, diluents, preservatives, dyes / colorants, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonicity agents, solvents, surfactants or emulsifiers in accordance with the above requirements. Representative pharmaceutical acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, formulations and compositions can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by the Office of Biologics Standards of the U.S. FDA and / or other relevant regulatory authorities in other countries.

[0188] Representative pharma- ceutically acceptable carriers include saline, buffered saline, saline, water, Hank's solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, Ill.), glycerol, ethanol, and combinations thereof. In certain embodiments, human serum albumin (HSA) or other human serum components or fetal bovine serum can be added to the carrier. In certain embodiments, the carrier for infusion comprises buffered saline with 5% HSA or dextrose. Alternative tonicity agents include polyhydric sugar alcohols, including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, and the like.

[0189] The carrier may contain a buffer such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer and / or a trimethylamine salt.

[0190] Stabilizers refer to a wide variety of excipients that can have various functions, from additives that can prevent cell adhesion to the walls of the container to bulking agents. Typical stabilizers include polyhydric sugar alcohols, amino acids, organic sugars, sugar alcohols, PEG, sulfur-containing reducing agents, bovine serum albumin, gelatin, immunoglobulins, polyvinylpyrrolidone, and sugars.

[0191] Where necessary or beneficial, the formulation may include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0192] Representative preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides, hexamethonium chloride, alkylparabens, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0193] The therapeutically effective amount of cells contained in the recombinant preparation is 10 2 More than 10 3 More than 10 4 More than 10 5 More than 10 6 More than 10 7 More than 10 8 More than 10 9 More than 10 10 More than 10 11 The number may be more than one.

[0194] In the recombinant formulations disclosed herein, the cells are typically contained in a volume of 1 L or less, 500 ml or less, 250 ml or less, or 100 ml or less. Thus, the density of cells administered is typically less than 10 4 Density >10 7 Density greater than 10 8 The density exceeds 50 / ml.

[0195] The therapeutically effective amount of the active ingredient (vector or nanoparticle) contained in the recombinant derivative formulation may be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dosage may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1-5 mg / kg or 0.5-1 mg / kg. In another example, the dosage may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0196] The therapeutically effective amount of the drug molecule contained in the composition of the present invention may be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dose may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1-5 mg / kg or 0.5-1 mg / kg. In another example, the dose may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0197] In certain embodiments, the recombinant preparation may include one or more genetically modified cells (e.g., modified T cells, NK cells, or stem cells) or genetically modified cells expressing one or more activity-inducible fusion proteins. Multiple genetically modified cell populations can be provided in various ratios. Furthermore, the recombinant preparation may deliver nucleic acids capable of genetically modifying two or more cells and / or expressing different types of activity-inducible fusion proteins.

[0198] Certain recombinant preparations include immune cells expressing two or more types of CARs. For example, immune cells (e.g., T cells) can be engineered to express different types of CARs with different types of ligand binding domains. Different types of ligand binding domains can bind to different epitopes on a cancer antigen or can bind to different cancer antigens. In this case, multiple CARs that each bind to a different epitope or antigen can further include different types of hsp90 binding domains so that their activation states can be individually controlled by administering different types of drug molecules (e.g., small molecule estrogen analogs).

[0199] In certain formulations, multiple types of activity-inducible fusion proteins are expressed, and each of these multiple types of activity-inducible fusion proteins can be activated or inactivated individually because they incorporate different types of EBDs. These different types of EBDs can include EBD (E353A) that can be activated by administration of ES8, EBD (L384M, M421G, G521R) that can be activated by administration of CMP8, and EBD ERT2 that can be activated by administration of 4-OHT. Based on the description of the present disclosure, various alternative combinations can be used.

[0200] Representative combinations of cancer antigens that are bound by immune cells expressing different CARs and different hsp90 binding domains include (i) CD19, CD22 and / or BAFF-R; (ii) CD19 and CD22; (iii) Her2, B7H3, EGFR and / or IL13Ra2; and (iv) CD33 and CD123. Other relevant cancer antigen groupings based on cancer type are described elsewhere in this disclosure. Other EBD / drug molecule combinations include EBD(E353A) and ES8, EBD(L384M, M421G, G521R) and CMP8, and EBD(ERT2) with G400V, M543A and L544A mutations and 4-OHT. This approach of utilizing different binding domains in combination with different hsp90 binding domains is referred to herein as "CAR combination therapy."

[0201] In certain examples, administration of the formulation results in the expression of a CAR and a costimulatory immune molecule (e.g., CD28, 4-1BB, OX40, ICOS), where the CAR and the costimulatory immune molecule each contain a different hsp90 binding domain. In certain examples, administration of the formulation results in the expression of a CAR and two costimulatory immune molecules (e.g., CD28, 4-1BB, OX40, ICOS), where the CAR and the two costimulatory immune molecules each contain a different hsp90 binding domain. In certain examples, administration of the formulation results in the expression of two CARs and two costimulatory immune molecules (e.g., CD28, 4-1BB, OX40, ICOS), where the two CARs each have a different binding domain and a different hsp90 binding domain, but the two costimulatory immune molecules each contain the same hsp90 binding domain. In a particular example, administration of the formulation results in the expression of two CARs and two costimulatory immune molecules (e.g., CD28, 4-1BB, OX40, ICOS), each of which has a different binding domain and a different hsp90 binding domain, and each of which contains a different hsp90 binding domain. In this example, the hsp90 binding domain contained in the costimulatory molecule may be the same as the hsp90 binding domain contained in the CAR, or may be different from the hsp90 binding domain contained in the CAR.

[0202] The recombinant formulation may further include different types of immune cells, each expressing a different type of CAR. For example, an individual recombinant immune cell expresses only one type of CAR, but is formulated with other immune cells that are engineered to express other types of CARs (e.g., CARs that contain different ligand binding domains that bind to different EBD and drug molecule combinations). The immune cells may be of the same type (all T cells) or may include a mixture of different types (e.g., T cells, NK cells, and / or HSPCs).

[0203] Recombinant inducer preparations can be prepared to induce immune cell populations in vivo with such characteristics (e.g., expression of different types of CARs by a single type of immune cell; expression of different types of CARs by different types of immune cells; expression of a single type of CAR by different types of immune cells; and / or expression of different types of CARs by different types of immune cells; and / or incorporation of activity-induced co-stimulatory or activity-induced inhibitory molecules).

[0204] The formulations and compositions can be prepared for administration, for example, by injection, infusion, irrigation, lavage or oral ingestion. The formulations and compositions can be formulated for bone marrow injection, intravenous injection, intradermal injection, intraarterial injection, intralymph node injection, intralymphatic injection, intraperitoneal injection, intralesional injection, intraprostatic injection, intravaginal injection, intrarectal injection, topical injection, intrathecal injection, intratumoral injection, intramuscular injection, intravesical injection and / or subcutaneous injection.

[0205] In some cases, it may be useful to cryopreserve the recombinant cell preparations of the present disclosure. As used herein, "cryopreserving" refers to preserving cells by cooling to subzero temperatures, such as (usually) 77K or -196°C (the boiling point of liquid nitrogen). To mitigate or prevent damage to cells caused by freezing at low temperatures or warming to room temperature, cryoprotectants are often used during storage at subzero temperatures. The use of cryoprotectants and optimal cooling rates can prevent cell damage. Cryoprotectants that can be used include dimethylsulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). In certain embodiments, the cooling rate is 1-3°C / min. After at least 2 hours, the cells reach a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) in a long-term cryogenic storage vessel or the like for permanent storage.

[0206] (xii) How to use The methods disclosed herein include treating a subject (human, companion animal (dog, cat, reptile, bird, etc.), livestock (horse, cow, goat, pig, chicken, etc.), or research animal (monkey, rat, mouse, fish, etc.) with (i) the recombinant and / or recombinant-derived formulations and (ii) the pharmaceutical composition disclosed herein. Treating a subject includes delivering a therapeutically effective amount. A therapeutically effective amount includes an amount that can provide effective, prophylactic and / or therapeutic treatment without causing undue toxicity.

[0207] An "effective amount" is the amount of a formulation or composition required to obtain a desired physiological effect. Effective amounts are often administered for research purposes. The effective amounts disclosed herein can induce chromium release or cytokine release in cell activation assays.

[0208] "Prophylactic treatment" includes treatment administered to a subject who does not exhibit signs or symptoms of a disease (e.g., cancer or an infectious disease) or who exhibits only early signs or symptoms of the disease, with the intent of reducing or decreasing the risk of the disease developing further. Thus, prophylactic treatment serves as a treatment that prevents the disease. In certain embodiments, prophylactic treatment inhibits, slows, or prevents the progression of the disease.

[0209] "Therapeutic treatment" includes treatment administered to a subject who exhibits symptoms or signs of a disease, and is administered to a subject with the intent of reducing or eliminating the signs or symptoms of the disease. Therapeutic treatment can prevent, control, or eliminate the presence or activity of the disease, and / or can prevent, control, or eliminate the side effects of the disease.

[0210] The terms effective amount, functioning as a prophylactic treatment or a therapeutic treatment are not mutually exclusive, and in certain embodiments, an administered dose may accomplish more than one treatment.

[0211] A therapeutically effective amount can be achieved by a single administration or multiple administrations during the course of a treatment regimen (e.g., daily, every other day, every third day, weekly, biweekly, monthly, bimonthly, 4 months, six months, yearly, etc.).

[0212] As described herein, the formulations and compositions of the present invention can be administered by injection, infusion, placement or implantation. Recombinant derived formulations and drug compositions can also be administered orally or by inhalation. In certain embodiments, the formulations and compositions of the present invention are administered parenterally. "Parenteral administration" and "administering parenterally" refer to any method of administration other than enteral or topical administration, usually by injection, including intravascular injection and infusion, intravenous injection and infusion, intramuscular injection and infusion, intraarterial injection and infusion, intrathecal injection and infusion, intracapsular injection and infusion, intratumoral injection and infusion, intraperitoneal injection and infusion, and subcutaneous injection and infusion. In certain embodiments, the formulations and compositions described herein are administered to a subject by direct injection into a tumor, lymph node or disease site. In certain embodiments, the drug composition is administered orally.

[0213] In certain instances, the disclosure provides a method of cellular immunotherapy in a subject having a disease or disorder, comprising administering a recombinant or recombinant-derived formulation that expresses a CAR in vivo in a constitutively "off" activation state. In this instance, the method further comprises administering a drug composition that can activate the CAR upon binding to an antigen. In some embodiments, the drug composition is delivered prior to administration of the recombinant or recombinant-derived formulation, delivered simultaneously with administration of the recombinant or recombinant-derived formulation, or delivered at a time point after administration of the recombinant or recombinant-derived formulation.

[0214] In some embodiments, the drug composition is administered together with a recombinant or recombinant-derived formulation, and if toxicity from the recombinant or recombinant-derived formulation is observed, administration of the drug composition is withheld until the toxicity has subsided. After the toxicity symptoms have subsided, administration of the drug composition can be resumed.

[0215] In certain examples, the disclosure provides a method of cellular immunotherapy in a subject with a disease or disorder, comprising administering a recombinant or recombinant-derived formulation that expresses at least two CARs in vivo in a constitutively "off" activation state. The two CARs each bind to a different cancer antigen, each have a different hsp90 binding domain, and each of the different hsp90 binding domains bind to a different drug molecule. In this example, the method further comprises selectively administering one or more drug molecule compositions that can selectively activate the different types of CARs by binding to the antigen. In some embodiments, the one or more drug molecule compositions are delivered prior to administration of the recombinant or recombinant-derived formulation, delivered simultaneously with administration of the recombinant or recombinant-derived formulation, or delivered at a time point after administration of the recombinant or recombinant-derived formulation.

[0216] In certain examples, the disclosure provides a method of cellular immunotherapy in a subject having a disease or disorder, comprising administering a recombinant or recombinant-derived formulation that expresses in vivo at least one CAR in a constitutively "off" state and at least one costimulatory molecule in a constitutively "off" state. The CAR and costimulatory molecule each have a different hsp90 binding domain, and the different hsp90 binding domains each bind to a different drug molecule. In this example, the method further comprises selectively administering one or more drug molecule compositions that can selectively activate the CAR and / or the costimulatory molecule upon binding to an antigen. In some embodiments, the one or more drug molecule compositions are delivered prior to administration of the recombinant or recombinant-derived formulation, delivered simultaneously with administration of the recombinant or recombinant-derived formulation, or delivered at a time point after administration of the recombinant or recombinant-derived formulation.

[0217] In some embodiments, one or more drug compositions are administered together with recombinant or recombinant-derived formulations, and if toxicity of the recombinant or recombinant-derived formulations is observed, administration of one or more drug compositions is discontinued until the toxicity is reduced.After the toxicity symptoms are alleviated, administration of one or more drug compositions is resumed.Toxicity can be observed, for example, based on the amount of TNFα or IFNγ exceeding a clinically significant threshold.

[0218] In some embodiments, the pharmaceutical composition is administered together with the recombinant or recombinant-derived preparation, and if a reduction in cancer cells or virus-infected cells is observed in the subject's body, administration of the pharmaceutical composition can be discontinued for a period of time to allow the recombinant cells to rest. If the cancer goes into remission or the infection is eliminated, administration of the pharmaceutical composition can also be discontinued.

[0219] In another embodiment, the method of the invention comprises administering to a subject a recombinant cytotoxic T lymphocyte preparation that provides a cellular immune response, the cytotoxic T lymphocyte preparation comprises CD8+ T cells expressing a CAR comprising a ligand binding domain; a spacer domain; a transmembrane domain; and an intracellular signaling domain under the control of an hsp90 binding domain as described herein; the ligand-binding domain being specific for a ligand; and said ligand being a tumor-specific molecule, a viral molecule, or other molecule expressed on a target cell population and capable of inducing recognition, regulation, suppression and / or elimination by lymphocytes; and / or The method of the present invention comprises administering to a subject a genetically modified helper T lymphocyte preparation that induces direct recognition of a tumor and enhances the ability of the genetically modified cytotoxic T lymphocyte preparation to mediate a cellular immune response; the helper T lymphocyte preparation comprises CD4+ T cells expressing a CAR comprising a ligand binding domain; a spacer domain; a transmembrane domain; and an intracellular signaling domain under the control of an hsp90 binding domain as described herein; the ligand-binding domain being specific for a ligand; and the ligand is a tumor-specific molecule, a virus-specific molecule, or other molecule expressed on a target cell population and capable of inducing recognition, regulation, suppression and / or elimination by lymphocytes; The methods of the invention further include administering a pharmaceutical composition that activates the CAR.

[0220] Cancers that can be treated with the recombinant or recombinant-derived formulations and drug compositions disclosed herein include carcinomas such as bladder cancer, head and neck cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, and skin cancer (including squamous cell carcinoma); lymphomas such as leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkitt's lymphoma. myeloid hematopoietic tumors such as acute myeloid leukemia, chronic myeloid leukemia, and promyelocytic leukemia; mesenchymal tumors such as fibrosarcoma and rhabdomyosarcoma; other tumors such as neuroblastoma and glioma; tumors of the central and peripheral nervous system such as astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tumors such as fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors such as malignant melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma. Other representative cancers that can be treated according to the present disclosure include lymphoid hematopoietic malignancies, e.g., T-cell and B-cell tumors, including T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell T-prolymphocytic leukemia and cerebro-like T-prolymphocytic leukemia; T-cell large granular lymphocyte leukemia (LGL); Sézary syndrome (SS); adult T-cell leukemia-lymphoma (ATLL); hepatosplenic T-cell lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angioimmunoblastic T-cell lymphoma; hematocentral (nasal) T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinal T-cell lymphoma; and T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL).

[0221] Based on the cancer experienced by the subject, a CAR having a ligand binding domain that binds to the cancer antigen can be selected. Representative cancer antigens include bladder cancer antigens: MUC16, PD-L1, EGFR; breast cancer antigens: HER2, ERBB2, ROR1, PD-L1, EGFR, MUC16, FOLR, CEA; cholangiocarcinoma antigens: mesothelin, PD-L1, EGFR; colorectal cancer antigens: CEA, PD-L1, EGFR; glioblastoma antigens: EGFR variant III ( EGFRvIII), IL13Ra2; Lung cancer antigens: ROR1, PD-L1, EGFR, mesothelin, MUC16, FOLR, CEA, CD56; Merkel cell carcinoma antigens: CD56, PD-L1, EGFR; Mesothelioma antigens: mesothelin, PD-L1, EGFR; Neuroblastoma antigens: ROR1, glypican 2, CD56, disialoganglioside, PD-L1, EGFR; Ovarian cancer antigens: EpCam, L1-CAM, MUC16, folate receptor (FOLR), Lewis Y, ROR1, mesothelin, WT-1, PD-L1, EGFR, CD56; melanoma antigen: tyrosinase-related protein 1 (TYRP1 / gp75); GD2, PD-L1, EGFR; multiple myeloma antigen: B-cell maturation antigen (BCMA), PD-L1, EGFR; pancreatic cancer antigen: mesothelin, CEA, CD24, ROR1, PD-L1, EGFR, MUC16; prostate cancer antigen: PSMA, WT1, prostate stem cell antigen (PSCA), SV40 T, PD-L1, EGFR; renal cell carcinoma antigen: carbonic anhydrase IX (CAIX); PD-L1, EGFR; and stem cell cancer antigen: CD133, PD-L1, EGFR. Other examples are known to those skilled in the art. These groupings can be used to configure CAR combination therapy, as described elsewhere herein. CAR combination therapy can include activity-induced costimulatory molecules and / or activity-induced inhibitory molecules.

[0222] Particular CAR combination therapies include (i) CARs with binding domains that bind to CD19, CD22, and / or BAFF-R (e.g., CD19 and CD22) for the treatment of acute lymphoblastic leukemia (ALL); (ii) CARs with binding domains that bind to Her2, B7H3, EGFR, and / or IL13Ra2 for the treatment of brain tumors; or (iii) CARs with binding domains that bind to CD33 and CD123 for the treatment of acute myeloid leukemia (AML). Drug molecules that can selectively activate CARs expressing ligand binding domains for these antigens include ES8, CMP8, and 4-OHT.

[0223] In certain examples, the cancer sample obtained from the subject can be evaluated for the presence or absence of a particular biomarker or cell surface marker.For example, the breast cancer cells obtained from the subject can be positive or negative for Her2Neu, estrogen receptor and / or progesterone receptor, respectively.Tumor antigens or cell surface molecules present on the tumor cells of individual subjects are selected, and CARs with binding domains that bind to these antigens are selected.Various combinations of such tumor antigens or cell surface molecules and CARs can be selected to form CAR combination therapy.

[0224] In certain embodiments, the therapeutically effective amount of the formulation and the drug composition can provide anti-cancer effects, including reducing the number of malignant cells, reducing the number of metastases, reducing tumor volume, increasing life expectancy, inducing chemotherapy or radiosensitivity in cancer cells, inhibiting angiogenesis in the vicinity of cancer cells, inhibiting the proliferation of cancer cells, inhibiting tumor proliferation, preventing or reducing metastasis, increasing the life span of a subject, inhibiting cancer-related pain, and / or reducing relapse or recurrence of cancer after treatment.

[0225] Infectious diseases that can be treated by the formulations and compositions of the present disclosure include bacterial, viral, fungal, parasitic and arthropod infections.In certain embodiments, the infectious disease is a chronic infectious disease.In certain embodiments, the bacterial infection includes infections caused by Staphylococcus spp., Streptococcus spp., Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Escherichia coli, Listeria monocytogenes, Salmonella spp., Vibrio spp., Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum. In certain embodiments, the viral infection includes an infection caused by a rhinovirus, influenza virus, respiratory syncytial virus (RSV), coronavirus (e.g., MERS, SARS, SARS-CoV-2), herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV), hepatitis A virus, norovirus, rotavirus, human papillomavirus (HPV), hepatitis B virus, human immunodeficiency virus (HIV), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV), West Nile virus (WNV), enterovirus, hepatitis C virus, human T-lymphotropic virus type 1 (HTLV-1), or Merkel cell polyomavirus (MCV). In certain embodiments, the fungal infection includes an infection caused by a Trichophyton or Candida genus. In certain embodiments, the parasitic infections include infections caused by Giardia, Toxoplasma gondii, Pinworm, Trypanosoma cruzi, Echinococcus, Cysticercosis, Toxocara, Trichomonas, and Entamoeba histolytica. In certain embodiments, the arthropod infections include infections transmitted by arthropods infected with viruses or bacteria, such as California encephalitis, Chikungunya, Dengue, Eastern equine encephalitis, Powassan encephalitis, St. Louis encephalitis, West Nile fever, Yellow fever, Zika, Lyme disease, or Babesiosis.

[0226] In certain embodiments, the therapeutically effective amounts of the formulations and drug compositions can provide an anti-infective effect, including a reduction in the amount or level of infectious pathogens, a reduction in fatigue, a reduction in appetite, an inhibition of weight loss, a reduction in fever, a reduction in night sweats, a reduction in chills, a reduction in aches and pains, a reduction in diarrhea, a reduction in bloating, a reduction in abdominal pain, a reduction in rash, a reduction in coughing, and / or a reduction in runny nose.

[0227] In certain embodiments, the administration of the drug composition is discontinued to obtain an anti-side effect, which can reduce or eliminate the negative effects of administration of the formulation, such as transplant-induced cytokine storm (cytokine release syndrome), tumor lysis syndrome (TLS), and B cell depletion.

[0228] When administering, the therapeutically effective amount (also referred to herein as "dosage") can be estimated initially based on the results of in vitro assays and / or animal model studies. Such information can be used to more accurately determine the dosage that is useful for the intended subject. The actual dosage administered to a particular subject can be determined by a physician, veterinarian or researcher, taking into account parameters such as physical and physiological factors, including target, body weight, disease severity, disease type, disease stage, previous or concurrent therapeutic interventions, subject's idiopathic disease and route of administration.

[0229] The therapeutically effective amount of the recombinant preparation to be administered is 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than 10 cells or 11 Examples include cells having more than one cell.

[0230] A useful dose for administration of a recombinant induction formulation or drug composition may be, for example, in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dose may be 1 μg / kg, 15 μg / kg, 30 μg / kg, 50 μg / kg, 55 μg / kg, 70 μg / kg, 90 μg / kg, 150 μg / kg, 350 μg / kg, 500 μg / kg, 750 μg / kg, 1000 μg / kg, 0.1-5 mg / kg or 0.5-1 mg / kg. In another example, the dose may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0231] (xiii) Kit The present disclosure also includes kits comprised of materials useful for carrying out aspects of the present disclosure, which may include, for example, cells (e.g., immune cells), nucleic acids encoding activation-inducible fusion proteins (e.g., CARs, costimulatory molecules, and / or inhibitory molecules), transfection reagents, assay reagents, drug molecules, buffers, cell nutrients and growth media, cell sorting molecules (e.g., Dynabeads), tubes, wells, and small molecule estrogen analogs (e.g., Tamoxifen, 4-OHT, ES8, CMP8).

[0232] (xiv) HSP90 clients The activity-inducible fusion proteins disclosed herein may contain a binding domain derived from another client molecule or domain of hsp90. The client proteins of hsp90 described herein are categorized as transcription factors, kinases and "other" as shown at picard.ch / downloads / hsp90 interactors.

[0233] Examples of client transcription factors of hsp90 include the 12(S)-HETE receptor; AF9 / MLLT3; all vertebrate steroid receptors (GR, MR, ERα, ERβ, PR, AR); AGL24; ATF3; BBX; BCL-6; Bclaf1; BES1; BrZ7; BZR1; C20orf194; CAR; CEBPE; Cwt1; CXXC1; cytoplasmic v-erbA; DLX6; DMRTA1; EcR; FOXD4L6; FOXM1; FOXP 2;GTF2IRD2;Hap1;HCFC1;HMGA1, HMGA2;HNF4A;HP1BP3;HSF-1;HsfA1, HsfA2, HsfB1;IRF2;IRF3;ISX;LFY;MAFG;Mal63;Ma lR;MAX;Met1;MeWRKY20;MKX;mod(mdg4);c-Myc;Nanog;NFIC;FRKB;Notch1(ICN1);NR1H3;NR1I2;Oct4;p53;p73;PAS family Members of the dioxin receptor (AhR), Sim, HIF-1α, HIF-2α, HIF-3α; PCGF6; POGK; PPARα, PPARβ, PPARγ; PRDM1; PREB; PXR; REST; REV-ERBα; (Aspergillus) RlmA; SETDB1; SIM2; SLFN11; SOC1; SOX11; Sp1; SREBF1; SREBP1; SREBP2; Stat2; Stat3 (caveolin-1 complex in lipid rafts) merging; Stat3; Stat5; SUP; TADA2A; TBX22; TCF25; TDP-43; TEAD2; TFDP3; THAP4; TonEBP / OREBP; TRIM32; Tup1; Twist1; Ure2; USP1; VDR; VP16; steroid (antheridiol) receptor of the aquatic fungus Gonorrhoeae; WT1; ZBED4; ZBTB17; ZBTB20; ZC3H7B; ZNF215; ZNF509; and ZNF74.

[0234] Examples of hsp90 client kinases include ACVR1B; ACVR1C; ACVR2B; Akt / PKB; AKT2; ALK; ALK1, ALK5; ALPK1; AMHR2; AMPKα, AMPKγ; ARAF; ASK1; ATM; AURKC; Aurora B;AXL;Bcr-Abl;BCR-FGFR1;BGLF4 of EBV;BLK;BMPR1A;BMX;BTK;c-Abl;c-Kit;c-Mos;CAMK1G;CAMK2A;CAMK2B;CAMK2D;CAMK2G;CAMK4;CAMKK1;CAMKK2;CAMKV;casein kinase IIα catalytic subunit;Cdc2 (=Cdk1);CDK11B;CDK14;CDK15;CDK18;Cdk2, Cdk4, Cdk6, Cdk9, Cdk11;CDK3 ;CheA (E. coli);Chk1;Cla4;CLK2;CLK3;Cot (=Tpl-2);CSF1R;CSNK1A1;DCLK2;DDR1;DDR2;cell death-related kinases DAPK, DAPK2, DAPK3;DLK;DMPK;DYRK1B;DYRK2;DYRK4;eEF-2 kinase;EGF receptor (mutant and wild type);eIF2-α kinases HRI, Gcn2, Perk, PKR;Eml4-Alk;EPHA1;EphA2;EPHA4;EPHB1;EPHB 6;ErbB2;ERBB3;ERBB4;ERK5;FASTK;FGFR1;FGFR3 and FGFR4;Flt3;FLT4;FOP2-FGFR1;FRK;Fused;FYN;Gal1;GRK2 and GRK6;GRK4;GRK7;GSK3A;GSK3β;HCK;HER3;HIPK4;HopBF1 effector;ICK;INSRR;insulin receptor;insulin-like growth factor 1 receptor;integrin-linked kinase;IP6K2;IRAK-1;IRAK2;I RAK3;Ire1α;ITK;IκB kinase (IKK)α, IKKβ, IKKγ, IKKε;JAK1;JNK;KSR;LATS1, LATS2;LCK;LIMK1;LIMK2;Lkb1;LMTK3;LRRK2;LYN;MAP2 K5;MAP2K7;MAP3K12;MAP3K15;MAP3K2;MAP3K6;MAP3K9;MAP4K1;MAP4K2;MAP4K4;MAPKKK(MEKK)YODA;MAPK15;MAPK4;MAPK6;MAPK7;MAST1;MAST2;MATK;MEK;MEKK1 and MEKK3;MERTK;MET;Mik1;MINK1;MLK3;MLKL;MOK, MAK, MRK;MpkA (Aspergillus);Mps1;mTOR;MUSK;MYLK2;MYLK3;MYLK4;NEK11;NEK8;NEK9;NIK;NPM-Alk;NPR2;NTRK1;NTRK2;NTRK3;NUAK2;nucleophosmin-anaplastic lymphoma kinase;p38;p90RSK;PAK6;PASK;Pbs2 ;PDGFRB;PDIK1L;PDK1;PGK1;PI4KIIβ;Pim-1;PIM2;PIM3;Pink1;PKCλ, PKCε and other PKCs;PKM2;PKN1;PKN2;platelet-derived growth factor receptor α;Plk1;Plk3;Pnck;pp60v-src, c-src;PRKAA2;PRKACB;PRKCA;PRKCB;PRKCG;PRKCH;PRKCI;PRKCQ;PRKCZ;PRKD1;PRKD2;PRKDC;PRKG2;PRKX;PRKY;PSKH1;P SKH2;PTK2;PTK2B;PTK6;PTK6;Raf-1, B-Raf, Ste11;RET;RET / PTC1;RIP1;RIP3;Ron;ROR2;RPS6KA1;RPS6KA2;RPS6KA3;RPS6KA5;RPS6KA6; RPS6KB1;RPS6KC1;RPS6KL1;Ryk;SGK-1;SGK2;SGK223;SGK3;Slt2;src-related tyrosine kinases: fer, fes, fgr, fps, lck, yes;SRPK1;SRPK3;SSCMK1;STK 32B; STK32C; STK33; STK38; STK38L; STYK1; SYK; TAK1; TAOK3; TBK1; TESK1; TESK2; TGFβ receptor I and TGFβ receptor II; TIE1; TNK1; TNK2; TNNI3K; TP53RK; TrkAI and TrkAIII; TrkB; TSSK1B; TSSK2; TSSK3; TSSK4; TSSK6; Tyk2; TYRO3; Ulk1; VEGFR1, VEGFR2; Wee1, Swe1; WNK4; and ZAP-70.

[0235] Examples of hsp90 client molecules classified as "other" include Act1 (=TRAF3IP2); adenosine A2A receptor; α2C adrenergic receptor; AID; AIP56; aldo-ketoreductase 1B10; ANAPC2; ANKMY2; annexin A2; ANP receptor; ANP32C / D; Apaf-1; apoB; APOBEC-3B, APOBEC-3C, APOBEC-3G; Arb1; ARD1; Argonaute 1 (Ago1); Argonaute 2 (=Ago2=GERp95); Argonaute 4 (Ago4); ARMC5; ArtAB; ASB17; ASB2; ASB3; ASB4; ASB6; ATG8 (GABARAP) protein; Axin 1; BCAP (PIK3AP1); BALF5 of EBV; Bcl-2; Bcl-xL; Beclin 1;Bid;BIN2;BLM helicase;Bms1;BPIFB4;BRAT1;BRCA1;BRCA2;BRMS1;BTRC;c-IAP1;calcineurin (Cna2; catalytic subunit);calmodulin;calmodulin methyltransferase;calpain 1;calponin;CARM1;caspase 8;β-catenin;CB2 cannabinoid receptor;Ccp1;CCDC117;type III CD38;CD79a;Cdc13;Cdc14;Cdc25a and Cdc25c;Cdk5 activator p35;CPEB1, CPEB2, CPEB3;CFTR (nascent and mutant polypeptides);ChAT;CheZ (E. coli);Chl1;Chronophin;cineole synthesis Enzyme 1; Clathrin heavy chain; CLC-1 chloride channel; CLC-2 chloride channel; Clostridial CDT toxin; Clostridial iota toxin; Clusterin; COG complex; COI1; Complement component 9 (C9); Cry toxins; CTA1 (=CtxA1); Ctf13 / Skp1 component of CBF3; CUL1; CUL2; CUL3; CUL4A; CUL4B; Cup; Cyclin B; Cyclophilin D (in mitochondria); Cyr1; Cytoskeletal proteins: actin, tubulin (including ciliary β4 tubulin), myosin (including Myo3B); DBC2; DEDD; Dengue virus E protein; Dengue virus NS1 / 2B / 3 / 4B / 5 proteins; DET1; Diphtheria toxin A;DNA helicase Ssl2;DNA polymerase α;DNA polymerase λ;DNA polymerase η;DnaA (E. coli);DNMT1;Dsn1;DTX4;E6^E7;EBAX-1;Emc2;ENC1;eNOS, nNOS(?);human delayed rectifier potassium ion channel (ERG=HERG=KCNH2);EZH2;F1F0-ATP synthase;FANCA;FBXL12;FBXL13;FBXL14;FB XL15;FBXL18;FBXL2;FBXL3;FBXL6;FBXL8;FBXO10;FBXO17;FBXO18;FBXO24;FBXO25;FBXO27;FBXO28;FBXO3;FBXO 34;FBXO38;FBXO4;FBXO40;FBXO6;FBXO9;FBXW11;FBXW2;FBXW5;FBXW7;FGAMS;Fibronectin;FliN, FliI (Escherichia coli);FLIP; S and FLIP L ;Folliculin;Free βγ subunit of G protein;FtsZ;G2E3;GAN;Gln1;GLT-1;GluR1;Glutathione S-transferase subunit 3 (KS type);Soluble guanylate cyclase;Gα0, Gα 12;glucocerebrosidase;GREB1;HAX-1;HDAC1;HDAC6;HECTD3;Hepatitis B virus core protein;Hepatitis C virus NS3 protein;Hepatitis E virus capsid protein;HERC4;HERC6;Histone H1, histone H2A, histone H2B, histone H3 and histone H4;HMGCR;Hsp27;Humanin;Huntingtin;Importin 4 (IPO4);Importin α1;Importin α6 (KPNA5);Ino80;Inositol 1,4,5-trisphosphate receptor 3;Integrin α2;Integrin α4;Integrin αL;IL-1β; IRS-2; E protein of Japanese encephalitis virus; JlpA; KAP1; KAT5; KBTBD4; KBTBD7; KCNA5; KCNA6; KCNG1; KCNS3; KCNQ4; KCTD8; KDM3A / JMJD1A; KDM4B / JMJD2B; KEAP1; KIAA0317; Kir6.2; KLHL1; KLHL10; KLHL13; KLHL14; KLHL15; KLHL22; KLHL23; KLHL25; KLHL26; KLHL29; KLHL32; KLHL34; KLHL36; KLHL38; KLHL6; knob complex (on the plasma membrane of Plasmodium-infected erythrocytes); KSHV K1;KSR1;KSR2;L protein of HRSV;lamin A / C;LAMP-2A;LANA of KS-HSV;LAP;LARP4B;legumain;LGALS3BP;LIS1;LNX1;LOC440248;LOX1(OLR1);LOXL2;Lpl1(Staphylococcus aureus);LRP1(=CD91);LRP5;LRSAM1;LSD1;LSM8;macromolecular aminoacyl-tRNA synthetase complex;macrophage scavenger receptor;MAP1B;MARCH9;Mdm2;MDM4;MeCatalase1;Mg 2+ dependent phosphatidic acid phosphohydrolase;MIF;misfolded VHL;MMP2, MMP3, MMP9;μ opioid receptor;MRE11 / Rad50 / NBS1 (MRN) complex;MRP1;Msps / XMAP215 / ch-TOG;MTA1;MTG8;MUC1;myoglobin;N-myc downstream regulated gene 1 (NRDG1);N-WASP;Na + -K + -Cl-Symporter 1;NadA;NAP1;NB-LRR proteins: RPM1 and RPS2, Nod1, Nod2, NALP2, NALP3, NALP4, NALP12, IPAF, RPP4;NCC;NDRG2;NELF-E;Nervous necrosis virus capsid protein;Neuraminidase;Neuropeptide Y;NHE1;NHLRC1;Nibrin;NleH1 and NleH2;NMNAT2;Norovirus capsid protein VP1;Nox1, Nox2, Nox3, Nox5;NS1;Nsl1;Chikungunya virus nsP3 and nsP4;MERS-CoV nucleoprotein (NP);Nup62;OGT;OsCERK1;P protein (rabies virus);P1 (picornavirus capsid precursor protein P1);p14ARF;P2X7 purinergic receptor;p300;P450 CYP2E1;PARK2;PARK7 (DJ-1);PB1 and PB2 subunits of influenza RNA polymerase;PCGF1;PCGF3;PCNA;Peli1;perilipin;PfCRT;PIDD;Piwi;PIWIL2;PLCγ;PLN;polysomal ribonuclease 1 (PMR1);PPAT;PRDM14;PRMT5;proDcp1;prolactin receptor;prostacyclin synthase;proteasome;PRPF8;PRPF19;PTPN22;Ptx;R protein I-2;Pih1-mediated R2TP complex;Rab-αGDI;Rab3a;Rab11a;RAB40A;Rac / Rop GTPase Rac1 (rice);Rac1;Rad51;Rad52;RAG1;Ral-binding protein 1 (RalBP1);RanBP9;Rapsyn;Raptor;RCBTB1;RCBTB2;Reovirus σ1 protein;REV1;Hepatitis B virus reverse transcriptase;RFWD3;RGS11;RGS6;RGS7;RGS9;RHOBTB1;Ribosomal protein L2 (Escherichia coli);Ribosomal proteins S3 and S6;Ricin catalytic A chain;RIG-I;RNA-dep;(Bamboo mosaic virus) RNA polymerase;RNF10;RNF111;RNF19B;RNF40;RNGTT;Rnr4;Rpb1;SCAP;SDF2;SENP3;SERCA2a;SERT (SLC6A4);SF3B3;SH3RF2;Sicily;SIR2 (SIR2RP1 in Leishmania spp.);SIRT1;SIRT2;SKP2;SKP2 complex;SLC6A14;SMYD1, SMYD2, SMYD3;snoRNP complex;SNRNP200;SOCS6;SPSB1;SPSB3;SREC-I;STING;SUR1 (subunit of the ATP-sensitive potassium channel in beta cells);survivin;SV40 large T antigen;Swr1;α-synuclein;Tab2 / 3;Tas3;tau protein;Tax;TCL1A;telomerase;TFR1;thiopurine S-methyltransferase;thrombin receptor (PAR-1);thromboxane synthase;TilS;TIMP2;TIR1;tissue plasminogen activator (tPA);titin;TLR4 / MD-2 complex;TLR7;TLR9;Tm-2; 2 ;TNFAIP3;TOM40;TRIM10;TRIM17;TRIM2;TRIM36;TRIM37;TRIM41;TRIM49;TRIM56;TRIM7;TRIM73;TRIM74;TRIM8;triosephosphate isomerase;Trithorax (and orthologue MLL);Trx1;TrxR;TSG101;tyrosine hydroxylase;UCH-L1;UHRF1;Ulp1;uPA;Ura 2; URI complex; uroporphyrinogen decarboxylase (HemE) [of cyanobacteria]; Us11 (of HSV-1); USP19; Utp21; vaccinia core protein 4a; vFLIP (of KSHV); vimentin; VIP1; VPS18; VPS41; WASF3; WSB2; WTAP; WWP1; XPO1; XPORT; XRCC1; ZEITLUPE; and ZMYND10.

[0236] (xv) Variant Variants of the sequences disclosed and referenced herein are also included herein. Guidelines for determining which amino acid residues can be substituted, inserted or deleted without losing biological activity can be determined using computer programs well known in the art, such as DNASTAR. TMSoftware (Madison, WI, USA) can be used to find. The amino acid changes in the protein variants are preferably conservative amino acid changes, i.e., substitutions of similarly charged amino acids with each other or substitutions of uncharged amino acids with each other. Conservative amino acid changes include substitutions with members of a family of amino acids whose side chains are related.

[0237] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such conservative substitutions can be made without generally altering the biological activity of the resulting molecule.Those skilled in the art will be familiar with the fact that generally, a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter the biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into conservative substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu), and ketone (K). Group 7 (polar uncharged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp) and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu and Ile; Group 10 (small aliphatic residues that are non-polar or slightly polar): Ala, Ser, Thr, Pro and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0238] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein is widely understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity index of each amino acid is Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamic acid (-3.5); Gln (-3.5); aspartic acid (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0239] It is well known in the art that substitution of a particular amino acid with another amino acid having a similar hydrophobicity index or hydrophobicity degree can also result in a protein with similar biological activity, i.e., a protein with biologically equivalent functionality. When making such changes, substitution of amino acids with hydrophobicity indices within ±2 is preferred, substitution of amino acids with hydrophobicity indices within ±1 is particularly preferred, and substitution of amino acids with hydrophobicity indices within ±0.5 is even more particularly preferred. Furthermore, it is well known in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity.

[0240] As detailed in U.S. Patent No. 4,554,101, each amino acid residue is assigned a hydrophilicity value, which is as follows: Arg (+3.0); Lys (+3.0); Aspartic acid (+3.0±1); Glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is well known that certain amino acids can be substituted with other amino acids having a similar hydrophilicity value, and that such substitutions will result in biologically equivalent proteins, and in particular immunologically equivalent proteins. When making such changes, substitutions between amino acids whose hydrophilicity values ​​are within the range of ±2 are preferred, substitutions between amino acids whose hydrophilicity values ​​are within the range of ±1 are particularly preferred, and substitutions between amino acids whose hydrophilicity values ​​are within the range of ±0.5 are even more particularly preferred.

[0241] As outlined above, amino acid substitutions may be made on the basis of the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0242] A functional variant includes one or more residue additions or substitutions that do not substantially affect the physiological activity of the protein. A functional fragment includes one or more deletions or truncations that do not substantially affect the physiological activity of the protein. The lack of substantial effect can be confirmed by observing experimentally comparable results in activation or binding tests. When in the activated state described in this disclosure, functional variants and functional fragments of the intracellular domain (e.g., the intracellular signaling domain) transmit activation or inhibition signals to the same extent as the wild-type reference intracellular domain. Also, functional variants and functional fragments of the binding domain bind to their cognate antigens or ligands at the same level as the wild-type reference binding domain.

[0243] In certain embodiments, the VH region of the binding domain is derived from or generated based on the VH region of a known antibody and may contain, compared to the VH region of the known antibody, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of these changes. The insertions, deletions or substitutions may be made anywhere in the VH region, including at the amino or carboxy terminus or both, so long as each CDR contains no changes or at most one, two or three changes and the binding domain comprising the recombined VH region is capable of specifically binding to a target with an affinity similar to that of the wild-type binding domain.

[0244] In certain embodiments, the VL region of the binding domain is derived from or generated based on the VL region of a known antibody and may contain, compared to the VL region of the known antibody, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of these changes. The insertions, deletions or substitutions may be made anywhere in the VL region, including at the amino or carboxy terminus or both, so long as each CDR contains no changes or at most one, two or three changes and the binding domain comprising the recombined VL region is capable of specifically binding to a target with an affinity similar to that of the wild-type binding domain.

[0245] Such considerations apply equally to TCR chains and other binding domains derived therefrom.

[0246] As described elsewhere herein, variants of a gene sequence include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that have no statistically significant effect on the function of the encoded product.

[0247] Variants of protein, nucleic acid and gene sequences also include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity to the protein, nucleic acid or gene sequences disclosed herein.

[0248] "Percent sequence identity" refers to the relatedness of two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of relatedness between protein, nucleic acid or gene sequences, as determined by the matching between strings of protein, nucleic acid or gene sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods of determining identity are designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and identity calculations may be performed using the Megalign program (DNASTAR, Inc., Madison, Wis.) in the LASERGENE suite of bioinformatics computing software.Multiple alignment of sequences can also be performed using the Clustal format alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty=10, gap length penalty=10)). Related programs further include the GCG suite of programs (Wisconsin package version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). In this disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted as being based on the "default values" that are the basis of the program. In this specification, "default values" refers to a set of numerical values ​​or parameters that are preregistered in the software at the time of initialization of the software.

[0249] Variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20μg / ml denatured salmon sperm DNA that has been fragmented, followed by washing the filter at 50°C with 0.1xSSC. Hybridization stringency and signal detection are altered primarily by adjusting the concentration of formamide (lower percentage of formamide results in lower stringency), salt conditions, or temperature. For example, moderately stringent conditions include overnight incubation at 37° C. in 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml blocking salmon sperm DNA, followed by washing with 1×SSPE and 0.1% SDS at 50° C. Even lower stringency is achieved by performing stringent post-hybridization washes at high salt concentrations (e.g., 5×SSC). The above conditions can be varied by adding and / or substituting other blocking reagents used to reduce the background of hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary preparations. The addition of certain blocking reagents may require some modification of the hybridization conditions described above due to compatibility issues.

[0250] (xvi) Representative embodiments 1. An activity-inducible fusion protein containing an hsp90 binding domain. 2. The activity-inducible fusion protein of embodiment 1, wherein the hsp90 binding domain binds to hsp90 with an affinity lower than that which binds to a drug molecule. 3. The activity-inducible fusion protein of embodiment 1 or 2, wherein the hsp90 binding domain comprises a hormone binding domain. 4. The activity-inducible fusion protein of embodiment 3, wherein said hormone binding domain is a recombinant binding domain (EBD) of the estrogen receptor. 5. The activity-inducing fusion protein of embodiment 4, wherein the EBD comprises a binding domain portion of an estrogen receptor and mutations selected from G521R; E353A; a combination of L384M and M421G; a combination of L384M, M421G and G521R; and a combination of G400V, M543A and L544A. 6. The activity-inducible fusion protein of embodiment 5, wherein the EBD has a sequence as set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. 7. The activity-inducible fusion protein of any one of embodiments 2 to 6, wherein the drug molecule comprises a small molecule estrogen analog. 8. The activity-inducible fusion protein of embodiment 7, wherein the small molecule estrogen analog comprises tamoxifen, a salt of tamoxifen, a metabolite of tamoxifen, or a compound structurally similar to tamoxifen. 9. The activity-inducible fusion protein of embodiment 7 or 8, wherein the small molecule estrogen analog comprises tamoxifen, 4-OHT, (Z)-endoxifen, ES8 or CMP8. 10. The activity-inducing fusion protein is a chimeric antigen receptor (CAR), and when expressed in a cell, it is expressed as a chimeric antigen receptor comprising an extracellular portion and an intracellular portion linked via a transmembrane domain; the extracellular portion comprises a ligand-binding domain; and the intracellular portion comprises an intracellular signaling domain and an hsp90 binding domain; 10. The activity-inducing fusion protein according to any one of embodiments 1 to 9, 11. The activity-induced fusion protein of embodiment 10, wherein the ligand-binding domain binds to a cancer antigen or a viral antigen. 12. The activity-induced fusion protein of embodiment 10 or 11, wherein the ligand binding domain comprises an scFv that binds to HER2, CE7, hB7H3, EGFR, EGFRvIII, CD19, CD20, CD22, EphA2, IL13Ra2, L1CAM, oaGD2, B7H3, CD33, mesothelin, ROR1, FITC, or VAR2CSA. 13. The activity-induced fusion protein of embodiment 12, wherein the scFv has a sequence as set forth in SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:342 or SEQ ID NO:43. 14. The activity-inducing fusion protein of embodiment 10, wherein the ligand-binding domain binds to an immune cell antigen. 15. The activity-inducible fusion protein of embodiment 14, wherein said immune cell antigen is an antigen expressed by B cells, T cells, natural killer cells, natural killer T cells, MAIT cells, myeloid cells, macrophages, monocytes or dendritic cells. 16. The activity-inducible fusion protein of embodiment 10, wherein the ligand-binding domain binds to a hapten. 17. The activity-inducible fusion protein of embodiment 16, wherein the hapten comprises fluorescein, urushiol, quinone, biotin, or dinitrophenol. 18. The activity-induced fusion protein of embodiment 16 or 17, wherein the ligand-binding domain is an scFv having the sequence set forth in SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65. 19. The activity-inducible fusion protein according to any one of embodiments 10 to 18, wherein the extracellular portion further comprises a spacer region. 20. The activity-induced fusion protein of embodiment 19, wherein the spacer region comprises an IgG4 hinge. 21. An activity-inducible fusion protein according to any one of embodiments 10 to 20, wherein the intracellular portion comprises the signal transduction domain of CD3ζ. 22. The activity-inducible fusion protein of any of embodiments 10 to 21, wherein the intracellular signaling domain comprises the signaling domain of 4-1BB. 23. An activity-inducible fusion protein according to any one of embodiments 10 to 22, wherein the intracellular signaling domain comprises the signaling domain of CD3ζ and the signaling domain of 4-1BB. 24. An activity-inducing fusion protein according to any one of embodiments 10 to 23, further comprising a linking amino acid sequence consisting of one glycine, two glycines or three glycines adjacent to the hsp90 binding domain, or no linker adjacent to the hsp90 binding domain. 25. The activity-inducible fusion protein of embodiment 24, wherein the linking amino acid consisting of one, two or three glycines, if present, is located between the 5' end of the hsp90 binding domain and the 3' end of the intracellular signaling domain. 26. The activity-inducible fusion protein according to any one of embodiments 10 to 25, wherein the hsp90-binding domain is located at the 3' end of the intracellular signaling domain. 27. An activity-inducible fusion protein according to any one of embodiments 10 to 25, wherein the hsp90 binding domain is located between the 5' end of the intracellular signaling domain and the 3' end of the transmembrane domain. 28. An activity-inducible fusion protein according to any one of embodiments 23 to 25, wherein the hsp90 binding domain is located between the 5' end of the CD3ζ portion of the intracellular signaling domain and the 3' end of the 4-1BB portion of the intracellular signaling domain. 29. An activity-inducible fusion protein according to any of embodiments 10 to 25, wherein the intracellular portion does not have a linker or a linking amino acid. 30. An activity-inducible fusion protein according to any one of embodiments 10 to 29, wherein the transmembrane domain comprises the transmembrane domain of CD28. 31. An activity-inducing fusion protein according to any one of embodiments 10 to 30, having the protein sequence shown in SEQ ID NO: 118. 32. The activity-inducible fusion protein of any of embodiments 1-31, wherein said hsp90 binding domain is linked to an intracellular portion of a costimulatory or inhibitory immune molecule. 33. The activity-inducible fusion protein of embodiment 32, wherein said costimulatory immune molecule comprises 4-1BB, OX40, CD40, CD30, CD27, DR3, SLAMF1, ICOS, GITR, CD25, CD28, CD79A, CD79B, CD226, CARD11, DAP10, DAP12, DR3, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, LIGHT, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TIM1, TRIM, Zap70, or PTCH2. 34. The activity-inducible fusion protein of embodiment 32, wherein the inhibitory immune molecule comprises PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD80, CD86, CD160, 2B4, B7-H3 (CD276), B7-H4 (VTCN1), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine or TGFR-β. 35. A nucleotide encoding an activity-inducible fusion protein according to any one of embodiments 1 to 34. 36. The nucleotide of embodiment 35, comprising a coding sequence as set forth in SEQ ID NO:7, SEQ ID NO:14 or SEQ ID NO:15. 37. The nucleotide of embodiment 36, further comprising the coding sequence set forth in SEQ ID NO: 17. 38. The nucleotide of embodiment 36 or 37, further comprising a coding sequence as set forth in SEQ ID NO:79. 39. The nucleotide of any one of embodiments 36 to 39, further comprising the coding sequence set forth in SEQ ID NO: 124. 40. The nucleotide of any one of embodiments 36 to 39, further comprising the coding sequence set forth in SEQ ID NO: 123. 41. The nucleotide according to any one of embodiments 36 to 40, further comprising the coding sequence set forth in SEQ ID NO: 122. 42. A nucleotide according to any one of embodiments 35 to 41, having the sequence shown in SEQ ID NO: 125. 43. A cell genetically engineered to express an activity-inducible fusion protein according to any one of embodiments 1 to 34. 44. The cell of embodiment 43, wherein the cell has been genetically engineered to express at least two activity-inducible fusion proteins of any one of embodiments 1 to 29, each of which has a different hsp90 binding domain, and each of which binds a different drug molecule. 45. The cell of embodiment 44, wherein the at least two activity-inducing fusion proteins further comprise different ligand-binding domains. 46. ​​The cell of embodiment 45, wherein the different ligand-binding domains each bind to a different antigen. 47. The cell of embodiment 46, wherein the different antigens are associated with a common cancer, a common infectious disease, a common immune cell type or a common hapten, or the different antigens are associated with different cancers, different infectious diseases, different immune cell types or different haptens. 48. The cell of embodiment 47, wherein the common cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) or brain cancer. 49. A cell according to any of embodiments 43 to 48, which is a T cell or a natural killer (NK) cell. 50. The cell of embodiment 49, wherein the T cell is a CD4+ T cell or a CD8+ T cell. 51. The cell according to any of embodiments 43 to 48, which is an induced pluripotent stem cell (iPSC), a tumor infiltrating lymphocyte (TIL), a bone marrow infiltrating lymphocyte (MIL), a natural killer T cell (NKT), a mucosal-associated invariant T (MAIT) cell, a dendritic cell, a monocyte or a macrophage. 52. A system for controlling the activation state of an activity-inducible fusion protein in vivo, comprising: comprising an activity-inducible fusion protein and a drug molecule, The system, wherein the activity-inducible fusion protein comprises an hsp90 binding domain that binds to the drug molecule. 53. The system of embodiment 52, wherein the hsp90 binding domain binds to hsp90 with an affinity lower than the affinity for binding to the drug molecule. 54. The system of embodiment 52 or 53, wherein the hsp90 binding domain comprises a hormone binding domain. 55. The system of embodiment 54, wherein the hormone binding domain comprises a recombinant binding domain (EBD) of the estrogen receptor. 56. The system of embodiment 55, wherein the EBD comprises a binding domain portion of an estrogen receptor and mutations selected from G521R; E353A; a combination of L384M and M421G; a combination of L384M, M421G and G521R; and a combination of G400V, M543A and L544A. 57. The system described in embodiment 55 or 56, wherein the EBD has a sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. 58. The system described in any of embodiments 52 to 57, wherein the drug molecule comprises a small molecule estrogen analogue. 59. The system of embodiment 58, wherein the small molecule estrogen analog comprises tamoxifen, a salt of tamoxifen, a metabolite of tamoxifen, or a compound structurally similar to tamoxifen. 60. The system of embodiment 58 or 59, wherein the small molecule estrogen analog comprises tamoxifen, 4-OHT, (Z)-endoxifen, ES8 or CMP8. 61. The activity-inducing fusion protein is a chimeric antigen receptor (CAR), and when expressed in a cell, it is expressed as a chimeric antigen receptor comprising an extracellular portion and an intracellular portion connected via a transmembrane domain; the extracellular portion comprises a ligand-binding domain; and the intracellular portion comprises an intracellular signaling domain and an hsp90 binding domain; A system described in any one of embodiments 52 to 60, characterized by: 62. The system of embodiment 61, wherein the ligand binding domain binds to a cancer antigen or a viral antigen. 63. The system of embodiment 61 or 62, wherein the ligand binding domain comprises an scFv that binds to HER2, CE7, hB7H3, EGFR, EGFRvIII, CD19, CD20, CD22, EphA2, IL13Ra2, L1CAM, oaGD2, B7H3, CD33, mesothelin, ROR1, FITC or VAR2CSA. 64. The system of embodiment 63, wherein the scFv has a sequence as set forth in SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 or SEQ ID NO:43. 65. The system of embodiment 61, wherein the ligand binding domain binds to an immune cell antigen. 66. The system of embodiment 65, wherein the immune cell antigen is an antigen expressed by B cells, T cells, natural killer cells, natural killer T cells, MAIT cells, myeloid cells, macrophages, monocytes or dendritic cells. 67. The system of embodiment 61, wherein the ligand binding domain binds to a hapten. 68. The system of embodiment 67, wherein the hapten comprises fluorescein, urushiol, quinone, biotin or dinitrophenol. 69. The system of embodiment 67 or 68, wherein the ligand binding domain is an scFv having the sequence shown in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65. 70. The system of any of embodiments 61-69, wherein the extracellular portion further comprises a spacer region. 71. The system of embodiment 70, wherein the spacer region comprises an IgG4 hinge. 72. A system described in any of embodiments 61 to 71, wherein the intracellular portion comprises the signaling domain of CD3ζ. 73. A system described in any of embodiments 61 to 72, wherein the intracellular signaling domain comprises the signaling domain of 4-1BB. 74. A system described in any of embodiments 61 to 73, wherein the intracellular signaling domain comprises the signaling domain of CD3ζ and the signaling domain of 4-1BB. 75. The system of any of embodiments 61-74, further comprising a linking amino acid sequence consisting of one glycine, two glycines, or three glycines adjacent to the hsp90 binding domain, or no linker adjacent to the hsp90 binding domain. 76. The system of embodiment 75, wherein, when a linking amino acid consisting of one, two or three glycines is present, the linking amino acid is located between the 5' end of the hsp90 binding domain and the 3' end of the intracellular signaling domain. 77. A system described in any of embodiments 61 to 76, wherein the hsp90 binding domain is located 3' to the intracellular signaling domain. 78. A system described in any of embodiments 61 to 76, wherein the hsp90 binding domain is located between the 5' end of the intracellular signaling domain and the 3' end of the transmembrane domain. 79. The system of any of embodiments 74-76, wherein the hsp90 binding domain is located between the 5' end of the CD3ζ portion of the intracellular signaling domain and the 3' end of the 4-1BB portion of the intracellular signaling domain. 80. The system of any of embodiments 61-79, wherein the intracellular portion does not have a linker or a linking amino acid. 81. A system described in any of embodiments 61 to 80, wherein the transmembrane domain comprises the transmembrane domain of CD28. 82. The system of any of embodiments 61 to 81, wherein the CAR has a protein sequence as set forth in SEQ ID NO: 118. 83. A system described in any of embodiments 52 to 82, wherein the activity-inducible fusion protein is a costimulatory immune molecule or an inhibitory immune molecule. 84. The system of embodiment 83, wherein the costimulatory immune molecule comprises 4-1BB, OX40, CD40, CD30, CD27, DR3, SLAMF1, ICOS, GITR, CD25, CD28, CD79A, CD79B, CD226, CARD11, DAP10, DAP12, DR3, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, LIGHT, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TIM1, TRIM, Zap70, or PTCH2. 85. The system of embodiment 83, wherein the inhibitory immune molecule comprises PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD80, CD86, CD160, 2B4, B7-H3 (CD276), B7-H4 (VTCN1), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine or TGFR-β. 86. A system described in any of embodiments 52 to 85, wherein the system comprises at least two activity-inducible fusion proteins, each of which has a different hsp90 binding domain, and each of which binds to a different drug molecule. 87. The system of embodiment 86, wherein the at least two activity-inducible fusion proteins further comprise different ligand-binding domains. 88. The system of embodiment 87, wherein the different ligand binding domains each bind to a different antigen. 89. The system of embodiment 88, wherein the different antigens are associated with a common cancer, a common infectious disease, a common immune cell type, or a common hapten, or the different antigens are associated with different cancers, different infectious diseases, different immune cell types, or different haptens. 90. The system of embodiment 89, wherein the common cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) or brain cancer. 91. A system described in any of embodiments 52 to 90, formulated for administration to a subject. 92. The formulated system, (i) ex vivo produced cells expressing said activity-inducible fusion protein in the form of a recombinant formulation in a pharma- ceutically acceptable carrier; and / or (ii) a cell-targeted viral vector and / or a cell-targeted nanoparticle in the form of a recombinant derivative formulation in a pharma- ceutically acceptable carrier; (iii) the drug molecule in the form of a drug composition formulated in a pharma- ceutically acceptable carrier; Including, the cell-targeted viral vector and / or the cell-targeted nanoparticle comprises a genetic recombination-inducing component that induces expression of the activity-inducible fusion protein in targeted cells in vivo after administration; The system described in embodiment 91. 93. A method of treating a subject in need of treatment, comprising administering to the subject a system described in embodiment 92, thereby treating the subject. 94. The method of embodiment 93, wherein the administering comprises administering the recombinant formulation and the pharmaceutical composition. 95. The method of embodiment 93, wherein said administering comprises administering said recombinant induction preparation and said pharmaceutical composition. 96. The method of any of embodiments 93-95, further comprising the step of discontinuing administration of the drug molecule to prevent side effects due to administration of the system. 97. The method of any of embodiments 93-96, further comprising discontinuing administration of the drug molecule when the subject no longer requires treatment. 98. The method of any of embodiments 93-97, wherein the subject is in need of treatment for cancer or a viral infection. 99. A method according to any of embodiments 93 to 98, wherein the system comprises at least two activity-inducible fusion proteins, each of which has a different hsp90 binding domain, and each of which binds to a different drug molecule. 100. The method of embodiment 99, comprising administering at least two drug molecules, one of which binds to the hsp90 binding domain of one activity-inducible fusion protein contained in the system, and another of which binds to the hsp90 binding domain of another activity-inducible fusion protein of the system. 101. The method of embodiment 100, further comprising discontinuing administration of at least one drug molecule. 102. The method of embodiment 100 or 101, further comprising discontinuing administration of all drug molecules. 103. The method of any one of embodiments 99-103, wherein the at least two activity-inducing fusion proteins further comprise different ligand-binding domains. 104. The method of embodiment 103, wherein the different ligand binding domains each bind to a different antigen. 105. The method of embodiment 104, wherein the different antigens are associated with a common cancer, a common infectious disease, a common immune cell type, or a common hapten, or the different antigens are associated with different cancers, different infectious diseases, different immune cell types, or different haptens. 106. The method of embodiment 105, wherein the common cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) or brain cancer. 107. An activity-inducible fusion protein according to any one of embodiments 1 to 34, which does not contain a degron sequence. 108. An activity-inducing fusion protein according to any one of embodiments 1 to 34 and 107, wherein the activity is induced within 1 hour after administration of a drug molecule that binds to the HSP binding domain. 109. An activity-inducing fusion protein according to any one of embodiments 1 to 34 and 107, wherein the activity is induced within 30 minutes after administration of a drug molecule that binds to the HSP binding domain. 110. An activity-inducing fusion protein according to any of embodiments 1 to 34 and 107, wherein the activity is induced within 15 minutes after administration of a drug molecule that binds to the HSP binding domain. 111. An activity-inducible fusion protein according to any of embodiments 1 to 34 and 107, wherein the activity is induced within 5 minutes after administration of a drug molecule that binds to the HSP binding domain.

[0251] (xvii) Experimental Examples Described in Figures 9 to 17 Further evaluation of the CAR-EBD constructs was performed to investigate the location of the EBD domain in the CAR construct and the linker linking the EBD domain to the CAR construct. Various CAR-EBD constructs were investigated, including a glycine linker consisting of one, two or three glycines (also referred to herein as "linking amino acids") located after (i.e., 3') CD3ζ (data shown). In addition, CAR-EBD constructs without a glycine linker, constructs in which the EBD domain, flanked by a three glycine linker, is located between 4-1BB and CD3ζ, and constructs in which the EBD domain is fused directly between 4-1BB and CD3ζ (no glycine linker) were designed. Example experiments included: huCD19 CAR-Gly1-EBD-P2A-DHFRdm-Her2tG; huCD19 CAR-Gly2-EBD-P2A-DHFRdm-Her2tG; huCD19 CAR-Gly3-EBD-P2A-DHFRdm-Her2tG; huCD19 CAR-EBD-P2A-DHFRdm-Her2tG; EGFR806scFv-IgG4hinge-CD28tm-4-1BB-Gly3-EBD-Gly3-CD3zeta-P2A-DHFRdm-CD19t; and EGFR806scFv-IgG4hinge-CD28tm-4-1BB-EBD-CD3zeta-P2A-DHFRdm-CD19t It included: The EBD may be, for example, EBD(4-OHT), EBD(CMP8) or EBD(ES8).

[0252] 9-17 present data for two EBD mutants shown below. EBD(4-OHT) (SEQ ID NO: 13): Responsive to 4-OHT and (Z)-endoxifen. EBD(CMP8) (sequence number 11): Responsive to 4-OHT, (Z)-endoxifen, and CMP8.

[0253] Figure 9 (top) shows a schematic diagram of Jurkat iSynPro eGFP:ffluc reporter cells. The Jurkat iSynPro eGFP:ffluc reporter cells are a Jurkat cell line transduced with lentivirus to stably express an inducible synthetic promoter (iSynPro; WO2018213332A1) that controls the expression of enhanced green fluorescent protein (eGFP) fused to the bioluminescence reporter firefly luciferase (ffluc). Clones were then selected from this cell line by limiting dilution. Upon activation in T cells, the Jurkat iSynPro eGFP:ffluc line induces the expression of GFP detectable by flow cytometry, and luciferase activity can be measured by addition of D-luciferin as a substrate to catalyze the luciferase reaction.

[0254] The results of the Jurkat iSynPro eGFP:ffluc assay are shown in Figure 9 (bottom). Clones of the Jurkat iSynPro eGFP:ffluc reporter cell line were transduced with the huCD19 CAR as described above, various huCD19 EBD(4-OHT)CAR constructs with different glycine linkers (Gly1-3), B7H3 CAR, B7H3 EBD(4-OHT)CAR or B7H3 EBD(CMP8)CAR and selected with MTX when possible. 24 hours prior to the assay, transduced T cells were co-cultured with the target cell line (K562+CD19 was used as target cells for the huCD19 CAR and K562 parental cells were used as target cells for the B7H3 CAR) in the presence of various concentrations of drug (4-OHT or CMP8). After 24 hours, cells were harvested and evaluated by flow cytometry. First, cells were stained with CD33 to gate out the target cell lines, and then GFP expression was assessed by positivity rate and / or mean fluorescence intensity (MFI). Next, each cell line was stained for transduction markers to confirm expression of the various CAR-EBD constructs.

[0255] In Figure 10, Figure 14 and Figure 15, we present the results of a study on huCD19-EBD(4-OHT) CAR with a glycine linker. Evaluation of activation curves plotting GFP expression by various huCD19-EBD(4-OHT) CARs with different glycine linkers showed that activation (on / off state) occurred in a drug-dependent manner and that different linkers had different effects on inducibility and leaky expression of the construct. For example, in this study, it was observed that the Gly3 linker mutant could be turned on at a lower drug concentration and reached maximum induction at a lower drug concentration compared to the Gly1 linker mutant and the Gly2 mutant.

[0256] The tamoxifen metabolite (Z)-endoxifen was further added to the experiment, and the ability to activate in the absence of target was investigated by repeating the experiment with a wider range of drug concentrations and a drug-added control group without tumor cells.Evaluation of mean fluorescence intensity (MFI) showed that both glycine linker constructs could be switched on / off by 4-OHT or (Z)-endoxifen, yielding similar activation curves.

[0257] Figures 11-13, 16 and 17 show the results of various B7H3-EBD CAR mutant studies. The first study was performed with the aim of confirming the functionality of the B7H3 CAR ERT2 (EBD(4-OHT)) mutant responsive to 4-OHT, or the EBD(CMP8) mutant responsive to both 4-OHT and CMP8 (EBD[L348M, M431G, G521R] mutant). Drug concentrations were chosen to be 0 nM, 500 nM or 1000 nM. For both EBD mutants (EBD(4-OHT) or EBD(CMP8)), a clear on / off state was observed, indicating responsiveness to 4-OHT. Furthermore, responsiveness to CMP8 was observed only in the CMP8-responsive EBD(CMP8) mutant ([L348M, M431G, G521R]), and no responsiveness to CMP8 was observed in the EBD(4-OHT) mutant (not shown).

[0258] Next, we performed a broad-range activation curve study in which the B7H3-EBD(4-OHT) CAR showed clear on / off states, indicating drug-dependent activation, whereas the CMP8-responsive EBD(CMP8) mutant showed both 4-OHT-induced and CMP8-induced expression even at a drug concentration of 0.49 nM.

[0259] Broad-range activation curve studies were repeated by adding the tamoxifen metabolite (Z)-endoxifen to the experiment and using a wider range of drug concentrations. Mean fluorescence intensity (MFI) assessment confirmed the responsiveness of the CMP8 (EBD(CMP8)[L348M, M431G, G521R]) mutant to 4-OHT or CMP8, and demonstrated that (Z)-endoxifen could also induce the system. Testing of EBD(4-OHT)-responsive mutants also confirmed that 4-OHT or (Z)-endoxifen, but not CMP8, could induce the system.

[0260] These studies demonstrated that all constructs were capable of inducing on / off states with each drug.

[0261] (xviii) Conclusion The nucleic acid and amino acid sequences provided herein are represented by the abbreviations used for nucleotide bases and amino acid residues as set forth in 37 CFR 1.822 and as set forth in Tables 1 and 3 of WIPO Standard ST.25 (1998) Supplement 2. Only one strand is shown for each nucleic acid sequence, although the complementary strand, if appropriate, is also included in the embodiments.

[0262] As used herein, the terms "specific binding affinity," "specifically binds," "specific binding," or "specific target" refer to one molecule binding to another molecule with a binding affinity higher than background binding. 5 M -1 A binding domain "specifically binds" to a target molecule if it binds to or associates with the target molecule with an affinity or Ka (i.e., the equilibrium binding constant of a particular binding interaction, expressed in units of 1 / M) of 10 or more. 6 M -1 That's it, 10 7 M -1 That's it, 10 8 M -1 That's it, 10 9 M -1 That's it, 10 10 M -1 That's it, 10 11 M -1 That's it, 10 12 M -1 Greater than or equal to 10 13 M -1 A "high affinity" binding domain binds to a target with a Ka of at least 10 7 M -1 At least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or higher.

[0263] Alternatively, affinity can be expressed as the equilibrium dissociation constant (Kd) (units: M) of a particular binding interaction (e.g., 10 -5 M~10 -13M or less). The affinity of the binding domain and CAR protein according to the present disclosure can be readily measured using conventional techniques, such as, for example, competitive ELISA (enzyme-linked immunosorbent assay); binding (association); displacement assays using labeled ligands; surface plasmon resonance instruments such as the Biacore T100 available from Biacore, Inc. (Piscataway, NJ); optical biosensor technology such as the EPIC system available from Corning and EnSpire available from PerkinElmer (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; U.S. Patent Publication No. 5,283,173; U.S. Patent Publication No. 5,468,614).

[0264] In certain embodiments, the affinity of specific binding is 2 times background binding, 5 times background binding, 10 times background binding, 20 times background binding, 50 times background binding, 100 times background binding, 1000 times background binding, or more.

[0265] As used herein, "derived from" refers to the relationship between a first molecule and a second molecule. The term "derived from" generally refers to the structural similarity between a first molecule and a second molecule, and does not mean or include any limitation on the process or source for obtaining a first molecule derived from a second molecule. For example, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure to perform the required function, i.e., the ability to generate a signal, under appropriate conditions. This does not mean or include any limitation on the specific process for obtaining the intracellular signaling domain, and does not mean, for example, that the intracellular signaling domain is prepared by starting with the CD3ζ sequence and deleting undesirable sequences or introducing mutations to create the intracellular signaling domain.

[0266] As will be appreciated by those skilled in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the particular components, steps, materials, or ingredients described. Thus, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consists essentially of, or consists of." The transitional phrase "comprise" means, but is not limited to, that the components, steps, materials, or ingredients not described are included, even if in large amounts. The transitional phrase "consisting of" excludes all components, steps, materials, or ingredients not described. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the components, steps, materials, or ingredients described and those components, steps, materials, or ingredients that do not significantly affect the embodiment. A significant effect refers to an effect that statistically significantly reduces the ability to activate cells expressing the activity-inducing fusion protein (e.g., binding of an antigen to a CAR; binding of a ligand to a costimulatory or inhibitory molecule) in the presence of the drug molecule involved and the physiological conditions involved.

[0267] Unless otherwise indicated, all numerical values ​​expressing quantities or properties of materials, such as molecular weight and reaction conditions, in the specification and claims are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding procedures. For clarity, the term "about," when used in conjunction with a stated value or range, has a meaning that would be reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; within ±19% of the stated value; within ±18% of the stated value; within ±17% of the stated value; within ±16% of the stated value; within ±15% of the stated value; within ±14% of the stated value; within ±13% of the stated value; within ±12% of the stated value; within ±11% of the stated value; within ±10% of the stated value; within ±9% of the stated value; within ±8% of the stated value; within ±7% of the stated value; within ±6% of the stated value; within ±5% of the stated value; within ±4% of the stated value; within ±3% of the stated value; within ±2% of the stated value; or within ±1% of the stated value.

[0268] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation associated with their respective testing measurements.

[0269] In the description of the present invention (particularly in the description of the claims below), the terms "a", "an", "the" and similar modifiers are intended to include both the singular and the plural unless otherwise indicated or the context clearly indicates otherwise. Numerical ranges described herein are intended to be a shorthand way of referring to each numerical value falling within the range individually. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any method described herein can be performed in any suitable order unless otherwise indicated or the context clearly indicates otherwise. The use of any examples or language of examples (e.g., "etc.") provided herein is intended to be for the purpose of illustrating the invention only and does not limit the scope of the invention as described in the claims. No term described herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0270] Groupings of other elements of the invention disclosed herein or of various embodiments of the invention should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be added to another group, or one or more members may be deleted from a group. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0271] Specific embodiments of the present invention are described herein, including those embodiments known to the inventors to be the best mode for carrying out the invention. Of course, those skilled in the art will readily appreciate that the embodiments described herein may be modified in various ways upon review of the above detailed description. The inventors anticipate that such modifications may be adopted by those skilled in the art, and intend that the present invention may be practiced in other ways than as specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter of the present invention to the extent permitted within the scope of applicable law. Moreover, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless otherwise indicated or the context clearly dictates otherwise.

[0272] Additionally, throughout this specification, various patents, publications, journal articles and other documents are cited (references herein). Each reference cited herein is individually incorporated herein by reference for the teachings thereof as if it were a part of this specification.

[0273] Finally, the embodiments of the invention disclosed herein are to be considered as illustrative of the principles of the invention. Other modifications may be adopted within the scope of the invention. Thus, by way of example, but not by way of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not to be limited to what has been precisely shown and described herein.

[0274] The details described herein are by way of example and are presented solely for the purpose of illustrating preferred embodiments of the present invention, to provide what is believed to be the most useful, and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no structural details of the present invention are described in more detail than is necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by reading the description of the present invention in conjunction with the drawings and / or examples.

[0275] The definitions and explanations used in this disclosure are intended to control future interpretations, unless obvious and unambiguous changes are made in the following examples, or the meaning of the terms makes the interpretation meaningless or substantially meaningless. If the definition of a term does not make sense or substantially meaningless from the interpretation of the term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. An activity-inducible fusion protein comprising an intracellular signaling domain of (i) a costimulatory immune molecule or (ii) an inhibitory immune molecule and an hsp90-binding domain.

2. The activity-inducible fusion protein of claim 1, wherein the hsp90 binding domain binds to a drug molecule and the hsp90 binding domain binds to hsp90 with an affinity lower than the affinity for binding to the drug molecule.

3. The activity-inducible fusion protein of claim 1 , wherein the hsp90 binding domain comprises a recombinant estrogen binding domain (EBD) of the estrogen receptor.

4. The activity-inducible fusion protein of claim 3, wherein the recombinant EBD comprises an estrogen receptor binding domain portion and mutations selected from G521R; E353A; a combination of L384M and M421G; a combination of L384M, M421G and G521R; and a combination of G400V, M543A and L544A.

5. The activity-inducible fusion protein of claim 3, wherein the recombinant EBD has the sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:

13.

6. The activity-inducible fusion protein of claim 2 , wherein the drug molecule comprises a small molecule estrogen analog.

7. 7. The activity-inducible fusion protein of claim 6, wherein the small molecule estrogen analog comprises tamoxifen, a salt of tamoxifen, a metabolite of tamoxifen, or a compound structurally similar to tamoxifen.

8. The activity-inducible fusion protein of claim 6 , wherein the small molecule estrogen analog comprises tamoxifen, 4-OHT, endoxifen, ES8 or CMP8.

9. the activity-inducing fusion protein is a chimeric antigen receptor (CAR), and when expressed in a cell, it is expressed as a chimeric antigen receptor comprising an extracellular portion and an intracellular portion linked via a transmembrane domain; the extracellular portion comprises a ligand-binding domain; and the intracellular portion comprises an intracellular signaling domain and an hsp90 binding domain of a costimulatory immune molecule; The activity-inducing fusion protein according to claim 1 ,

10. The activity-inducing fusion protein of claim 9 , wherein the ligand-binding domain binds to a cancer antigen, a viral antigen, an immune cell antigen, or a hapten.

11. The activity-induced fusion protein described in claim 9, wherein the transmembrane domain comprises the transmembrane domain of CD28 and the intracellular signaling domain comprises the intracellular signaling domain of 4-1BB and the intracellular signaling domain of CD3ζ.

12. The activity-inducing fusion protein of claim 9, further comprising a linking amino acid sequence consisting of one glycine, two glycines or three glycines adjacent to the hsp90 binding domain, or comprising no linker or linking amino acids adjacent to the hsp90 binding domain.

13. The activity-inducible fusion protein of claim 1 , which does not contain a degron sequence.

14. 2. The activity-inducible fusion protein of claim 1, wherein the costimulatory immune molecule comprises 4-1BB, OX40, CD40, CD30, CD27, DR3, SLAMF1, ICOS, GITR, CD25, CD28, CD79A, CD79B, CD226, CARD11, DAP10, DAP12, DR3, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, LIGHT, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TIM1, TRIM, Zap70 or PTCH2.

15. The activity-inducing fusion protein of claim 1, wherein the inhibitory immune molecule comprises PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD80, CD86, CD160, 2B4, B7-H3 (CD276), B7-H4 (VTCN1), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine or TGFR-β.

16. Nucleotides encoding an activity-inducible fusion protein, the activity-inducible fusion protein comprising an intracellular signaling domain of (i) a costimulatory immune molecule or (ii) an inhibitory immune molecule, and an hsp90 binding domain.

17. 17. The nucleotide of claim 16, comprising the coding sequence shown in SEQ ID NO:7, SEQ ID NO:14 or SEQ ID NO:

15.

18. The nucleotide of claim 16 having the sequence shown in SEQ ID NO:

125.

19. A pharmaceutical composition for treating cancer or a viral infection, comprising the nucleotide described in claim 16 as an active ingredient.

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