Activity-inducible fusion protein with a heat shock protein 90 binding domain

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 of related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 149,131, filed on 12 February 2021, and U.S. Provisional Patent Application No. 63 / 226,554, filed on 28 July 2021, the contents of which are incorporated herein by reference in their entirety.

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

[0003] Significant progress has been made in methods of activating immune system cells and using these immune cells to kill cancer cells and infected cells through genetic engineering. For example, T cells can be genetically modified to express molecules that have an extracellular portion that binds to a specific target antigen and an intracellular portion that induces the action of T cells when the extracellular portion binds to the target antigen. As an example, the extracellular portion can be designed to bind to a target antigen found on cancer cells or infected cells. When the target antigen of cancer cells or infected cells binds to this extracellular portion, the intracellular portion activates T cells, and the cells bound to the extracellular portion are destroyed by the T cells. An example of such a molecule is the chimeric antigen receptor (CAR).

[0004] While T cells expressing CARs can exhibit potent antitumor activity, they may also exhibit significant toxicity. For example, transplanted cells can induce cytokine storms (cytokine release syndrome), tumor lysis syndrome (TLS), and chronic B cell reduction, all of which are caused by the unregulated functioning of constitutively expressed active CARs. Such toxicity can limit the applicability of CAR-based therapies.

[0005] While methods to eliminate CAR-T cells, such as using suicide genes to remove them, can mitigate such toxicity, this method risks reducing antitumor activity before it can be fully exerted, thus significantly impacting therapeutic effectiveness. Therefore, it is necessary to find ways to control CAR activity after it has been expressed by genetically modified immune cells.

[0006] Besides CARs, there are many other beneficial proteins whose activation state can be controlled. Further examples include transmembrane receptors, such as those that transmit co-stimulatory and inhibitory signals in immune cells. [Overview of the Initiative] [Means for solving the problem]

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

[0008] An example of a fusion protein of this disclosure is a CAR whose ability can be activated by antigen binding in vivo, and whose ability can be controlled by the administration of a drug molecule. If activation can be controlled after in vivo expression, safety, which is important in immunotherapy using CARs, can be improved.

[0009] This disclosure achieves such progress by incorporating an hsp90-binding domain into the intracellular portion of the CAR. In the absence of drug molecules, hsp90 can bind to this hsp90-binding domain, preventing other important intracellular molecules necessary for the activation of the CAR after antigen binding from interacting with the CAR.

[0010] When a drug molecule is present, it displaces the bound HSP90 from its HSP90-binding domain and / or causes a conformational change, leading to antigen binding and subsequent intracellular signal transduction.

[0011] In certain embodiments disclosed herein, a hormone-binding domain is used as the hsp90-binding domain. This hormone-binding domain may be an estrogen receptor-binding domain (EBD). The EBD may be derived from a natural estrogen receptor, but it must contain at least one mutation so that it binds to a drug molecule with higher affinity than hsp90 without binding to estrogen. Examples of drug molecules include tamoxifen or its derivatives or metabolites with fewer side effects, such as 4-hydroxytamoxifen (4-OHT), CMP8, and ES8.

[0012] In the absence of tamoxifen or its derivatives or metabolites, hsp90 binds to EBD and CAR is in the "off" state. When nanomolar concentrations of tamoxifen enter the cytoplasm, tamoxifen acts actively to overcome hsp90 in binding to EBD and / or induce a conformational change, and when CAR binds to the antigen, an activation signal is transmitted by CAR.

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

[0014] One of the advantages of this disclosure is that the activity of single-chain proteins can be controlled without relying on dimerization or polymerization with other proteins, and without relying on protein stabilization / destabilization, such as the incorporation of degron sequences. [Brief explanation of the drawing]

[0015] Some of the drawings submitted in this application are considered easier to understand in color. The applicants consider color versions of these drawings to be part of the original application and reserve the right to submit color images of the drawings in subsequent proceedings.

[0016] [Figure 1A-1C] The figures visually demonstrate that EBD (e.g., ERT2, EBD(CMP8), or EBD(ES8)) plays a specific role in CAR signaling. (Figure 1A) In the absence of drug molecules, heat shock protein 90 (hsp90) binds to EBD, sterically repressing the interaction between the signaling domains of CD3ζ and 4-1BB and lymphocyte-specific protein tyrosine kinase (LCK), a SRC family kinase, thereby suppressing LCK-mediated phosphorylation of CD3ζ and TCRζ chain-related protein kinase (ZAP70). (Figures 1B, 1C) However, upon addition of a drug molecule (e.g., 4-hydroxytamoxifen (4-OHT), CMP8, or ES8), hsp90 dissociates, and the CAR binding domain (e.g., scFv) interacts with the relevant antigen, allowing the two signaling domains (CD3ζ and 4-1BB) to perform normal signaling.

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

[0018] [Figure 3] This shows Jurkat-Dual NF-κB-Luc reporter T cells. Jurkat-Dual cells are characterized by a Lucia luciferase gene (a secreted luciferase reporter gene) driven by a promoter fused to five copies of the NF-κB consensus transcription reaction element. Secreted Lucia luciferase is measured as an indicator of the NF-κB response mediated by CAR activation.

[0019] [Figure 4A-4B]Jurkat-Dual NF-κB reporter cells were transduced with either 806EGFR CAR or 806EGFR-ERT2 CAR, and selected with MTX if possible. Transduced cells and K562+EGFRvIII cells were co-cultured in the presence of 4-OHT at various concentrations (0-16,000 nM) 24 hours prior to each assay. After 24 hours, 10 μl of the supernatant was collected and mixed with 50 μl of QUANTI-Luc, and immediately read using SpectraMax. Before each assay, transduced Jurkat-DUAL cells were collected by flow cytometry. (Figure 4A) Flow cytometry results for CD19t (CD19 APC) and (Figure 4B) Flow cytometry results for EGFRt (cetuximab APC) confirmed that 806EGFR CAR or 806EGFR-ERT2 CAR were expressed in Jurkat-DUAL cells, respectively.

[0020] [Figure 5A-5B] The Jurkat Dual NF-κB-Luc reporter assay is shown. Secreted Lucia luciferase was measured by an endpoint luminescence assay. This assay was performed with an effector-to-target ratio of 2:1 (Figure 5A) or 1:1 (Figure 5B). Prior to this assay, Jurkat-DUAL cells transduced with 806EGFR CAR or 806EGFR-ERT2 CAR were stimulated with K562+EGFRvIII cells and a predetermined concentration of 4-OHT for 24 hours. Both cell lines showed an 80% CAR positivity rate. 4-OHT was added 24 hours prior to the assay. Selection of cells not sensitive to MTX was performed the day before the assay. Jurkat-DUAL cells transduced with 806EGFR-ER2T CAR showed an increase in secreted Lucia luciferase concentration with increasing 4-OHT concentration, indicating that these CAR T cells are activated in a dose-dependent manner. As mentioned above, ERT2 is also referred to as EBD(4-OHT) in this specification, and its sequence is shown in Sequence ID No. 13.

[0021] [Figure 6A-6B] The image shows cells on day 6 after rapid expansion culture following stimulation (S1R1D6). Post-stimulation culture (S1): Mock cells, 806EGFR CAR T cells (Figure 6B), and 806EGFR-ERT2 CAR T cells (Figure 6A) were grown by rapid expansion culture (REP) using irradiated feeder cells (20 × 10⁶ TM-LCL and 100 × 10⁶ PBMC) in the presence of rhIL-2, rhIL-15, and exogenous OKT3 antibody. On day 6 of the REP method, the CAR positivity rate and the proportion of CD4 T cells and CD8 T cells were measured by flow cytometry. The results were: Mock (40% CD4 / 60% CD8); 806 CAR (30% CD4 / 70% CD8); 806-ERT2 (46% CD4 / 53% CD8).

[0022] [Figures 7A-7B] The 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 EGFR806 CAR (Figure 7A) or primary T cells expressing EGFR806-ERT2 CAR (Figure 7B). Lymphocytes / single cells / live cells / CD4 / CD19t were gated, and CD4 T cells containing CARs were evaluated. 4-OHT was added 24 hours before the assay, and incubation was performed for 4 hours with the addition of 4-OHT. 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)-endoxiphene (on state).

[0023] [Figure 7C]The intracellular cytokine staining (ICCS) results are shown. The CD107a degranulation assay is also shown. Primary T cells expressing EGFR806 CAR or EGFR806-ERT2 CAR were used for a 4-hour intracellular cytokine staining (ICCS) assay. Lymphocytes / single cells / live cells / CD8 / CD19t were gated, and CD8 T cells containing CARs were evaluated. 4-OHT at each dose level (20 nM, 100 nM, and 500 nM) was added 24 hours before the assay, and the cells were incubated with the addition for 4 hours. In the case of EGFR806-ERT2 CAR, the expression of the CD107a marker increased with increasing 4-OHT concentration, indicating increased degranulation.

[0024] [Figure 7D] The intracellular cytokine staining (ICCS) is shown. The early activation marker Nur77 is shown. A 4-hour intracellular cytokine staining (ICCS) assay was performed using primary T cells expressing either the EGFR806 CAR or the EGFR806-ERT2 CAR. Lymphocytes / single cells / live cells / CD8 / CD19t were gated, and CD8 T cells containing the CAR were evaluated. 4-OHT was added 24 hours before the assay, and incubation was performed for 4 hours with the addition of 4-OHT. The EGFR806-ERT2 CAR expresses the early activation marker Nur77 in EGFRvIII+ target cells only in the presence of 500 nM 4-OHT or (Z)-endoxiphene (on state).

[0025] [Figures 8A-8D]The chromium release assay is described below. A 4-hour chromium release assay was performed using primary T cells expressing either EGFR806 CAR or EGFR806-ERT2 CAR. EGFR806-ERT2 CAR T cells were added to ensure consistency with 806EGFR CAR T cells, resulting in a CAR positivity rate of 78%. 4-OHT was added 18 hours prior to the assay, and a 4-hour incubation was performed with the 4-OHT still present. EGFR806-ERT2 CAR was only able to lyse EGFRvIII+ target cells in the presence of 4-OHT or (Z)-endoxiphene. 4-OHT or (Z)-endoxiphene was added 24 hours prior to the assay and during incubation. Figure 8A shows K562 parental cells on day 10 (S1R1D10) after rapid expansion culture following stimulation; Figure 8B shows K562 + OKT3 (positive control); Figure 8C shows K562 + EGFRvIII; and Figure 8D shows chromium release assays. Figure 8D is a graph showing each dose classified into 4-OHT or (Z)-endoxiphene.

[0026] [Figure 9] The top panel shows a schematic diagram of the Jurkat iSynPro:GFP-ffluc model, and the bottom panel shows results related to this model.

[0027] [Figure 10] The activation curves of multiple huCD19-EBD (ERT2, also referred to herein as EBD(4-OHT) (SEQ ID NO: 13)) with different glycine linkers are shown.

[0028] [Figure 11]This paper outlines the design of a B7H3 CAR EBD mutant study. The study aimed to investigate the EBD incorporated into B7H3CAR and to examine the activation efficacy of two EBD mutants (EBD(4-OHT) and EBD(CMP8)) when interacting with estrogen analogs (4-OHT or CMP8), and to investigate the differences between the two EBD mutants in this regard. The following Jurkat iSynPro strains were used: 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)). K562 parental cells were used as the target cells. Each cell line was tested with concentrations of 4-OHT and CMP8 at 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 CARs using EBD(4-OHT (SEQ ID NO: 13)) or EBD(CMP8) (containing L384M, M421G, and G521R mutations (SEQ ID NO: 11)), indicating that activation was regulated. In this figure, EBD(CMP8) is shown as B7H3 ERT2-(L384M|M421G|G521R(CMP8)).

[0030] [Figure 13] The activation curves for the B7H3 CAR ERT2 mutant (SEQ ID NO: 13 (shown in medium gray and diamond shape)) and the B7H3 CAR EBD(CMP8) mutant (SEQ ID NO: 11 (shown in light gray and square, or dark gray and circular shape)) are shown.

[0031] [Figure 14]The design of the activation curve study is presented. This study aimed to investigate the activation curves of CAR EBD Jurkat iSynPro strains as the concentration of 4-OHT or (Z)-endoxiphene was gradually increased. The following Jurkat iSynPro strains were used: 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 used as targets. This study was conducted using a 1:2.5 drug dilution series. For all drugs used in the study, the highest concentration was set at 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] This paper presents the dose-response relationship and comparisons of various huCD19 CAR-EBD linker variants.

[0033] [Figure 16]The design of the activation curve study is presented. This study aimed to investigate the activation curves of CAR EBD Jurkat iSynPro strains as the concentrations of 4-OHT, (Z)-endoxifene, or CMP8 were increased stepwise. The Jurkat iSynPro strains 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 constructed by starting with a concentration of 1000 nM for 4-OHT and CMP8, and with a concentration of 6250 nM for (Z)-endoxifene.

[0034] [Figure 17] This paper presents the 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)).

[0035] [Figure 18]This study demonstrates drug-dependent specific cell lysis by various B7H3CAR-3Gly-EBD mutants in the presence of estrogen analogs 4-OHT, CMP8, or (Z)-endoxiphene. Specific cell lysis was evaluated using a chromium release assay. First, cells containing the B7H3CAR-3Gly-EBD(4OHT) mutant or the B7H3CAR-3Gly-EBD(CMP8) mutant were cultured for 24 hours in the presence of three estrogen analogs (4-OHT, CMP8, or (Z)-endoxiphene) at various drug concentrations (0nM, 1nM, 50nM, or 500nM). These cells were then co-cultured for 4 hours with antigen-expressing tumor lines pre-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. The measurements showed that no specific cell lysis was observed in any of the CAR-EBD mutants when no estrogen analog was added. It was found that the activity of B7H3CAR-3Gly-EBD(4OHT) is regulated by 4OHT or (Z)-endoxifene, but not by CMP8. This result was demonstrated by the 0% specific lysis observed in the presence of 500 nM CMP8. The activity of B7H3CAR-3Gly-EBD(CMP8) is regulated by all three estrogen analogs. Typically, 1 nM estrogen analog is insufficient to induce maximum activity. Antigen-specific cell lysis was most frequently observed with both 50 nM and 500 nM estrogen analogs, regardless of the type of EBD mutant. When the concentrations of each estrogen analog were gradually increased during culture, it was shown that specific cell lysis induced by B7H3CAR-EBD activity is dose-dependent.

[0036] [Figures 19A-19B](Figure 19A) Shows drug-dependent cytokine release by various B7H3CAR-3Gly-EBD mutants in the presence of estrogen analogs 4-OHT, CMP8, or (Z)-endoxiphene. (Figure 19B) Shows drug-dependent cytokine release by various B7H3CAR-3Gly-EBD mutants in the presence of estrogen analogs 4-OHT, CMP8, or (Z)-endoxiphene. (Figures 20A and 20B) Cytokine release was evaluated using an assay from Meso Scale Diagnostics. First, cells containing the B7H3CAR-3Gly-EBD(4OHT) mutant or the B7H3CAR-3Gly-EBD(CMP8) mutant were cultured for 24 hours in the presence of three estrogen analogs (4-OHT, CMP8, or (Z)-endoxiphene) at various drug concentrations (0nM, 1nM, 50nM, or 500nM). These cells were then co-cultured for 24 hours with various tumor lines expressing B7H3 (the target antigen) or with B7H3 knocked out (KO). After 24 hours of incubation, the supernatant was collected from each co-culture and measured using a MESO QuickPlex SQ 120 instrument to confirm cytokine release. The results showed that, when co-cultured with a tumor line not expressing the antigen (K562 B7H3 KO), cytokines were not released by any of the B7H3CAR-EBD mutants, regardless of the drug conditions. The activity of B7H3CAR-3Gly-EBD(4OHT) was found to be regulated by 4OHT or (Z)-endoxifen, but not by CMP8. The activity of B7H3CAR-3Gly-EBD(CMP8) is regulated by any of the three estrogen analogs. Typically, 1 nM estrogen analogs are insufficient to induce maximum activity. With estrogen analogs ranging from 50 nM to 500 nM, antigen-specific cytokine release was most abundant, regardless of the type of CAR-EBD mutant. When the concentrations of each estrogen analog were gradually increased and cultured, cytokine release by all CAR-EBD mutants was shown to be dose-dependent.

[0037] [Figures 20A-20B] (Figure 20A) Shows the difference in glycine linker-dependent target cell lysis when huCD19CAR-EBD(4-OHT) targeting K562+CD19 tumor cells is combined with a glycine linker. (Figure 20B) Shows the difference in glycine linker-dependent target cell lysis when huCD19CAR-EBD(4-OHT) targeting Raji tumor cells is combined with a glycine linker. (Figures 20A and 20B) Specific cell lysis was evaluated using a chromium release assay. First, various huCD19CAR-EBD(4OHT) effector strains, each having one, two, or three glycine molecules between the CAR and EBD linker, were cultured for 24 hours in the presence of two estrogen analogs (4-OHT or (Z)-endoxiphene) at various drug concentrations (0 nM, 1 nM, 50 nM, or 500 nM). These were then co-cultured for 4 hours with antigen-expressing tumor cells (K562+CD19 or Raji parental cells) pre-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. It was observed that cell lysis was least pronounced in any CAR-EBD(4OHT) strain with one, two, or three glycine molecules in the absence of estrogen analogs. In the absence of inducible drugs, the highest activity was observed in the CAR-2Gly-EBD(4OHT) construct. In huCD19CAR-EBD(4OHT), specific cell lysis was least pronounced in the absence of estrogen analogs when a linker consisting of one glycine molecule was present, while specific cell lysis was equally pronounced in the presence of 50 nM or 500 nM estrogen analogs when linkers consisting of two or three glycine molecules were present. When cells were cultured with gradually increasing concentrations of various estrogen analogs, it was shown that specific cell lysis due to CAR-EBD activity remained dose-dependent.

[0038] [Figure 21]Drug-dependent specific cell lysis is reproducible even in multiple T cell donors. Specific cell lysis was evaluated using a chromium release assay with huCD19CAR-EBD(4OHT) effector cell lines derived from two T cell donors, each containing a linker consisting of one, two, or three glycine molecules. Effector cell lines from the two donors were cultured for 4 hours in the presence of two estrogen analogs (4-OHT or (Z)-endoxiphene) at various drug concentrations (only the lowest and highest drug conditions are shown from 0nM, 1nM, 50nM, or 500nM). These cells were then co-cultured for 4 hours with an antigen-expressing tumor cell line (K562 CD19) pre-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. The measurement results showed that drug-dependent CAR-EBD-induced specific cell lysis was reproducible even with different T cell donors.

[0039] [Figure 22]This study demonstrates the differences in cytokine release dependent on a glycine linker when huCD19CAR-EBD(4-OHT) targeting K562+CD19 tumor cells is combined with a glycine linker. Cytokine release was evaluated using an assay from Meso Scale Diagnostics. First, various huCD19CAR-EBD(4OHT) effector strains, each containing a single glycine linker, a double glycine linker, or a triple glycine linker between the CAR and EBD, were cultured for 24 hours in the presence of two estrogen analogs (4-OHT or (Z)-endoxiphene) at various drug concentrations (0nM, 1nM, 50nM, or 500nM). These strains were then co-cultured with antigen-expressing tumor cells (K562 CD19) for 24 hours. After 24 hours of incubation, supernatants were collected from each co-culture and measured using a MESO QuickPlex SQ 120 instrument to confirm cytokine release. The amount of cytokines released in the absence of estrogen analogs was extremely small. When the concentrations of various estrogen analogs were gradually increased and the cells were cultured, cytokine release in CAR-EBD strains with one, two, or three glycine linkers was shown to be dose-dependent. Typically, 1 nM estrogen analogs are insufficient to induce maximum activation. With 50 nM to 500 nM estrogen analogs, antigen-specific cytokine release was most abundant, regardless of the type of CAR-EBD strain with one, two, or three glycine linkers.

[0040] [Figure 23]This study demonstrates the differences in cytokine release dependent on a glycine linker when huCD19CAR-EBD(4-OHT) targeting Raji tumor cells is combined with a glycine linker. Cytokine release was evaluated using an assay from Meso Scale Diagnostics. First, various huCD19CAR-EBD(4OHT) effector strains, each containing a single glycine linker, a double glycine linker, or a triple glycine linker between the CAR and EBD, were cultured for 24 hours in the presence of two estrogen analogs (4-OHT or (Z)-endoxiphene) at various drug concentrations (0nM, 1nM, 50nM, or 500nM). These strains were then co-cultured with antigen-expressing tumor cells (Raji parent cells) for 24 hours. After 24 hours of incubation, the supernatant was collected from each co-culture and measured using a MESO QuickPlex SQ 120 instrument to confirm cytokine release. The amount of cytokine released in the absence of estrogen analogs was extremely low. When various estrogen analogs were cultured with gradually increasing concentrations, cytokine release in CAR-EBD strains with one, two, or three glycine linkers was shown to be dose-dependent. Typically, 1 nM estrogen analogs are insufficient to induce maximum activity. With 50 nM to 500 nM estrogen analogs, antigen-specific cytokine release was most abundant, regardless of the type of CAR-EBD strain with one, two, or three glycine linkers.

[0041] [Figure 24]Drug-dependent cytokine release is reproducible in multiple T-cell donors. Cytokine release was evaluated using a Meso Scale Diagnostic assay with huCD19CAR-EBD(4OHT) effector strains derived from two T-cell donors, each containing a linker consisting of one, two, or three glycine molecules between the CAR and EBD. Effector strains from each donor were co-cultured for 24 hours with various tumor strains expressing CD19 (the target antigen). After 24 hours of incubation, supernatant was collected from each co-culture and measured using a MESO QuickPlex SQ 120 instrument to confirm cytokine release. In Donor 1, huCD19CAR was tested under all drug conditions, while in Donor 2, huCD19CAR was evaluated only under drug-free conditions and conditions with 500 nM of drug present. In all donors, an increase in estrogen analog concentration was associated with increased release of IFN-γ, IL-2, and TNFα, indicating that drug-dependent cytokine release induced by CAR-EBD is reproducible even with different T cell donors.

[0042] [Figure 25]This study demonstrates drug-dependent specific cytolysis by two huCD19CAR-EBD(4OHT) variants with different linkers between CAR and EBD(4OHT) (either without a glycine linker or with a linker made from one glycine molecule) in the presence of the estrogen analogues 4-OHT or (Z)-endoxiphene. Specific cytolysis was evaluated in donor 3 using a chromium release assay. First, huCD19CAR-EBD(4OHT) linker variants (either a variant without a glycine linker (NoGly) or a variant with a linker made from one glycine molecule (1Gly)) were cultured for 24 hours in the presence of two estrogen analogues (4-OHT or (Z)-endoxiphene) at various drug concentrations (0nM, 1nM, 50nM, or 500nM). Next, each variant was co-cultured for 4 hours with an antigen-expressing tumor cell line pre-labeled with radioactive Cr-51. After a 4-hour incubation, the amount of Cr51 released into each well along with tumor lysis was quantified using a PerkinElmer TopCount. The results showed that this huCD19CAR-EBD(4OHT) system exhibited higher stringency than initially observed with huCD19CAR-EBD(4OHT) derived from donor 1 or donor 2, in the absence of inducible drugs (Figure 22: direct comparison with huCD19CAR-EBD(4OHT) with a single glycine linker). Both variants without a glycine linker and those with a single glycine linker still showed a small amount of killing. Both variants could be adjusted with 4OHT or (Z)-endoxiphene, and complete killing (comparable to constitutive huCD19CAR) was achieved with each drug at 50 nM and 500 nM conditions. In this assay, neither drug performed better than the other. Measurements from the chromium release assay showed that both variants lacking a glycine linker and those with a single glycine linker conferred similar regulatory capacity to the CAR-EBD system in terms of antigen-specific cell lysis.

[0043] [Figure 26] This paper presents anti-ζ chain Western blots evaluating various huCD19CAR-EBD(4OHT) cells with different glycine linkers in the presence of 0 nM or 500 nM 4OHT or (Z)-endoxiphene. Anti-CD3ζ Western blot analysis was used to evaluate the protein processing and expression of huCD19CAR-EBD cells with varying linker configurations between the CAR and EBD domains. Whole cell lysates were prepared from huCD19CAR-EBD(4OHT) T cell lines, each containing a single glycine linker, a double glycine linker, or a triple glycine linker between the CAR and EBD domains, after incubation for 24 hours under drug conditions of 0 nM or 500 nM 4OHT or (Z)-endoxiphene. Whole cell lysates were also prepared from mock T cell lines and T cell lines containing only CAR under the same conditions. Western blots were captured and analyzed using LiCOR's Odyssey system. Furthermore, protein band quantification was performed using LiCOR's Odyssey system. CAR ζ-chain expression was observed in all CAR-EBD strains, regardless of whether they possessed a linker consisting of one, two, or three glycine molecules, both in the absence of the drug and under maximum drug concentration conditions. Quantification of the CAR-EBD ζ-chain band revealed that the expression of CAR-EBD protein increased approximately threefold upon the addition of 500 nM 4OHT or 500 nM (Z)-endoxiphene. Such a change was not observed in the constitutive CAR ζ-chain band.

[0044] [Figure 27]This study demonstrates changes in the cell surface expression of huCD19CAR-1Gly-EBD(4OHT) upon drug induction / removal. CD4 T cells and CD8 T cells expressing the huCD19CAR-1Gly-EBD(4OHT) construct (a 1:1 mixture) were cultured for various periods in a medium containing 1 μM 4-OHT, or without 1 μM 4-OHT for various periods. Each culture was then stained with a fluorescent dye-labeled antibody specific to the scFv portion of the huCD19CAR-1Gly-EBD(4OHT) construct. The median fluorescence intensity (MFI) of each cell population was then measured at each time point shown in the graph. Addition or removal of the drug altered the cell surface expression of the CAR-EBD construct. The dynamics of this change were very slow in both directions (with increasing day-to-day rates), showing no difference from known degron, indicating a different regulatory mechanism.

[0045] [Figures 28A-28B](Figure 28A) Shows CD107a activation assays of various huCD19CAR-EBD(4OHT) linker variants applied to K562+CD19t. (Figure 28B) Shows CD107a activation assays of various B7H3CAR-3Gly-EBD variants applied to K562+CD19t. (Figures 29A and 29B) CD4 T cells and CD8 T cells (in a 1:1 ratio mixture) expressing various huCD19CAR-EBD constructs or B7H3CAR-EBD constructs, which differ in the linker between the CAR domain and the EBD domain (a linker consisting of one glycine molecule (1Gly), a linker consisting of two glycine molecules (2Gly), or a linker consisting of three glycine molecules (3Gly)) and the type of EBD domain (EBD(4OHT), EBD(CMP8), or EBD(ES8)), were analyzed to investigate a) the regulatory ability of these cells (e.g., on / off switching by various drugs) and b) the dose-response of activity to drug induction. To perform this experiment, T cells (including mocks and constitutively active CAR controls) were pre-treated for 24 hours in the presence of various concentrations of target drugs before being exposed to tumor lines expressing the target antigen (CD19 or B7H3). After co-culturing T cells and target tumors for 4 hours at various drug concentrations, a) CD107a was stained and flow cytometry was performed to evaluate T cell activation and degranulation, and b) human scFv (capable of detecting both B7H3CAR and huCD19CAR) was stained and flow cytometry was performed to evaluate CAR-EBD on the surface of T cells. The median fluorescence intensity (MFI) for each stain of each sample under each condition was measured and plotted logarithmically against drug concentration. EC10 (10% effective concentration) and EC90 (90% effective concentration) for CD107a expression were calculated using Graphpad Prism and used as estimates of the drug concentrations required to switch each construct "on" and "off". Positive and negative controls for each CAR are shown in bar graphs.A slight shift was observed between mock T cells and uninduced CAR-EBD T cells in the CD107a MFA, suggesting the presence of a small amount of uncontrolled degranulation in all EBD constructs, although this degranulation was more pronounced in the huCD19CAR-EBD construct than in the B7H3CAR-EBD construct. EBD(4OHT) responded to 4-OHT and (Z)-endoxiphene, but not to CMP8 or ES8. The degranulation response to target antigens in the presence of inducing molecules was roughly the same across various glycine linker variants. EBD(4OHT) showed very high sensitivity to the intended inducing molecules, initiating activation at single-digit nanomolar concentrations (EC10) and fully activating at double-digit nanomolar concentrations (EC90). EBD(4OHT) can be used to modulate both huCD19CAR and B7H3CAR, and despite the higher background value of the huCD19CAR construct, these two CARs exhibit similar dose-response characteristics (similar EC10 / EC90). EBD(CMP8) is less sensitive to drug induction than EBD(4OHT). EBD(ES8) showed very high sensitivity 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. On the other hand, EBD(CMP8) is not responsive to ES8, and EBD(ES8) is not responsive to CMP8, indicating that multiple types of EBD domains can be modulated independently.

[0046] [Figures 29A-29B](Figures 29A and 29B) Show CAR expression by various huCD19CAR-EBD linker variants acting on K562+CD19t. CD4 T cells and CD8 T cells (in a 1:1 ratio mixture) expressing various huCD19CAR-EBD constructs or B7H3CAR-EBD constructs differ in the linker between the CAR domain and the EBD domain (1-glycine linker (1Gly), 2-glycine linker (2Gly), or 3-glycine linker (3Gly)) and the type of EBD domain (EBD(4OHT), EBD(CMP8), or EBD(ES8)). We analyzed these cells to investigate a) their regulatory ability (e.g., on / off switching by various drugs) and b) their dose-response activity to drug induction. To perform this experiment, T cells (including mocks and constitutively active CAR controls) were pretreated for 24 hours in the presence of various concentrations of target drugs, and then exposed to tumor lines expressing the target antigen (CD19 or B7H3). After co-culturing T cells and tumors for 4 hours at 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 median fluorescence intensity (MFI) for each stain of each sample under each condition was measured and plotted logarithmically against drug concentration. It was found that the changes in cell surface CAR-EBD observed in the previous experiment (Figure 25) were not only dependent on time but also on drug concentration, as shown in Figure 30A. Changes in CAR-EBD on the cell surface are directly correlated with the ability to induce EBD, regardless of the presence of nonspecific estrogen analogs (e.g., CMP8) (i.e., EBD increases when cell activation is possible, but cell activation is not necessarily required for the increase in EBD). (Figure 29B) Relative activation and cell surface expression of CAR-EBD using identical cells are shown (each point in the stacked line graph represents two fluorescent dyes from the same cell population).The drug-induced changes in CAR-EBD cell surface expression were unrelated to drug-induced CAR-EBD activation (a higher concentration of the drug was required to increase CAR expression than the concentration needed to enhance activation). These results suggest that while CAR-EBD activation correlates with its cell surface expression, the activation mechanism of CAR-EBD is distinct from simple cell surface expression.

[0047] [Figures 30A-30C] This describes the incucyte cytotoxicity assay of huCD19CAR-1Gly-EBD(4OHT). CD4 T cells and CD8 T cells expressing huCD19CAR-EBD-1Gly-EBD(4OHT) (a 1:1 mixture) were pre-incubated for 24 hours with 500 nM of the inducible drug, or subjected to the assay without pre-incubation. These T cells were then seeded on plates with CAR-targeted tumor cells constitutively expressing mCherry (for detection and quantification) ((Figure 30A) K562+CD19, (Figure 30B) Raji and / or (Figure 30C) Be2+CD19). The tumor cells were seeded at a density of 10,000 cells / well and co-cultured in the presence or absence of 500 nM 4-OHT in a ratio of 8:1 or 4:1 (ratios are noted in footnotes where necessary). Each sample was placed in the Incucyte instrument and imaged every 3-4 hours for several days. Next, the mCherry signal was quantified and plotted as a function of time. Constitutively active, appropriate CAR T cells were used as the positive control, and mock T cells were used as the negative control. No difference in functionality was observed between CAR-EBD T cells pre-incubated with the drug ("24-hour pre-treatment") and unpre-incubated CAR-EBD T cells ("0-hour pre-treatment"). This result further demonstrates that CAR-EBD is mechanistically different from degron. Degron technology takes time (typically 3-6 hours) to accumulate a sufficient amount and exhibit activity. In contrast, the "on" dynamics of CAR-EBD killing in the Incucyte cytotoxicity assay can be observed almost immediately.

[0048] [Figure 31A-31B](Figure 31A) This shows the Incucyte cytotoxicity assays of various B7H3CAR-3Gly-EBD variants. CD4 T cells and CD8 T cells expressing B7H3CAR-3Gly-EBD(4OHT), B7H3CAR-3Gly-EBD(CMP8), or B7H3CAR-3Gly-EBD(ES8) (in a 1:1 ratio mixture) were pre-incubated with 500 nM inducible drugs for 24 hours or subjected to the assay without pre-incubation. These T cells were then seeded on plates with CAR-targeted tumor lines (K562 and / or Be2) constitutively expressing mCherry (for detection and quantification). Tumor cells were seeded at a density of 10,000 cells / well and co-cultured in a 4:1 ratio (T cells:tumor cells =) in or without the presence of 500 nM of each drug (500 nM 4OHT was added to B7H3CAR-3Gly-EBD(4OHT), 500 nM CMP8 was added to B7H3CAR-3Gly-EBD(CMP8), and 500 nM ES8 was added to B7H3CAR-3Gly-EBD(ES8)). Each plate was placed in an Incucyte instrument and imaged every 3-4 hours for several days. Next, the mCherry signal was quantified and plotted as a function of time. Constitutively active appropriate CAR T cells were used as a positive control, and mock T cells were used as a negative control. All three B7H3CAR-3Gly-EBD variants exhibited both a potent "on" state of antitumor activity and a well-controlled "off" state, demonstrating that each B7H3CAR-3Gly-EBD variant possesses high activity regulation capabilities. (Figures 31A and 31B) The B7H3CAR-EBD (CMP8) constructs acted on K562+mCherry are shown. Figure 31A compares the drug-free condition with the drug-assisted condition, and Figure 31B compares the drug-free condition with the condition after 24 hours with the drug added (black perpendicular line). The amount of tumor growth by 24 hours was insufficient to overcome by the donor itself or the B7H3CAR-3Gly-EBD (CMP8) T cells at this ratio, but when B7H3CAR-3Gly-EBD (CMP8) T cells were activated by CMP8, the dynamics of tumor growth changed immediately.

[0049] [Figures 32A-32C](Figures 32A-32C) CD4 T cells and CD8 T cells expressing B7H3CAR-3Gly-EBD(4OHT), B7H3CAR-3Gly-EBD(CMP8), or B7H3CAR-3Gly-EBD(ES8) (mixture in a 1:1 ratio) were pre-incubated for 24 hours with 500 nM inducible drugs or subjected to assays without pre-incubation (500 nM 4OHT was added to B7H3CAR-3Gly-EBD(4OHT), 500 nM CMP8 was added to B7H3CAR-3Gly-EBD(CMP8), and 500 nM ES8 was added to B7H3CAR-3Gly-EBD(ES8)). These T cells were then seeded onto plates with CAR-targeted tumor cells (K562 and / or Be2) constitutively expressing mCherry (for detection and quantification). Tumor cells were seeded at a density of 10,000 cells / well and co-cultured in a 4:1 ratio (T cells:tumor cells) in or without the presence of a suitable 500 nM drug. Each sample was placed in an Incucyte instrument and imaged every 3-4 hours for several days. The mCherry signal was then quantified and plotted as a function of time. Constitutively active, suitable CAR T cells were used as a positive control, and mock T cells were used as a negative control. In one experiment, T cells were repeatedly challenged with tumor cells to evaluate differences in the effect on functional lifespan across different constructs. (Figure 32A) This shows an Incucyte cytotoxic assay evaluating the effect 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) Shows the Incucyte cytotoxicity assay of the B7H3CAR-3Gly-EBD variant. It was shown that there was no difference in the killing kinetics of CAR-EBD T cells between those pretreated with the inducible drug for 24 hours and those added at the start of the experiment. (Figure 32C) Shows the Incucyte cytotoxicity assay of various B7H3CAR-3Gly-EBD variants.Both CAR-EBD(4OHT) cells and CAR-EBD(ES8) cells demonstrated highly regulated killing of Be2+ mCherry tumor cells. Be2 cells were attached to the plate 24 hours before T cells were added.

[0050] [Figure 33]Representative sequences supporting this disclosure are shown below: Estrogen receptor (SEQ ID NO: 1), wild-type estrogen-binding domain (EBD) with underlined N-terminal and C-terminal cleavage sites (SEQ ID NO: 2), estrogen receptor with G521R mutation (SEQ ID NO: 3), EBD with G521R mutation (SEQ ID NO: 4), estrogen receptor with E353A mutation (SEQ ID NO: 5), EBD(E353A) (SEQ ID NO: 6), coding sequence of EBD(E353A) (SEQ ID NO: 7), estrogen receptor with L384M and M421G mutations (SEQ ID NO: 8), EBD with L384M and M421G mutations (SEQ ID NO: 9), L384M mutation, M421G mutation and G521R Estrogen receptors with mutations (SEQ ID NO: 10), EBD (L384M, M421G, G521R) used in combination with 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 of ERT2 (SEQ ID NO: 14), coding sequence of EBD(CMP8) (L384M, M421G, G521R) (SEQ ID NO: 15), EGFRVIII(806) scFv (sequence number 16), EGFRVIII (806) scFv code sequence (sequence number 17), EGFR scFv (sequence number 18), huCD19 (G01S) scFv (sequence number 19), muCD19 (FMC63) scFv (sequence number 20), CD19 scFv (sequence number 21), CD19 scFv code sequence (sequence number 22), CD20 (Leu 16) scFv (sequence number 23), CD20 scFv code sequence (sequence number 24), CD22 (m971) scFv (sequence number 25), B7H3 (hBRCA84D) scFv (sequence number 26), L1CAM (CE7) scFv (sequence number 27), EphA2 (2A4) scFv (sequence number 28), EpHA2 (4H5) scFv (sequence number 29), FITC (E2) scFv(sequence number 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 zetakin) (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 (sequence code 64) of scFv(XC), VL-linker-VH (sequence code 65) of DNP scFv(XC), VH-linker-VL (sequence code 66) of DNP scFv(1BAF), DNPVL-linker-VH (sequence number 67) of scFv(1BAF), VH-linker-VL (sequence number 68) of DNP scFv(XC), VL-linker-VH (sequence number 69) of DNP scFv(Ab-3) (sequence number 70), DNP scFv;DNPThe VH-linker-VL (SEQ ID NO: 71), glycineserine linker (SEQ ID NO: 72), glycineserine linker (SEQ ID NO: 73), glycineserine linker (SEQ ID NO: 74), glycineserine linker (SEQ ID NO: 75), the code sequence for the (G4S)3 linker (SEQ ID NO: 76), the code sequence for the (G4S)4 linker (SEQ ID NO: 77), the short spacer (IgG4 (short hinge)) (SEQ ID NO: 78), and the code sequence for the short spacer (IgG4 (short hinge)) (Sequence No. No. 79), short spacer 2 (another IgG4 linker) (SEQ ID NO: 80), medium-length spacer (SEQ ID NO: 81), long spacer with single mutation (L235D) (SEQ ID NO: 82), long spacer with double mutation (L235D, N297Q) (SEQ ID NO: 83), glycine linker, coding sequence of glycine linker (SEQ ID NO: 84), P2A (SEQ ID NO: 85), coding sequence of P2A (SEQ ID NO: 86), T2A (SEQ ID NO: 87), coding sequence of 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), cleaved 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 (the sequences shown in the table combine to form SEQ ID NO: 1306), the coding sequence of EGFR806CAR-ERT2-P2A-DHFRdm-T2A-CD19t shown in the table (the sequences shown in the table combine to form SEQ ID NO: 125) (composes), CD8α hinge / transmembrane domain (SEQ ID NO: 126), another CD8 hinge / transmembrane domain (SEQ ID NO: 127), another CD8 hinge / transmembrane domain (SEQ ID NO: 128), another human CD8α chain hinge (SEQ ID NO: 129), intracellular signaling domain of 4-1BB / CD3ζ (SEQ ID NO: 130), coding sequence of the intracellular signaling domain of 4-1BB / CD3ζ (SEQ ID NO: 131), coding sequence of the intracellular signaling domain of another 4-1BB / CD3ζ (SEQ ID NO: 132), CD3ζ ICD (SEQ ID NO: 121), CD3ζ ICD coding sequence (SEQ ID NO: 124), T cell surface glycoprotein CD3ε chain (CD3ε) (SEQ ID NO: 133), CD3ε ECD (SEQ ID NO: 134), CD3ε ICD (SEQ ID NO: 135), CD3ε TM (SEQ ID NO: 136), T cell surface glycoprotein CD3δ chain (CD3δ) (SEQ ID NO: 137), CD3δ ECD (SEQ ID NO: 138), CD3δ ICD (SEQ ID NO: 139), CD3δ TM (SEQ ID NO: 140), T cell surface glycoprotein CD3ζ chain (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), 4-1BB ICD coding sequence (SEQ ID NO: 123), 4-1BB TM (SEQ ID NO: 173), Interleukin-2 receptor subunit α (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), Code sequence of human CD28 ICD (SEQ ID NO: 182), CD28 TM (SEQ ID NO: 183), Transmembrane domain of CD28 (SEQ ID NO: 119), Code sequence of CD28™ (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), Signal Transduction 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 ε receptor subunit α (SEQ ID NO: 213), High affinity immunoglobulin ε receptor subunit β (SEQ ID NO: 214), FcR-γ, i.e., high affinity immunoglobulin ε receptor subunit γ 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 α chain precursor (pTα) (SEQ ID NO: 228), T cell receptor α chain (TCRα) (SEQ ID NO: 229), T cell receptor β 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™(SEQ ID NO: 237), Programmed Cell Death 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 Ligand 1 (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™ (SEQ ID NO: 258), Carcinoembryonic Antigen-Associated 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™ (SEQ ID NO: 263), T-cell Activation Suppressor Containing Immunoglobulin Domains (VISTA) (SEQ ID NO: 264), VISTA ECD (SEQ ID NO: 265), VISTA ICD (SEQ ID NO: 266), VISTA™ (SEQ ID NO: 267), B Lymphocyte / T Lymphocyte Atenuator (BTLA) (SEQ ID NO: 268), BTLA ECD (SEQ ID NO: 269), BTLA ICD (SEQ ID NO: 270), BTLA™ (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™ (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™ (SEQ ID NO: 279), T lymphocyte-activating 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 activating 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 (SEQ ID NO: 299), killer cell immunoglobulin-like receptor (SEQ ID NO: 300), adenosine receptor A2a (SEQ ID NO: 301), galectin 9 (GAL9) (SEQ ID NO: 302), transforming growth factor β receptor I (TGFRβ) (SEQ ID NO: 303), MHC class I (SEQ ID NO: 304) and some MHC class II (SEQ ID NO: 305). [Modes for carrying out the invention]

[0051] Significant progress has been made in methods of activating immune system cells and using these immune cells to kill cancer cells and infected cells through genetic engineering. For example, T cells can be genetically modified to express molecules that have an extracellular portion that binds to a specific target antigen and an intracellular portion that induces the action of T cells when the extracellular portion binds to the target antigen. As an example, the extracellular portion can be designed to bind to a target antigen found on cancer cells or infected cells. When the target antigen of cancer cells or infected cells binds to this extracellular portion, the intracellular portion activates T cells, and the cells bound to the extracellular portion are destroyed by the T cells. An example of such a molecule is the chimeric antigen receptor (CAR).

[0052] As described herein, CARs include synthetically designed 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 for a receptor (e.g., a cytokine or chemokine).

[0053] While T cells expressing CARs can exhibit potent antitumor activity, they may also exhibit significant toxicity. For example, transplanted cells can induce cytokine storms (cytokine release syndrome), tumor lysis syndrome (TLS), and chronic B cell reduction, all of which are caused by the unregulated functioning of constitutively expressed active CARs. Such toxicity can limit the applicability of CAR-based therapies.

[0054] While methods to eliminate CAR-T cells, such as using suicide genes, can mitigate such toxicity, this method risks reducing antitumor activity before it can be fully exerted, thus significantly impacting therapeutic efficacy. Another method to control protein activity is to use degron sequences to control protein degradation or stability. However, the effect of degron sequences is delayed because it depends on intracellular protein degradation. Therefore, there is a need to find a rapid and controllable method to regulate the activity of CARs and other proteins expressed in vivo.

[0055] This disclosure provides a CAR that can be activated by antigen binding after being expressed in vivo, and whose ability can be controlled by the administration of a drug molecule. If activation can be controlled without relying on protein degradation, safety, which is important in CAR-based immunotherapy, can be improved.

[0056] This disclosure achieves such progress by incorporating a heat shock protein 90 (hsp90) binding domain into the intracellular portion of the CAR. In the absence of drug molecules, hsp90 can bind to this hsp90 binding domain, preventing other important intracellular molecules necessary for the activation of the CAR after antigen binding from interacting with the CAR.

[0057] When a drug molecule is present, it displaces the bound HSP90 from its HSP90-binding domain and / or causes a conformational change, leading to antigen binding and subsequent intracellular signal transduction.

[0058] This mechanism is shown 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 innate estrogen receptor, but it must contain at least one mutation so that it binds to the drug molecule without binding to estrogen. In the embodiments shown in Figures 1A-1C, the drug molecule is 4-OHT. In the absence of the drug molecule, hsp90 binds to the EBD, and the CAR is in the "off" state (Figure 1A). When the drug molecule enters the cell, it acts actively to overcome hsp90 in binding to the EBD and / or cause a conformational change, and when the CAR binds to the antigen, the CAR transmits an activation signal (Figures 1B, 1C).

[0059] Figures 2A and 2B show typical schematic diagrams of CARs that can be used in carrying out various embodiments of this disclosure. As shown in Figure 2A, this CAR includes a ligand-binding domain, a spacer, a transmembrane domain, a signaling domain, and an estrogen-binding domain (EBD). This CAR may further include a selection / transduction marker. Figure 2B shows a specific CAR named 806-ERT2 CAR. This CAR incorporates an IgG4 hinge between EGFR806 Vh / Vl scFv and the transmembrane domain. In this CAR, the signaling domain and the ERT2 EBD are linked by a Gly3 linker consisting of three glycine molecules. Behind this ERT2 domain are linked P2A-DHFRdm as a selection marker and T2A-CD19t as a transduction marker. Another CAR disclosed herein includes a Gly2 linker or a Gly1 linker (hereinafter also referred to herein as a linking amino acid) instead of a Gly3 linker, and yet another CAR does not include a linker between the signaling domain and the EBD. Furthermore, the EBD may be contained 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 the intracellular space portion on the 5' side of the transmembrane domain. 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 to cortisol, androgens, progesterone, or aldosterone. Furthermore, the hsp90 binding domain may be derived from various other proteins that bind to hsp90, and such proteins are generally called hsp90 clients. Common examples of hsp90 clients include hormone receptors, transcription factors and kinases, as well as other types of molecules.

[0061] One of the advantages of this disclosure is that the activity of a single-chain protein can be controlled without relying on dimerization or polymerization with another protein, and without relying on protein stabilization / destabilization, for example, by incorporating a degron sequence. In this specification, "degron sequence" refers to an amino acid sequence linked to a fusion protein by recombinant technology for the purpose of controlling the stability / degradation of the protein. Degron sequences are usually linked to the C-terminus of the fusion protein. Examples of degron sequences are described, for example, in U.S. Patent Publication 2014 / 0255361, and include RRRG and RRRGN (SEQ ID NO: 306).

[0062] Various aspects of this disclosure, along with further options, are described in more detail below. These various aspects of this disclosure are described in accordance with the following topics: (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) other transmembrane receptors; (viii) cells genetically modified to express inducible fusion proteins; (ix) methods for recombining cells ex vivo and in vivo; (x) production of inducible fusion proteins; (xi) recombinant formulations, recombinant inducers, 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) conclusions. These headings are provided for systematic purposes only and do not limit the scope or interpretation of this disclosure.

[0063] (i) Drug molecule and hsp90 binding domain Drug molecules used with the inducible activity fusion proteins disclosed herein can overcome hsp90 in binding to the hsp90 binding domain present on the inducible activity fusion protein and / or induce conformational changes that result in intracellular signaling. In certain examples, the binding domain present on the inducible activity fusion protein is located on the intracellular segment of the CAR. Overcoming hsp90 in binding to the hsp90 binding domain and / or conformational changes enable 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 low-molecular-weight estrogen analog. Examples of low-molecular-weight estrogen analogs include 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 marketed as an FDA-approved drug. Tamoxifen has a proven safety profile, a favorable pharmacokinetic profile, good tissue distribution, and a low partition coefficient between the extracellular space and cytoplasm. Tamoxifen is often administered orally as a pharmaceutically 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 adjunct agent in the treatment of breast cancer in women who have undergone mastectomy, axillary lymph node dissection, and breast irradiation. Tamoxifen citrate is also indicated for reducing 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-dimethylethaneamine or (Z)-2-(p-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethylamine (IUPAC), and its molecular formula is C 26 H 29 It is denoted by NO, and its molecular weight (MW) is 371.52 g / mol.

[0067] Potentially useful metabolites of tamoxifen in some of the approaches described herein include the main metabolite N-desmethyltamoxifen (RN 31750-48-8, MW357.494), 4-hydroxytamoxifen (4-OHT) (RN 68392-35-8, MW387.52, 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). Other metabolites useful in some of the approaches described herein include cis-4-hydroxytamoxifen (RN 174592, MW387.52; 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, Figure 1), and 4'-hydroxytamoxifen ((Z)-4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-phenylbuta-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-methyltamoxifen (RN 73717-95-5, MW 385.548), N-desmethyltamoxifen (RN 31750-48-8, MW 357.494), (Z)-desethylmethyltamoxifen (RN 15917-50-7, MW 357.494), (E)-desethylmethyltamoxifen (RN 31750-45-5, MW 357.494), trans-4-hydroxytamoxifen (RN 68047-06-3, MW 387.52), and afimoxifen (RN 68392-35-8, MW 357.494). (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), desethyltamoxifen (RN 19957-51-8, MW 343.47), (E)-desethyltamoxifen (RN 97151-10-5, MW 343.47), (Z)-desethyltamoxifen (RN 97151-11-6, MW 343.47), Miproxyfen (RN 129612-87-9, MW 429.6), 2-(p-(β-ethyl-α-phenylstyryl)phenoxy)triethylamine (RN 749-86-0, MW 399.575), Droloxifen (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 429.6), 385.548), and 4-hydroxytoremifene (RN 110503-62-3, MW)421.965); and their pharmaceutically acceptable salts, hydrates, or solvates.

[0069] Citrates 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), droloxifen citrate (RN 97752-20-0, MW 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-ethaneamine 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.Examples include 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, tamoxifen, 4-OHT, ES8, or CMP8 are used as the drug molecule. In certain embodiments, fulvestrant or raloxifene are used as the drug molecule.

[0071] A representative hormone-binding domain is the estrogen receptor having at least one mutation that suppresses or blocks binding to endogenous estrogen / estradiol. The protein sequence of this estrogen receptor is shown in Figure 33 as SEQ ID NO: 1. This point mutation in the estrogen receptor (G521R (SEQ ID NO: 3)) suppresses binding to endogenous estrogen but confers nanoscale specificity to tamoxifen metabolites such as 4-OHT, fulvestrant, and other estrogen analogs. In this particular embodiment, the G521R mutation-binding domain (EBD) of the estrogen receptor shown in SEQ ID NO: 4 is utilized. In another particular embodiment, the EBD with the E353A mutation (SEQ ID NO: 6), as described in Shi & Koh, Chemistry & Biology 8 (2001) 501-510, is used in conjunction with the drug molecule ES8. In another embodiment, an EBD having 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. Therefore, in some embodiments, an EBD having the sequence shown in SEQ ID NO: 13 can be used. For additional information on drug systems using estrogen receptors, 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 dose of a drug that enables the activity of a fusion protein in the presence of the physiological events involved is the amount that increases the activity of the fusion protein compared to the uninducible state and / or basal activity. For example, in some embodiments, the effective dose of a drug that enables the activity of a CAR in the presence of antigen binding is the amount that increases the activity of the CAR compared to the uninducible state and / or basal activity. In another embodiment, an effective dose of a drug enables stimulative, co-stimulative, or inhibitory immune signaling activity in the presence of ligand binding compared to the uninducible state and / or basal activity. Such effective doses 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 profiles, tissue distribution, low distribution coefficient between extracellular space and 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. Typical ligand-binding domains include antibodies or their binding fragments, receptors (e.g., T cell receptors), and receptor ligands (e.g., cytokines and chemokines).

[0075] As is understood by those skilled in the art, a full-length antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of 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. A full-length antibody has a "Y" shape. 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, with a disulfide bond (interchain bond) formed at the hinge. The γ, α, and δ heavy chains have a constant region in which three immunoglobulin domains are linked in tandem (series) and a hinge region that provides flexibility; the μ and ε heavy chains have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and the "CH3 domain," respectively. Each arm of a Y-shaped full-length antibody consists of a variable region and a first constant region of a heavy chain linked to a light chain consisting of a variable region and a constant region. The variable regions of the light chain variable region and the heavy chain variable region are responsible for binding to the antigen.

[0076] The light chain variable region and the heavy chain variable region contain a "framework" region that is sandwiched between three hypervariable regions, also called the "complementarity determination region" or "CDR".The combination of CDRs is, for example, 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. The numbering scheme can be determined based on 406(2):228-256 ("North" numbering scheme); or other numbering schemes.

[0077] Various software programs and bioinformatics tools can be used to determine 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), and Antibody i-Patch (Krawczyk et al. (2013) Protein Eng Examples include Des Sel 26(10):621-629) and proABC-2 (Ambrosetti et al. (2020) Bioinformatics 36(20):5107-5108).

[0078] The sequences of various light chain or heavy chain framework regions are relatively conserved across biological species, including humans. The framework region of an antibody is formed by the linkage of multiple framework regions within the light and heavy chains that constitute the antibody, and it plays a role in arranging and aligning the CDRs in three-dimensional space. CDRs are primarily responsible for binding to the antigen's epitope. The CDRs of each chain are generally numbered sequentially from the N-terminus and called CDR1, CDR2, and CDR3, and are generally identified by the chain on which they are located. Therefore, CDRs in the variable domain of the antibody's heavy chain are called CDRH1, CDRH2, and CDRH3, while CDRs in the variable domain of the antibody's light chain are called CDRL1, CDRL2, and CDRL3. If the antibody has different specificity (i.e., different binding sites for various antigens), the CDRs it possesses will also differ. While the CDRs differ from antibody to antibody, only a limited number of amino acid positions within each CDR are directly involved in antigen binding. These amino acid positions within a CDR are called specificity-determining residues (SDRs).

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

[0080] Antibodies that specifically bind to cell surface molecules can be produced using methods for obtaining monoclonal antibodies, phage display, methods for producing human antibodies or humanized antibodies, or methods using transgenic animals or plants genetically modified to produce human antibodies. Phage display libraries of partially synthesized or totally synthesized antibodies are available, and from these libraries, antibodies or fragments capable of binding to target molecules can be screened. Phage display libraries of human antibodies are also available. Once the amino acid or polynucleotide sequence encoding the antibody is identified, these sequences can be isolated and / or sequenced. Numerous related antibodies are known and commercially available.

[0081] In some embodiments, the antibody specifically binds to surface molecules of cancer cells or virus-infected cells and does not cross-react with nonspecific 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 Fv (sdFv), Fd fragments containing a VH domain and a CH1 constant domain, linear antibodies, single-domain antibodies (e.g., sdAb(VL or VH)), domains consisting only of the heavy chain variable region of camelids (VHH), multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment containing two Fab fragments linked at a hinge region by disulfide crosslinking), and epitope-binding fragments of isolated CDRs or other antibodies (Harlow et al., 1999, published in *Using Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, published 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). Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, or bis-scFvs (see, for example, Hollinger and Hudson (2005) Nature Biotechnology 23:1126-1136).

[0083] In certain embodiments, the binding domain may include a humanized form of a non-human (e.g., mouse) antibody or an antigen-binding fragment thereof. Examples of humanized antibodies include antibodies in which the constant framework region and variable region of one or more human immunoglobulins are fused to the binding region (e.g., CDR) of an animal (non-human) immunoglobulin. Such humanized antibodies are designed to avoid immune responses to the non-human antibody while retaining the binding specificity of the non-human antibody from which the binding region originates. In certain embodiments, the binding domain may include a fully human antibody or an antibody fragment thereof, either entirely derived from a human molecule or containing the same amino acid sequence as the human form of the antibody or immunoglobulin.

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

[0085] There are various methods for identifying and selecting specific TCRs used in CARs. For example, many TCR sequences that bind to specific antigen fragments are known and publicly available.

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

[0087] In certain embodiments, it may be necessary to combine the TCRα and TCRβ chains after sequencing (i.e., to perform analysis of the combined TCR chains). Various methods can be used to combine the TCR chains as needed. For example, computer-aided methods may be used to combine the TCR chains in silico, 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, Washington) have also been developed.

[0088] In certain embodiments, recombinant TCRs include single-chain T cell receptors (scTCRs) that are specific to a target of interest (e.g., a peptide-MHC complex) and contain Vα / β chains and Cα / β chains (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ) or Vα-Cα pairs, Vβ-Cβ pairs, or Vα-Vβ pairs.

[0089] Cancer antigens are proteins produced by cancer cells, and viral antigens are proteins produced by virus-infected cells. The ligand-binding domains of the CARs disclosed herein can be selected to bind to cancer antigens or viral antigens. In some embodiments, cancer antigens or viral antigens are selectively expressed or overexpressed on cancer cells or infected cells compared to other cells of the same type of tissue. In some embodiments, cancer antigens or viral antigens are cell surface molecules present on cancer cells or virus-infected cells that are substantially absent on normal tissues or whose expression is restricted to normal tissues where it is not significant.

[0090] Representative cancer antigens include carcinoembryonic antigen (CEA), prostate-specific antigen, prostate stem cell antigen (PSCA), PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, 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 Examples include 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 skilled in the art.

[0091] In certain embodiments, ligand-binding domains that specifically bind to HER2, CE7, hB7H3, EGFR, EGFRvIII, CD19, CD20, CD22, EphA2, IL13Ra2, L1CAM, oaGD2, B7H3, CD33, mesothelin, ROR1, FITC, or VAR2CSA are utilized.

[0092] In certain embodiments, scFvs used in accordance with the teachings of this 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, and IL13Ra2(hu07)VhVl Examples of scFv amino acid sequences include oaGD2(8B6) VlVh, ROR1(R12) scFv, CD33(h2H12) VhVl scFv, CD33(h2H12) VlVh scFv, mesothelin(P4) scFv, VAR2CSA(ID1-DBL2Xb) scFv, and IL13Ra2 (IL13 zetakin). See Figure 33 for these representative scFv sequences.

[0093] Binding domains that bind to representative viral antigens can also be used. These representative viral antigens include: coronavirus antigen: spike (S) protein; cytomegalovirus antigen: envelope glycoprotein B and CMV pp65; Epstein-Barr virus antigen: EBV EBNAI, EBV P18 and EBV P23; hepatitis virus antigen: S protein, M protein and L protein of hepatitis B virus, pre-S antigen of hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C virus RNA, HCV NS3 and HCV NS4; herpes simplex virus antigen: pre-initial protein and glycoprotein D; HIV antigen: gene products of gag gene, pol gene or env gene, e.g., HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV Examples include 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 further 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 to ligands on B cells, specifically, the ligands on B cells to which the binding domain shows specificity are 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 These are 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] Furthermore, the CAR binding domain can bind to other immune cell antigens found, for example, natural killer T (NKT) cells, natural killer cells (also known as K cells or killer cells), tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), MAIT cells, macrophages, monocytes, and / or dendritic cells. These cells and representative cell surface antigens are described separately herein.

[0096] Furthermore, the binding domains described herein can bind to haptens. The hapten can be any small molecule that, when combined with a larger carrier such as a protein, induces the production of an antibody that specifically binds to the hapten (either in its free state or bound to a carrier). Examples of haptens include peptides, other larger chemical substances, and aptamers. In some embodiments, the hapten may be any hapten provided in the hapten database accessible from the World Wide Web at 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 (modifications: 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)); Ametrine (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine); Deethylatrazine (DEA) (structurally related s-triazine); Disopropylatrazine (DIA) (structurally related s-triazine); Deethyldisopropylatrazine (DEDIA) (structurally related s-triazine); Deethyldisopropylatrazine (DEDIA) (structurally related s-triazine); Hydroxyatrazine (HA) (structurally related s-triazine); Disopropylhydroxy Trazine (DIHA) (structurally related s-triazine); Deethyl disopropyl hydroxyatrazine (DEDIHA) (structurally related s-triazine); Simazine (structurally related s-triazine); Desmethrin (structurally related s-triazine); Promethrin (structurally related s-triazine); 2-Hydroxyatrazine (atrazine derivative); 2-Hydroxypropazine (structurally related s-triazine); 2-Hydroxysimazine; N-(4-amine-6-hydroxy-[1,3,5]triazine-2-yl)-4-aminobutanoic acid (modified: R 1 = NH2, R 2 = NH(CH2)3COOH, R 3= OH); Sulcoflon; 5-chloro-2-{4-chloro-2-[3-(3,4-dichlorophenyl)ureido]phenoxy}benzenesulfonic acid; Fulcoflon (1,3-bis(4-chloro-α,α,α-trifluoro-m-tolyl)urea); Agatha resinol; Sequilin C; Sugiresinol; Hydroxysugiresinol; Hinokiresinol; Coniferyl alcohol; Cinnamyl alcohol; p-coumaric acid; Cinnamic acid; p-coumaric acid; Cinnamic acid; Hinokinin; Guayacylglycerol-β-guaiasyl ether; Morphine-3-glucuronide (M3 G); Codeine; Norcodeine; 6-Monoacetylmorphine; (+)-Methamphetamine; Ceftazidime; Phenobarbital; p-Hydroxyphenobarbital; p-Aminophenobarbital; Cyclobarbital; 3'-Ketocyclobarbital; 3'-Hydroxycyclobarbital; Secobarbital; Barbital; Metalbital; Barbituric acid; Thiopental; Thiobalbituric acid; Primidone; Glutethymide; Pentobarbital; Heroin; Diacetylmorphine; Levalorphan; L-11-Allyl-1,2,3,9,10 ,10a-Hexahydro-4H-10,4a-Iminoethanophenanthrene-6-ol;Petidine (Demerol;Drantin;Meperidin;1-Methyl-4-phenylpiperidine-4-carboxylate ethyl;Isonipecaine);Methamphetamine;d-Desoxyephedrine;Mesedrine;Tolpropamine;Pratalgin;Pragman;Benzoylecgonine;3-Carboxymethylmorphine;Cocaine;5-Benzimidazolecarboxylic acid;ABA (4-Acetylbenzoic acid);Dexamethasone;Flumethasone;6α,9α-Difluoro- 11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione; 9α-fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione; 9-α-fluoroprednisolone; desoxymethazone; triamcinolone; 9α-fluoro-11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20-dione; fluocortone; 6α-fluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione; cortisol;11β,17,21-trihydroxypregna-4-ene-3,20-dione; prednisone; 17,21-dihydroxypregna-4-ene-3,11,20-trione; methylprednisolone; 11β,17,21-trihydroxy-6α-methylpregna-1,4-diene-3,20-dione; triamcinolone hexacetonide; 21-(3,3-dimethyl-1-oxobutoxy)-9α-fluoro-11-hydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione; carbofuran; me 2,3-dihydro-2,2-dimethyl-7-benzofuranyl tylcarbamate; BFNP(3-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]amino]propanoic acid); Carbofuran derivatives; 2,3-dihydro-2,2-dimethyl-7-benzofuranol; Benziocarb; Carbaryl; Methiocarb; Propoxul; Aldicarb; Methomyl; Benalaxyl; Methyl N-(phenylacetyl)-N-(2,6-xylyl)-DL-alaninate; Bn-Ba(4-[2-(N-phenylacetyl-N-2,6-xylyl) [Lylamino)propionamide]butyric acid); Bn-COOH (4-[2-(N-phenylacetyl-N-2,6-xylyl-DL-alanine); Benalaxyl derivative; Flalaxyl; Metalaxyl; Acetochlor; Dimetachlor; Metrachlor; 2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide; Dietatylethyl; Benzoylpropetyl; Benzoylpropetyl; 2,4,5-Trichlorophenoxyacetic acid; 2-Chloro-6'-ethyl-N-(2-methoxy-1-methylethyl)aceto-o-toluidide; Dietatylethyl Tatyl ethyl; benzoylpropetyl; propachlor; propachlor; 2,4,5-trichlorophenoxyacetic acid; 2,4,5-T; Weedone; 2,4-dichlorophenoxybutyrate (2,4-DB); 2,4-DB; butanoic acid; 4-(2,4-dichlorophenoxy)-; Butoxone; Embutone; MCPA; 2-methyl-4-chlorophenoxyacetic acid; Metaxone; dichlorprop (2,4-DP); 1-[(2-chloro)phenylsulfonyl]monamidosuccinate; chlorsulfuron; chlorbromulon;Amidosulfuron; Chlortoluron; Isoproturon; Diuron; Linuron; O-methyl-O-(4-nitrophenyl)-N-(4-carboxybutyl)-phosphoramidethioate; Parathion methyl; O,O-dimethyl O-4-nitrophenyl phosphorothioate; Metaphos; Wolfatox; Dimethylparathion; Metaside; Parathion ethyl; p-nitrophenylthiophosphate diethyl; O,O-diethyl O-(p-nitrophenyl)phosphorothioate; Fenitrothion; O,O-dimethyl O-4-nitro-m-tolylphosphorothioate; Fenthion, O,O-dimethyl O-4-methylthio-m-tolylphosphorothioate; Bromophos, O-4-bromo-2,5-dichlorophenyl O,O-dimethylphosphorothioate; Chlorpyrifos methyl, O,O-dimethyl O-3,5,6-trichloro-2-pyridyl Phosphothioates; methyl parathion oxide, paraoxone; phosphoric acid; O,O-diethyl O-(4-nitrophenyl) ester, diazinon, O,O-diethyl O-2-isopropyl-6-methylpyrimidine-4-yl phosphorothioate; azinomethyl; pyrimiphomethyl; O-2-diethylamino-6-methylpyrimidine-4-yl O,O-dimethyl phosphorothioate; methidathione; S-2,3-dihydro-5-methoxy-2-oxo-1,3,4-thiadiazole-3-ylmethyl O,O-dimethyl phosphorodithioate; dimethylchlorothiophosphate; 4-nitrophenol; p-nitrophenol; phenol derivatives (modification on the benzene ring: R; 1 = OH, R 2 = NO2, R 3 = H, R 4 = CH2COOH, R 5 = H, R 6= H); 2-nitrophenol; o-nitrophenol; 3-nitrophenol; m-nitrophenol; 2,4-dinitrophenol; 3,4-dinitrophenol; 2,5-dinitrophenol; 2,4-dinitro-6-methylphenol; 2,3,6-trinitrophenol; 2-chlorophenol; 4-chloro-3-methylphenol, fenitroxone; 3-methyl-4-nitrophenol; nonylphenol; HOM(3-[2-hydroxy-5-nitrobenzylthio]propionic acid; phenol, Delor 103; polychlorinated biphenyl; Delor 104; polychlorinated biphenyl; Delor 105, polychlorinated biphenyl, Delor 106; 4,4'-dichlorobiphenyl, PCBs; 2,4,4'-trichlorobiphenyl; PCBs, 2,4'-; PCBs; 2,2'-dichlorobiphenyl, PCBs; 2,4,5-trichlorobiphenyl, PCBs; 3,3',4,4'-tetrachlorobiphenyl, PCBs; PCBs; 2,2',4,4',5,5'-hexachlorobiphenyl; 2-(5-carboxypentanoylamino)-4,4'-dichlorobiphenyl; biphenyl derivatives; 4-chlorophenoxyacetic acid; 2-chlorophenoxyacetic acid; DDT, 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane; DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene; p-chlorophenol; 4-chlorophenol; m-chlorophenol; 3,4-dichloropheno 3,5-Dichlorophenol; 2,3,4-Trichlorophenol; 2,3,5-Trichlorophenol; 3-Methylindole; 3-Methylindole derivatives; 4-(3-Methylindole-5-yloxy)butanoic acid; 4-(3-Methylindole-5-yloxy)butanoic acid; 3-Methylindole derivatives; 6-[n-3-Methylindole-5-yloxycarbonyl)amino]hexanoic acid; 6-[n-3-Methylindole-5-yloxycarbonyl)amino]hexanoic acid; 3-Methylindole derivatives; 2-[4-(3-Methylindole-6-yl)buta-1-ylthuro]acetic acid; 2-[4-(3-Methylindole-6-yl)buta-1-ylthuro]acetic acid; 3-Methylindole derivatives; 4-(3-Methylindole-6-yl-4-oxo)butanoic acid;4-(3-methylindole-6-yl-4-oxo)butanoic acid; 3-methylindole derivatives; 6-(3-methylindole-7-yloxy)hexanoic acid; 6-(3-methylindole-7-yloxy)hexanoic acid; indole; indole-3-carboxylic acid; indole-3-acetic acid (an indole derivative); indole-3-acetic acid (an indole derivative); indole-3-propionic acid (an indole derivative); indole-3-propionic acid; indole-3-carbinol, indole-3-carbinol (an indole derivative); tryptophan; tryptamine; 5-methoxyindole-3-carboxyaldehyde, 5-methoxytryptamine; 5-methoxyindole; 6-methoxyindole; 7-methoxyindole, EB1089 (theocalcitol); EB1 089 (theocalcitol) derivatives; (22E,24E)-des-A,B-24-homo-26,27-dimethyl-8-[(E)-N-(2-carboxyethyl)-carbamoylmethylidene]-cholesta-22,24-dien-25-ol; 1α-25-dihydroxyvitamin D3; 25(OH)D3, 25-hydroxyvitamin D3, 24R,25(OH)2D3; 24R,25-dihydroxyvitamin D3; vitamin D2, ergocalciferol; vitamin D3; cholecalciferol; EB1446; EB1436; EB1445; EB1470; deethylhydroxyatrazine (DEHA) (structurally related s-triazines); irgarol 1051; fluorescein isothiocyanate; FITC, metanephrine, normetanephrine; propazine; Terbutyrazine; Terbutyrazine; 6-Chloro-N-(1,1-dimethylethyl)-N'-ethyl-1,3,5-triazine-2,4-diamine; (Structurally related s-triazines); Ametrin (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine (modified: iPr / SCH3 / Et, R 1 = (CH3)2-CH-NH-, R 2 = -SCH3, R 3 = -NH-CH2-CH3; Irgarol; Cyanazine (Modified: R) 1 = Cl, R 2 = NHCH2CH3, R3 = NHCCN(CH3)2); OH-terbutyrazine; terbutyrazine-2OH; hydroxytriazine (EQ-0027); disopropylatrazine (structurally related s-triazine); desethylterbutyrazine (structurally related s-triazine); desethyl-disopropylatrazine (structurally related s-triazine); atlatone; terbutrin (structurally related s-triazines); atrazine derivatives (modified: R 1 = -NHCH(CH3)2, R 2 = -S(CH2)2COOH, R 3 = -NHC2H5); cyanuryl chloride; trifluralin; (structurally related s-triazines) tBu / C4 / SCH3 (modification: R 1 = -NH-C-(CH3)3, R 2 = -NH(CH2)3COOH, R 3 = -SCH3); sulfamethazine; (structurally related s-triazines) 6-[[[4-chloro-6-(methylamino)]-1,3,5-triazin-2-yl]amino]hexanoic acid (modification: Me / Cl / C6, R 1 = -NHCH3, R 2 = -Cl, R 3 = -NH(CH2)5COOH); (Structurally related s-triazines) Procyazin (Modification: R 1 = -Cl, R 2 = -NH-cyclopropyl, R 3 = -NHCCN(CH3)2); (Structurally related s-triazines); prometone (modification: R 1 = -OCH3, R 2 = -NHCH(CH3)2, R 3 = -NHCH(CH3)2); (Structurally related s-triazines) Atrazine mercapturic acid (AM) (Modification: R 1 = -SCH2CH(NHAc)COOH, R 2 = -NHCH2CH3, R 3 = -NHCH(CH3)2); (structurally related s-triazines), desethylatrazine mercapturic acid (desethyl AM) (modification: R 1= -NAcCys, R 2 = -NH2, R 3 = -NHCH(CH3)2); (Structurally related s-triazines); disopropyl atrazine mercapturic acid (disopropyl AM) (modification: R 1 = -NAcCys, R 2 = -NHCH2CH3, R 3 = -NH2); (Structurally related s-triazines); Didealkylated atrazine mercapturic acid (didealkylated AM) (Modification: R 1 = -NAcCys, R 2 = -NH2, R 3 = -NH2); (Structurally related s-triazines); simazine mercapsule (modified: R 1 = -NAcCys, R 2 = -NHCH2CH3, R 3 = -NHCH2CH3); (Structurally related s-triazines) (Modification: R 1 = -S(CH2)2COOH, R 2 = -NHCH2CH3, R 3 = -NHCH2CH3); (Structurally related s-triazines) (Modification: R 1 = -Cl, R 2 = -NHCH(CH3)2, R 3 = -NH(CH2)2COOH); (Structurally related s-triazines) (Modification: R 1 = -Cl, R 2 = -NHCH2CH3, R 3 = -NH(CH2)2COOH); (Structurally related s-triazines); Atrazine mercapturate methyl ester (AM methyl ester) (Modification: R 1 = -NAcCysME, R 2 = -NHCH2CH3, R 3 = -NHCH(CH3)2); N-acetylcysteine; S-benzyl mercapturic acid; (structurally related s-triazines); simetryn (modified: R 1 = -SCH3, R 2 = -NHCH2CH3, R 3= -NHCH2CH3); Metrivudine; 4-amino-6-tert-butyl-4,5-dihydro-3-methylthio-1,2,4-triazine-5-one; Sulfonamide; N 4 -Acetylsulfamethazine (modified: N) 4-Acetylsulfamethazine); Sulfonamides; Sulfatiazole; Sulfatiazole; Sulfamerazine; Sulfamerazine; Sulfaquinoxaline; Sulfaquinoxaline; Sulfachlorpyridazine; Sulfachlorpyridazine; Sulfapyridine; Sulfadimethoxine; Sulfadimethoxine; Sulfamethoxazole; Sulfamethoxazole; Sulfisoxazole; Sulfamethazole; Sulfamethazole; Sulfanilamide; Sulfaguanidine; Sulfag Anidine; sulfadiazine; sulfadiazine; sulfamethoxypyrididine; sulfamethoxypyrididine; pentachlorophenoxypropionic acid; pentachlorophenol; PCP; 2,3,5,6-tetrachlorophenol; 1,2,4,5-tetrachlorobenzene; 2,4,6-trichlorophenol; 2-methoxy-3,5,6-trichloropyridine; 1,3,5-trichlorobenzene; 1,3-dichlorobenzene; 2,4,5-trichlorophenol; 2,6-dichlorophenol; 3,5,6-trichloro-2 -Pyridinoxyacetic acid; 3,5,6-trichloro-2-pyridinol; TCP; 2,4-dichlorophenol; 2,5-dichlorophenol; DNC; 4,4'-dinitrocarbanilide; (structurally related s-triazines); dichloroatrazine; (structurally related s-triazines); dichlorosimazine; 1-((6-chloropyridine-3-yl)methyl)imidazolidined-2-imine; pyridine derivatives; 6-chloropyridine-3-carboxylic acid; nicotinic acid; pyridine derivatives; N-((6-chloropyridine-3-yl)methyl )-N-methylacetamide; (6-chloropyridine-3-yl)-N-methylmethanamine; (6-chloropyridine-3-yl)methanol; imidacloprid; 1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidined-2-yrideneamine; acetamiprid; (E)-N1-[(6-chloro-3-pyridyl)methyl]-N2-cyano-N1-methylacetamidine; nitenpyram; deltamethrin; 1(R)-cis-α(S)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylic acid cyano(3-phenoxyphenyl)methyl ester; DON; deoxynivalenol;DON derivatives; 15-AcDON (15-acetyldeoxynivalenol); DON derivatives; -AcDON (3-acetyldeoxynivalenol); DON derivatives; 3,15-DiacDON (3,15-diacetyldeoxynivalenol); DON derivatives; 3,7,15-TriacDON (3,7,15-triacetyldeoxynivalenol); NIV (nivalenol); nivalenol; NIV derivatives; 4-AcNIV (fusarenone X); fluthranil; α,α,α-trifluoro-3'-isopropoxy-o-toluanilide; mepronil; mebenil; Benodanyl; 24,25(OH)2D3; (24R)-24,25-dihydroxyvitamin D3; 24S,25(OH)2D3; 24S,25-dihydroxyvitamin D3; 25R,26(OH)2D3; 25R,26-dihydroxyvitamin D3; 25S,26(OH)2D3; 25S,26-dihydroxyvitamin D3; 1,24,25(OH)3D3; 1,24,25-trihydroxyvitamin D3; 1,25-lactone; (23S,25R)-1,25(OH)2D3-26,23-lactone; 24,25(OH)2-7-DHC; 24,25(O H)2-7-dehydrocholesterol; 25(OH)D3-3S; 25(OH)D33-sulfate; 24,25(OH)2D3-hemiglutarate derivative; 11α-hemiglutaryloxy-(24R)-24,25-dihydroxyvitamin D3; 24,25(OH)2D3-hemiglutarate derivative; (24R)-24,25-dihydroxyvitamin D3-3-hemiglutarate; 24R,25(OH)2D2; 24S,25(OH)2D2; 25(OH)D2; 1,24(OH)2D3; 2,3,6-trichlorophenol; tetrachlorohydroquinone; pe Ntachloroaniline; pentachlorobenzene; 2,3-dinitrotoluene; ,4-dinitrotoluene; 2,4,5-trichloronitrobenzene; 3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid; 2,3,4,6-tetrachlorophenol; 2,4,6-trichloroanisole; 2,4,6-TCA; pentabromophenol; PBP; 2,4,6-tribromophenol; 2,4,6-TBP; 2-bromo-4-chlorophenol; 2-B-4-CP, 2,4-dibromophenol; 2,4-DBP; 2,6-dibromophenol;2,6-DBP; 4-bromophenol; 4-BP; furosemide; ampicillin; amoxicillin; 6-aminopenicillaneic acid (6-APA); azurocillin; bacampicillin; carbenicillin; epicillin; cloxacillin; dicloxacillin; methanepicillin; methicillin; moxalactam; oxacillin; penicillin G; benzylpenicillin; penicillin V; phenoxymethylpenicillin; pheneticillin; piperacillin; ticarcillin; ampicillin hydrolysate; Penicillin G hydrolysate; 3-phenoxybenzoic acid (3-PBAc); chlorpyrifos; chlorpyrifos derivatives; HCl01; derivatives directly synthesized by substituting the chlorine at position 6 of chlorpyrifos for research purposes with a 3-mercaptopropanoic acid spacer arm; chlorpyrifos derivatives; HTCP (modified: the TCP metabolite HTCP was prepared by hydrolysis of the thiophosphate ester of HCl01); zeatin riboside (trans isomer); zeatin (trans isomer); N; 6 -(2-isopentenyl)-adenosine; IPA; N 6-(2-isopentenyl)-adenine; 2-iP; benzyladenine; kinetin; monulon; monolinuron; fenuron; nebulon; propanil; profam; chloropropam; 4-chloroaniline; methylurea derivative; 1-(3-carboxypropyl)-3-(4-chlorophenyl)-1-methylurea; methylurea derivative; 1-(5-carboxypentyl)-3-(4-chlorophenyl)-1-methylurea; metobromulone; sennoside B; SB; sennoside B with an erythrogenic configuration between C-10 and C-10'; sennoside A ( Modified compounds: Sennoside A (where the C-10 terminals are in a threo configuration); lein; emodin; aloe-emodin; barbaroin; 1,4-dihydroxyanthraquinone; lapontisine; gallic acid; vanillic acid; caffeic acid; homogentisic acid; esculin; cinnamtannin B1; baicalin; naringin hydrate; ougonin; ougonin 7-O-β-glucuronide; curcumin; δ1-tetrahydrocannabinolic acid; δ1-tetrahydrocannabinol; (±)-cis-4-aminopermethrin; 3-(4-aminophenoxy)benzyl (±)-cis-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate; permethrin; trans-permethrin; cis-permethrin; cypermethrin; phenothrin; resmethrin; cyfluthrin; trans-permethrin esfenvalerate; fluvalinate; fenpropatrin; cis-permethrin; 4-phenoxybenzoyl alcohol; Diurone derivatives; 1-(3-carboxypropyl)-3-(3,4-dichlorophenyl)-1-methylurea; Sidurone; Terbutiurone; Barban; Trifluralin; 2,6-dinitro-N-propyl-N-(2-carboxyethyl)-4-(trifluoromethyl)benzeneamine; TR-13; 2-ethyl-7-nitro-1-propyl-5-(trifluoromethyl)-1H-benzimidazole; Benefin; 2,6-dinitro-N-butyl-N-ethyl-4-(trifluoromethyl)benzeneamine; TR-2; 2,6-dinitro-N-propyl -4-(trifluoromethyl)benzeneamine; ethalfluralin; 2,6-dinitro-N-ethyl-N-(2-methyl-2-propenyl)-4-(trifluoromethyl)benzeneamine; TR-40; N-(2,6-dinitro-4-(trifluoromethyl)phenyl)-N-propylpropanamide; TR-15; 2-ethyl-4-nitro-6-(trifluoromethyl)-1H-benzimidazole; TR-3; 2,6-dinitro-4-(trifluoromethyl)benzeneamine; TR-6; 3-nitro-5-(trifluoromethyl)-1,2-benze Diamine; TR-9; 5-(trifluoromethyl)-1,2,3-benzenetriamine; TR-21; 4-(dipropylamino)-3,5-dinitrobenzoic acid; TR-36M; 3-methoxy-2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzeneamine; oryzalin; 3,5-dinitro-4-(dipropylamino)benzenesulfonamide; pendimethalin; 2,6-dinitro-N-(1-ethylpropyl)-3,4-dimethylbenzeneamine; pentagalloyl glucose; pyrene; pyrene-1-carboxyaldehyde; f 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; acenaphthyrene; fluorantene; benzo(j,k)fluorene; indeno-1,2,3-cd-pyrene; 1,10-(1,2-phenylene)pyrene; benzo[a]anthracene; 1,2-benzanthracene; benzo(k)fluorantene; naphthalene; benzo[a]fluorantene; dibenzo[ah]anthracene;1,2,5,6-Dibenzanthracene; 2,3-Diaminonaphthalene; 2,6-Dinitroaniline; 17-β-Estradiol (ED); Estr-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 Body; N-(2,6-dinitro-4-trifluoromethylphenyl)-N-propyl-6-aminohexanoic acid; Trifluralin derivative; N-(2,6-dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid methyl ester; Trifluralin derivative; N-(2,6-dinitro-4-trifluoromethylphenyl)-6-aminohexanoic acid tert-butyl ester; Benfluralin; Etalfluralin; Trifluralin derivative; 2,6-dinitro-4-trifluoromethylphenol; Isopropaline; Aniline; 2-Hydroxybenzotri Fluoride; N-propyl-6-aminohexanoic acid; N-methyl-6-aminohexanoic acid; MHPG derivative; D-MHPG (D-3-methoxy-4-hydroxyphenyl glycol); MHPG derivative; L-MHPG (L-3-methoxy-4-hydroxyphenyl glycol); MHPG derivative; DL-MHPG (DL-3-methoxy-4-hydroxyphenyl glycol); mixture of isomers of D-MHPG and L-MHPG; MHPG derivative; DL-MHPG-SO4 (DL-3-methoxy-4-hydroxyphenyl glycol sulfate), modified form is D-MHP May contain isomer mixtures of G-SO4 and L-MHPG-SO4; serotonin; 5-HT; 5-hydroxydopamine (5-4HDA); 3,4-dihydroxyphenyl glycol (DOPEG); dopamine; 4-(2-aminoethyl)pyrocatechol; 3-hydroxytyramine; 3,4-dihydroxyphenethylamine; L-3,4-dihydroxyphenylalanine; L-DOPA; vanillomandelic acid; DL-VMA; homovanillic acid; norepinephrine; DL-NE; D-epinephrine; DE; 3-methoxytyramine; MTA; 3-methoxytyrosine;MTyr; 3,4-dihydroxymandelic acid; DL-DOMA; 3,4-dihydroxyphenylacetic acid; DOPAC; L-phenylalanine; tyramine; p-tyramine; 4-(2-aminoethyl)phenol; D-mandelic acid; homocatechol; octopamine; DL-octopamine; azinephosethyl; S-(3,4-dihydro-4-oxobenzo[d]-[1,2,3]-triazine-3-ylmethyl)O,O-diethylphosphodithioate; phosmet; O,O-dimethyl S-phthalimidomethylphosphorodithioate; forpet; N-[(trichloromethyl)thio]phthalimide; tetramethrin; (1-cyclohexen-1,2-dicarboximide)methyl-2,2-dimethyl-3-(2-methylpropenyl)-cyclopropanecarboxylate; N-(bromomethyl)phthalimide; N-(chloromethyl)benzazimide; 6-(N-phthalimidoylmethylthio)hexanoic acid (MFH); b Romasyl; 5-bromo-3-sec-butyl-6-methyluracil; bromacyl derivative; 5-bromo-6-(hydroxymethyl)-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione; bromacyl derivative; 5-bromo-3-(2-methylpropyl-6-methyl-2,4(1H,3H)-pyrimidinedione; metabolite of bromacyl; bromacyl derivative; 3-hydroxy-1-methylpropyl-6-methyl-2,4(1H [,3H]-pyrimidinedione (modified: bromacyl metabolite); bromacyl derivative; 6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified: bromacyl metabolite); terbacyl derivative; [5-chloro-3-(1,1-dimethylethyl)-6-(hydroxymethyl)-2,4(1H,3H)-pyrimidinedione; terbacyl; 3-tert-butyl-5-chloro-6-methyluracil; bromacyl derivative Conductor; ethyl-5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-1-yl)hexanoate; N-1 alkylated bromacyl derivative; bromacyl derivative 5-(5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-1-yl)hexanoic acid (modified: N-1 alkylated bromacyl derivative); bromacyl derivative;Bromo-6-(bromomethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione (modified: a bromacyl derivative with a methyl substituted at position 6); a bromacyl derivative [5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]-2-carboxylpropanoic acid (modified: a bromacyl derivative with a methyl substituted at position 6); 3-[5-bromo-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione-6-yl]propanoic acid (modified: a bromacyl derivative with a methyl substituted at position 6) Bromacyl derivatives; 5-bromo-1,6-dimethyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione, a bromacyl derivative; 5-bromo-1-butyl-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione, a bromacyl derivative; butachlor; N-butoxymethyl-2-chloro-2',6'-diethylacetanilide; amidochlor; N-[(acetylamino)methyl]-2-chloro-N-(2,6-diethylphenyl)acetamide; nicarbazine; 4,6 -N,N'-bis(4-nitrophenyl) compound having -dimethyl-2(1H)-pyrimidinone (modified: DNC+HDP); 2-hydroxy-4,6-dimethylpyrimidine; HDP; imazalil; [1-(β-allyloxy-2,4-dichlorophenethyl)imidazole]; imazalil derivative; EIT-0073 (modified imazalil having an -O(CH2)5-COOH group instead of the original -OCH2CH=CH2 group); penconazole; (RS)-1-(2,4-dichloro-β-propylphenethyl)-1H-1,2,4-tri Zol; Hexaconazole; (RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)hexane-2-ol; Propiconazole; cis-trans-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazol; 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 derivative; EIT-0183; imazalil derivative; EIT-0180; imazalil derivative; EIT-0111; imazalil derivative; EIT-0158; imazalil derivative; K-240; chlorothalonil; tetrachloroisophthalonitrile (modification on benzene ring: R; 1 = CN, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); 2,4,5,6-Tetrachloro-3-cyanobenzamide which is a chlorothalonil derivative (modification on benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = CN, R 4 = Cl, R 5 = Cl, R 6 = Cl); 2,5,6-Trichloro-4-hydroxyisophthalonitrile which is a chlorothalonil derivative (modification on benzene ring: R 1 = CN, R 2 = Cl, R 3 = CN, R 4 = OH, R 5 = Cl, R 6 = Cl); 3-Carbamoyl-2,4,5-trichlorobenzoic acid (modification on benzene ring: R 1 = CONH2, R 2 = Cl, R 3 = COOR, R 4 = H, R 5 = Cl, R 6 = Cl); Pentachloronitrobenzene (modification on benzene ring: R 1 = NO2, R 2 = Cl, R 3 = Cl, R 4 = Cl, R 5 = Cl, R<​1 = Cl, R 2 = Cl, R 3 = Cl, R 4 = Cl, R 5 = Cl, R 6 = Cl); 2,4,5,6-tetrachlorophenol (modification on the benzene ring: R 1 = OH, R 2 = Cl, R 3 = H, R 4 = Cl, R 5 = Cl, R 6 = Cl); carbaryl derivative; ethyl carbamate (modified: R) 1 = OCONHCH2CH3, R 3 = H); 1-Naphthol; 1-Naphthaleneacetamide; -(1-Naphthyl)acetamide; Carbaryl derivative; 1-Methyl carbonate (Modified: R) 1 = OCOOCH3, R 2 = H); Carbaryl derivative; 1-ethyl carbonate (modified: R 1 = OCOOCH2CH3, R 2 = H); 2-ethyl carbonate (modified: R), a carbaryl derivative. 1 = H, R 2 = OCOOCH2CH3); Carbaryl derivative; 1-Ethyl thiocarbonate (modified: R 1 = OCOSCH2CH3, R 2 = H); Carbaryl derivative; 2-ethylthiocarbonate (modified: R 1 = H, R 2 = OCOSCH2CH3); naptalam; N-1-naphthylphthalamiate; carbaryl derivative; 3-hydroxycarbaryl (modified: R 1 = OCONHCH3, R 2 = H, R 3 = OH, R 4 = H, R 5 = H); 4-hydroxycarbaryl (modified: R) is a carbaryl derivative. 1 = OCONHCH3, R 2 = H, R 3 = H, R 4 = OH, R5 = H); 5-hydroxycarbaryl (modified: R) is a carbaryl derivative. 1 = OCONHCH3, R 2 = H, R 3 = H, R 4 = H, R 5 = OH); Carbaryl derivative; 1-(5-carboxypentyl)-3-(1-naphthyl)urea (modification: R 1 = NHCONH(CH2)5COOH, R 2 = H); (Structurally related s-triazines: adiprothrin; 4-azido-N-isopropyl-6-methylthio-1,3,5-triazine-2-ylamine (modification: R) 1 = -SCH3, R 2 = -N3, R 3 = -CH(CH3)2); (Structurally related s-triazines); 2-(ethylamino)-4-(methylthio)-6-aminotriazine (modification: R 1 = -SCH3, R 2 = -NH-C2H5, R 3 = -NH2); (Structurally related s-triazines are 2-amino-4-(methylthio)-6-(isopropylamino)triazine (modification: R 1 = -SCH3, R 2 = -NH2, R 3 = -NH-CH(CH3)2); (Structurally related s-triazines are 2-amino-4-methoxy-6-(isopropylamino)triazine (modification: R 1 = -OCH3, R 2 = -NH2, R 3= -NH-CH(CH3)2); TCP derivative (3,5,6-trichloro-2-pyridinol derivative); 3-(3,5-dichloro-6-hydroxy-2-pyridyl)thiopropanoic acid; p-nitrosuccinialidic acid (PNA-S); PNA-S; PNA-C; p-nitro-cis-1,2-cyclohexanedicarboxylic acid; nitroaniline derivative; 2-nitroaniline; o-nitroaniline; 3-nitroaniline, a nitroaniline derivative; m-nitroaniline; 4-nitroaniline, a nitroaniline derivative; p-nitroaniline; aromatic alcohols; 4-nitrobenzyl alcohol; 4-nitrophenethyl alcohol, an aromatic alcohol; 2-nitrobenzyl alcohol, an aromatic alcohol; aromatic alcohols; 3-nitrobenzyl alcohol; 1-benzyl-3-(4-nitrophenyl)urea, a urea derivative; 1-(3-chlorophenyl)- 3-(2-methoxy-5-nitrophenyl)urea; urea derivative 1-(3-chlorophenyl)-3-(4-methoxy-3-nitrophenyl)urea; urea derivative 1-(4-chlorophenyl)-3-(4-nitrophenyl)urea; urea derivative (2-fluorophenyl)-3-(2-methoxy-4-nitrophenyl)urea; 1-(3-methoxyphenyl)-3-(3-nitrophenyl)urea; carbofuran derivative m-carbofranphenol; hydroxycarbofran; ketocarbofran; carbosulfan; (dibutylaminothio)methylcarbamate 2,3-dihydro-2,2-dimethylbenzofuran-7-yl; benfuracarb; N-[2,3-dihydro-2,2-dimethylbenzofuran-7-yloxycarbonyl(methyl)aminothio]-N-isopropyl-β-allinate; furathiocarb; 2,3-dihydro-2,2-dimethyl-7-benzofuranyl 2,4-dimethyl-5-oxo-6-oxa-3-thia-2,4-diazadecanoate; carbofuran derivative; 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]amino]butanoic acid (BFNB) (modified: n=3, X=CH2); endrin; nendorin;(1R,4S,4aS,5S,6S,7R,8R,8aR)-1,2,3,4,10,10-Hexachloro-1,4,4a,5,6,7,8,8a-Octahydro-6,7-Epoxy-1,4,5,8-Dimethanonaphthalene;Heptachlor;1,4,5,6,7,8,8-Heptachloro-3a,4,7,7a-Tetrahydro-4,7-Methanoindene;Chlordane;1,2,4,5,6,7,8,8-Octachloro-2,3,3a,4,7,7a-Hexahydro-4,7-Methanoindene;Endosulfan (Modified forms: α- and β-forms) Isomer mixture of; Endosulfan (modified: α-isomer); Endosulfan (modified: β-isomer); Endosulfan derivatives; Endosulfan sulfate (modified: sulfate); Endosulfan derivatives; Endosulfan diol; Endosulfan diol metabolites; Endosulfan derivatives; Endosulfan ether (modified: Endosulfan ether metabolites); Endosulfan derivatives; Hydroxy ether; Endosulfan hydroxyether metabolites; Endosulfan derivatives; Endosulfan lactone ( Modified compounds: lactone metabolites of endosulfan); aldrin; dieldrin; fenvalerate isomer (modified compound: 1S,2R isomer, R:Ph); fenvalerate isomer (modified compound: 1R,2S isomer, R:Ph); fenvalerate isomer (modified compound: 1R,2R isomer, R:Ph); fenvalerate isomer (modified compound: 1S,2R / S isomer, R:Ph); fenvalerate isomer (modified compound: 1R,2R / S isomer, R:Ph); fenvalerate isomer; fenvalerate (modified compound: 1R / S,2R / S isomer, R:Ph); Thiabendazole; 2-(thiazole-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-succinamidthiabendazole; thiabendazole derivatives; 2-succinamidthiabendazole; cambendazole; fenvalerate haptens;(S)-4-chloro-α-(1-methylethyl)benzene acetate cyano[3-(4-aminophenoxy)phenyl]methyl(4-aminoesfenvalerate); fenvalerate haptens; 4-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]benzenepropanoate benzyl; fenvalerate haptens; 3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetate benzyl; fenvalerate haptens; 3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxyacetate; fenvalerate haptens; 6-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]hexanoate benzyl; Fenvalerate haptens; 6-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]hexanoic acid (fenvalerate haptens); 4-[3-[cyano[(S)-2-(4-chlorophenyl)-3-methyl-1-oxobutanoxy]methyl]]phenoxy]benzenepropanoic acid; (S)-fenvalerate acid; (structurally related s-triazines); atrazine mercaptulate (modified: R; 1 = -SCH2CH(NHCOCH3)COOH, R 2 = -NHCH2CH3, R 3= -NHCH(CH3)2; fenthion hapten; methyl O-[3-methyl-4-(methylthio)phenyl] N-(3-carboxypropyl)phosphoramidethioate (modified form called hapten B); fenthion derivative; fenthion oxide; fenthion derivative; fenthion oxide; pyrimiphos-ethyl; 4-(methylthio)-m-cresol; chlorpyrifos derivative; chlorpyrifos-oxone; fenchlorphos; O,O-dimethyl O-2,4,5-trichlorophenyl phosphorothioate; trichloronate; O-ethyl O-2,4,5-trichlorophenyl ethylphosphonothioate; diclofenthion; O-2,4-dichlorophenyl O,O-diethyl phosphorothioate; parathion; O,O-diethyl O-4-nitrophenyl phosphorothioate; thiophos; chlorpyrifos derivative (modified form: synthesis of AR1 has been reported); chlorpyrifos derivative; O-ethyl O-(3,5,6-trichloro-2-pyridyl) O-(3-carboxypropyl) phosphorothioate; (PO); a chlorpyrifos derivative, O-ethyl O-(3,5,6-trichloro-2-pyridyl) N-(5-carboxyethyl) phosphoramidothioate; (PN1) (modified form: amide bond of thiophosphate reagent); chlorpyrifos derivative; O-ethyl O-(3,5,6-trichloro-2-pyridyl) N-(2-carboxyethyl)phosphoramidethioate; (PN1) (modified: amide bond of appropriate thiophosphate reagent); triadimephon; (RS)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one; GR151004; (4-[[5-[3-[2-(dimethylamino)ethyl]]-5-benzofuranyl]-3-pyridinyl]acetyl]morpholine dihydrochloride; diflubenzuron; 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea; (Structurally related s-triazines SprAAT (modified: 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 (modified: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH(CH2CH3); (structurally related s-triazines CDIT (modified: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH(CH(CH3)2)); (Structurally related s-triazines); CDDT (Modification: R 1 = Cl, R 2 = NH[C(O)CH3], R 3 = NH[C(O)CH3]); (Anmeline, a structurally related s-triazine; OAAT (modified: R 1 = OH, R 2 = NH2, R 3 = NH2); (Structurally related s-triazines such as ammerid; OOAT (modification: R 1 = OH, R 2 = OH, R 3 = NH2); (Structurally related s-triazine cyanuric acid; OOOT (modification: R 1 = OH, R 2 = OH, R 3 = OH); (structurally related s-triazines); melamine; AAAT (modification: R 1 = NH2, R 2 = NH2, R 3 = NH2); structurally related s-triazines, N-isopropylammeline; OIAT (modified: R 1 = OH, R2 = NH[CH(CH3)2], R 3 = NH2); structurally related s-triazines, N-ethylammeline; OEAT (modification: R 1 = OH, R 2 = NHCH2CH3, R 3 = NH2); structurally related s-triazines; N-ethyl ammerido; OOET (modification: R 1 = OH, R 2 = OH, R 3 = NHCH2CH3); structurally related s-triazines include cyromazine and CyPAAT (modified: R 1 = NH(C3H5), R 2 = NH2, R 3 = NH2); diamino-s-triazine, a structurally related s-triazine; HAAT (modified: R 1 = H, R 2 = NH2, R 3= NH2); PCBs; 2,5,3',4'-tetrachlorobiphenyl (modified: IUPAC no.: 70); PCBs 2,4,5,3',4'-pentachlorobiphenyl (modified: IUPAC no.: 118); PCBs 2,2',5,5'-tetrachlorobiphenyl (modified: IUPAC no.: 52); PCBs; 6-[3,3',4'-trichlorobiphenyl-4-yl)oxy]hexanoic acid; Methrazone; Trademark name: Mykrox, Zaroxolyn; Furfuryl benzoate; DDT metabolite; DDA; Paraquat; 1,1'-dimethyl-4,4'-bipyridinium ion; Diethylcarbamazine; THP; 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium; o-DNCP; Dinitrocarboxyphenol; PCBs; 3-chlorobiphenylol (modified: IUPAC No. 2); PCBs; 3,4'-dichlorobiphenyl (modified: IUPAC No. 13), PCBs; 3,5-dichlorobiphenyl (modified: IUPAC No. 14); PCBs; 3,4,5,3',4'-pentachlorobiphenyl (modified: IUPAC No. 126); 2,3,3',4'-tetrachlorobiphenyl (modified: IUPAC No. 56); 2',3,4,5-tetrachlorobiphenyl (modified: IUPAC No. 76); 3,3',5,5'-tetrachlorobiphenyl (modified: IUPAC No. 80); 2,4,5,2',5'-pentachlorobiphenyl (modified: IUPAC No. 101); 2,3,3',4,4'-pentachlorobiphenyl (modified: IUPAC No. 105); 2,3,6,3',4'-Pentachlorobiphenyl (Modified: IUPAC No. 110); 3,3',4,5,5'-Pentachlorobiphenyl (Modified: IUPAC No. 127); 3,4,5,3',4',5'-Hexachlorobiphenyl (Modified: IUPAC No. 169); 2,3,3',4,4',5-Hexachlorobiphenyl (Modified: IUPAC No.156); 3,4,3',4'-tetrabromoviphenyl; 3,4,5,3',4',5'-hexabromoviphenyl; 2,4,5,2',4',5'-hexabromoviphenyl; dibenzofurans and dioxins; 2,3,7,8-tetrachlorobenzofuran; 2,3,7,8-tetrachlorodibenzo-p-dioxin; 3,4',5-trichloro-4-biphenylol; 3,3',5,5'-tetrachloro-4,4'-biphenyldiol; 3,4,3',4'-tetrachlorodiphenyl ether; 1,2-dichlorobenzene; 1,4-dichlorobenzene; 1,2,4-trichlorobenzene; 3,4-dichloroaniline; DDT metabolites; 4,4'-DDT; 4,4'-DDD retronesin; 3,4-dichlorobiphenyl (modified: IUPAC No. 12); 3,4,3'-Trichlorobiphenyl (Modified: IUPAC No. 35); PCBs; 3,4,4'-Trichlorobiphenyl (Modified: IUPAC No. 37); 3,4,3',5-Tetrachlorobiphenyl (Modified: IUPAC No. 78); 3,4,3',5'-Tetrachlorobiphenyl (Modified: IUPAC No. 79); 3,4,4',5-Tetrachlorobiphenyl (Modified: IUPAC No.81); DDT metabolites; p,p'-DDT (modified: p,p'-dichlorodiphenyltrichloroethane); o,p'-DDT (modified: o,p'-dichlorodiphenyltrichloroethane); p,p'-DDE (modified: p,p'-DDE); o,p'-DDE (modified: o,p'-); p,p'-DDD (modified: p,p'-DDD); o,p'-DDD (modified: o,p'-DDD); dicofol Lu; 4,4-dichloro-α-(trichloromethyl)benzhydrol; cyprazine; 6-chloro-N-cyclopropyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine; structurally related s-triazines; dipropetrin; 6-(ethylthio)-N,N'-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine; trietazine; 6-chloro-N,N,N'-triethyl-1,3,5-triazine-2,4-diamine; 6-hydroxyatrazine; hexazinone; 3-cyclohexyl-6-dimethylamino-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione; TNT; 2,4,6-trinitrotoluene; tetraconazole (M14360); 1-[2-(2,4-dichlorophenyl)-3- (1,1,2,2-tetrafluoroethoxy)propyl]-1H-1,2,4-triazole; DTP; 2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazole-1-yl)propanol; imazalil; phenalimol; (RS)-2,4'-dichloro-α-(pyrimidine-5-yl)benzhydryl alcohol; rupanine metabolite; (+)-rupanine (modified: R = H); rupanine metabolite; (+)-13-hydroxyrupanine (modified: R = OH); rupanine metabolite; (+)-13-hydroxyrupanine hemysuccinate (modified: R = OCO-(CH2)2COOH); rupanine metabolite; (+)-13-hydroxyrupanine cis-hexahydrophthalate (modified: R = OCOC6H. 10 COOH); Rupanine metabolites; α-Isolupanine; Rupanine metabolites; Hydroxylupanine; Spartein; Cysteine; Multiflolin; Epirupinine; (Structurally related s-triazines); Cyanadic acid (modified: R 1 = Cl, R 2= NHCH2CH3, R 3 = NHCCOOH(CH3)2); structurally related s-triazines (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)3COOH); structurally related s-triazines (modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NHCH2COOH); (Structurally related s-triazines) (Modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)4COOH); norflurazone; 4-chloro-5-(methylamino)-2-[3-(trifluoromethyl)phenyl]-3(2H)-pyridazinone; norflurazone derivatives; desmethylnorflurazone; metoflurazone; chloro-5-(dimethylamino)-2-[(3-trifluoromethyl)phenyl]-3(2H)-pyridazinone; pyrazone; chloridazone; 5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone (active ingredient); dichlorophenyl-pyridazone; (structurally related s-triazines such as azidoatrazine (modified: R 1 = N3, R 2 = NHCH(CH3)2, R 3 = NHCH2CH3); alachlor, 2-chloro-2',6'-diethyl-N-methoxymethylacetanilide; trichothecolone (modified: R 1 = H, R 2 = OH, R 3 = H, R 4 = O, R 5= H); DON derivative; Acetyl-T-2; DON derivative; T-2 tetraacetate; Chlorpyrifos derivative; Monodechloro-CP; Bromophos derivative; Bromophos methyl; Bromophos derivative; Bromophos ethyl dicapton; 2-Chloro-4-nitrophenyl O,O-dimethyl phosphorothioate; Tetrachlorvinphos; Phosphate (Z)-2-chloro-1-(2,4,5-trichlorophenyl)vinyldimethyl; Triclopyr; 3,5,6-trichloro-2-pyridyloxyacetic acid; Pichloram; 4-amino-3,5,6-trichloropyridine-2-carboxylic acid; Formononetin; Biochanin A; 5,7-dihydroxy-4'-methoxyisoflavones (modified: 4'-methyl ether of genistein); Equol; (7-hydro Xy-3-(4'-hydroxyphenyl)-chroman; 2'-methoxyformononetin; daidzein; 7-hydroxy-3-(4'-hydroxyphenyl)-4H-1-benzopyran-4-one; genistein; quercetin; 3,3',4',5,7-pentahydroxyflavone; 3,5,7,3',4'-pentahydroxyflavone; matheucinol; coumestrol; (structurally related s-triazines); hydroxysimazine (modified: R 1 = OH, R 2 = NHCH2CH3, R 3 = NHCH2CH3); Angustiphorine; Alodan; 1-Methyl-4-phenyl-4-carboethoxypiperidine hydrochloride; Zearalenone; RAL; F-2 toxin; Fenpropimorph; (RS)-cis-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine; Tridemorph; 2,6-dimethyl-4-tridecylmorpholine; 2,6-dimethylmorpholine; Amorolphine; Fenpropidine; (RS)-1-[3-(4-tert-butylphenyl)-2-methylpropyl]piperidine; (Structurally related s-triazines) (Modification: R 1 = Cl, R 2 = Cl, R 3 = NHCH2CH3); (Structurally related s-triazines) (Modification: R 1 = Cl, R 2 = Cl, R3 = NHCH(CH3)2); (Structurally related s-triazines) (Modification: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NH(CH2)5COOH); (Structurally related s-triazines) (Modification: R 1 = Cl, R 2 = NHCH(CH3)2, R 3 = NHCH2COOH); (Structurally related s-triazines) (Modification: R 1 = Cl, R 2 = NHCH(CH3)2, R 3 = NH(CH2)5COOH); structurally related s-triazines; cyanazineamide (modified: R 1 = Cl, R 2 = NHCH2CH3, R 3 = NHCCONH2(CH3)2); Hydroxycyanadic acid (modification: R 1 = OH, R 2 = NHCH2CH3, R 3 = NHCCOOH(CH3)2); Deethylsimazine (Modification: R 1 = Cl, R 2 = NH2, R 3= NHCH2CH3); Albendazole sulfoxide; [5-(propylthionyl)-1H-benzimidazole-2-yl]-methyl ester; Albendazole sulfone; 5(6)-alkylbenzimidazoles; 2-amino-5-(propylthio)benzimidazole; 5(6)-alkylbenzimidazoles; 2-amino-5-(propylsulfonyl)benzimidazole; Oxybendazole; 5-propoxybenzimidazole-2-methylcarbamate; 5(6)-arylbenzimidazoles; Fenbendazole sulfone (modified: sulfone metabolite of fenbendazole); 5(6)-arylbenzimidazoles; 4'-H Droxyfenbendazole; 5(6)-arylbenzimidazoles; oxfendazole (modified: oxfendazole is a sulfoxide metabolite of fenbendazole); 5(6)-arylbenzimidazoles; flubendazole; benzimidazole metabolites; 2-aminobenzimidazole; benzimidazole metabolites; 5-aminobenzimidazole; benzimidazole metabolites; 2-acetylbenzimidazole; benzophenone; diphenylmethanone; phenyl ketone; diphenyl ketone; benzoylbenzene; benzaldehyde; benzaldehyde; 4-bromo-2,5-dichlorophenol; acephate; O,S-dimethyl Acetylphosphoramidethioate; methamidophos; O,S-dimethylphosphoramidethioate; dichlorvos; 2,2-dichlorovinyldimethyl phosphate; fentoate; S-α-ethoxycarbonylbenzyl O,O-dimethylphosphodithioate; EPN; ethyl p-nitrophenyl thionobenzenephosphonate; bioresmethrin; (1R,3R)-2,2-dimethyl-3-(2-methylpropa-1-enyl)cyclopropanecarboxylic acid 5-benzyl-3-furylmethyl (modification: the ISO general name for the unseparated mixture of isomers of this substance is resmethrin); flufenoxuron; 1-[4-(2-chloro-α,α,α-trifluoro-p-tolyloxy)-2-fluorophenyl]-3-(2,6-difluorobenzoyl)urea; amitorol; 1H-1,2,4-triazole-3-ylamine; molinate; S-ethyl Azepan-1-carbothioate;Molinate derivative (modified: S-2-carboxyethyl hexahydroazepine-1-carbothioate); Molinate derivative (modified: S-5-carboxypentyl hexahydroazepine-1-carbothioate); Molinate derivative (modified: molinate sulfone); Molinate derivative (modified: S-(p-aminobenzyl) hexahydroazepine-1-carbothioate); Molinate derivative (modified: S-2-(p-aminophenyl)ethyl hexahydroazepine-1-carbothioate); Hexamethyleneimine; Thiobencarb (Bolero); Butyrate (Sutan); EPTC (Eptam); Cycloate (Roneet); Pevlate (Tillam); Vernolate (Vernam); Aflatoxin M1; AFM1 (modified: AFM1); Aflatoxin B1; AFB1 (modified: AFB1); Aflatoxin G1; AFB1 (modified: AFG1); Aflatoxin M2; AFM2 (modified: AFM2); Aflatoxin B2; AFB2 (modified: AFB2); Aflatoxin G2; AFG2 (modified: AFG2); Aflatoxin B2α; AFB2α (modified: AFB2α); Aflatoxin G2α; AFG2α (modified: AFG2α); KB-6806; 6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl) (modified: R; 1 = NH2, R 2 = CH(CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: R 1 = NH2, R 2 = CH2CH(CH3)2, R 3 = CH3); Hapten name: KB-6806 (benzimidazole) derivative (modification: R 1 = NH2, R 2 = CH(CH2CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: R 1 = NHCOCH3, R 2 = CH(CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: R 1 = H, R 2= CH(CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: R 1 = NH2, R 2 = CH(CH3)2, R 3 = CH3);KB-6806 (benzimidazole) derivative (modified: R 1 = NH2, R 2 = CH(CH3)2, R 3 = =N(→O)CH3(N-oxide);KB-6806 (benzimidazole) derivative (modification: R 1 = NH2, R 2 = CH(CH3)2, R 3 = H);KB-6806 (benzimidazole) derivative (modified: R 1 = NH2, R 2 = CH2CH3, R 3= CH3); Aminopraoxon; Phosphate; O,O-Diethyl O-(4-aminophenyl) ester, Methylparathion; Phosphothioic acid; O,O-Dimethyl O-(4-nitrophenyl) ester; Diethylphenyl phosphate; Phenylphosphonic acid; O,O-Diethyl ester; Diethyl phosphate; Ethylphosphonic acid; O,O-Diethyl ester; p-Nitrophenyl phosphate; Phosphonic acid; O-(4-nitrophenyl) ester; Phorate; Phosphodithioic acid; O,O-Diethyl S-[(ethylthio)methyl] ester; Ethion; Bis(phosphodithioic acid); S,S'-Methylene O,O,O',O'-Tetraethyl ester; Carbophenothion; Phosphodithioic acid; O,O-Diethyl S-[[(4-chlorophenyl)thio]methyl] ester; Disulfone; Phosphodithioic acid; O,O-Diethyl S-[(2-ethylthio)ethyl]ester; TS; N-[4-(carboxymethyl)-2-thiazolyl)sulfanilamide; NS; N-(4-nitrophenyl)sulfanilamide; sulfamoxole; sulfacetamide; DNP-SL; spin-labeled dinitrophenyl (modification: synthesis of DNP-SL was reported by Balakrishnan et al. (1982); formula is described in the literature by Anglister et al. (1984)); β-ecdysone; benzimidazole derivative; 5(6)-[carboxypentyl)thio]-2-(methoxycarbonyl)amino]-benzimidazole; 2-hydroxybiphenyl; H BP; Atrazine caproic acid; Lysophosphatidic acid (LPA); 1-Acyl-2-hydroxy-sn-glycero-3-phosphate; Berberine; Palmatine; 9-Acetylberberine; Coridaline; Coptisin; Berberbine; 8-Oxoberbein; Papaverine; Berberine derivatives; 9-O-Carboxymethylberberine; Phencyclidine; 1-(1-Phenylcyclohexyl)piperidine; Methoxychlor; Endosulfan derivatives; 4-Oxobutanoic acid, 4-(4,5,6,7,8,8-Hexachloro-3a,4,7,7a-Tetrahydro-4,7-Methane-1H-Indenyl-1-oxy); Endosulfan derivatives;4-Oxybutanoic acid, 4-(1,3,4,5,6,7,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindanyl-2-oxy; Endosulfan derivative (modified: hemisuccinate of endosulfandiol); Triazole derivative; 5-(3-hydroxypropyl)-3-amino-2H-1,2,4-triazole; Triazole derivative; 5-(3-hydroxypropyl)-3-(2-nitrophenylsulfenyl)amino-2H-1,2,4-triazole; Triazole derivative; 3-amino-5-[(3-succinyloxy) Propyl]-2H-1,2,4-triazole; triazole derivative; 3-amino-1,2,4-triazole-5-thiol; triazole derivative; 3-[(2-nitrophenylsulfenyl)amino-2H-1,2,4-triazole-5-thiol; triazole derivative; 2H-1,2,4-triazole-5-thiol; triazole derivative; 4-methyl-1,2,4-triazole-3-thiol; triazole derivative; (1,2,4-triazole-2-yl)acetic acid; 1,2,4-triazole; 4-nitrophenyl4'-carboxymethylphenyl Phosphate; Triazole derivatives; 4-amino-1,2,4-triazole; Triazole derivatives; 3-acetamido-1H-1,2,4-triazole; Triazole derivatives; 3-amino-1,2,4-triazole-5-carboxylic acid hemihydrate; Triazole derivatives; 2-(4-chlorophenyl)-2-(1,2,4-triazole-1-yl)-methylhexanoic acid; Succinic acid; Imidazole; L-histidine; L-glutamic acid; Permethrin derivatives; 3-phenoxybenzyl 2,2-Dimethylcyclopropane-1,3-Dicarboxylate; 3-Phenoxybenzaldehyde; Flucitrinate; Chrysanthemum acid; 2,4-Dinitrophenyl; DNP; Thyramhaptens; 4-[Carbodithioate(methyl)-amino]butanoate disodium; Thyramhaptens; 5,11-Dimethyl-6,10-Dithioxo-7,9-Dithia-5,11-Diazadodecanoic acid; Thyramhaptens; 2-{[(dimethylamino)carbotioil]sulfanyl}ethaneic acid; Thyramhaptens; 4-{[(dimethylamino)carbotioil]sulfanyl}butanoic acid; Thyramhaptens;6-{[(dimethylamino)carbotiol]sulfanyl}hexanoic acid; thyram haptens; 11-{[(dimethylamino)carbotiol]sulfanyl}undecanoic acid; thyram haptens; 2-{[(dimethylamino)carbotiol]sulfanyl}ethaneic acid; thyram; tetramethylthiuram monosulfide; tetraethylthiuram disulfide; sodium dimethyldithiocarbamate; zinc dimethyldithiocarbamate; sodium diethyldithiocarbamate; N,N,N',N'-tetramethylthiourea; nabam; zineb; maneb; ethylenethiourea; chlorpyrifoshapten; O,O-diethyl O-[3,5-dichloro-6-[(2-carboxyethyl)thio]-2-pyridyl] Phosphothioates; 2-succinamidebenzimidazole; 2-benzimidazolecarbamate methyl; MBC; benzimidazole; 2-benzimidazolylurea; succinamide; ethyl carbamate; urea; N-methylurea; N,N'-dimethylurea; brevetoxin PbTx-3; organophosphorus haptens; O,O-diethyl O-(5-carboxy-2-fluorophenyl) Phosphothioate; Chlorpyrifos-ethyl; Anandamide hapten; N-arachidonyl-7-amino-6-hydroxyheptanoic acid; Anandamide; Arachidonic acid; Docosatetraenoylethanolamide; Dihomo-γ-linolenylethanolamide; 2-arachidonylglycerol; 2-arachidonylglycerol ether; Stearoylethanolamide; Heptadecanoylethanolamide; Prostaglandin E1; 3-Hydroxy-2-(3-hydroxy-1-octenyl)-5-oxocyclopentaneheptanoic acid; Alprostadil; PGE1; Prostaglandin D2; PGD2; Prostaglandin A2; PGA2; Prostaglandin B2; PGB2; Prostaglandin F; 2α 7-[3,5-dihydroxy-2-(3-hydroxy-1-octenyl)cyclopentyl]-5-heptenoic acid; dinoprost; PGF 2α ; Prostaglandin F 1α ;PGF 1α ;6-Keto-Prostaglandin F 1α ;6-keto-PGF 1α;13,14-dihydro-15-keto-prostaglandin E2;13,14-dihydro-15-keto-PGE2;13,14-dihydro-15-keto-prostaglandin F 2α 14-dihydro-15-keto-PGF 2α ;5α,7α-dihydroxy-11-ketotetranorpostan-1,16-diacid;15-keto-PGF 2α;TXB2;Prostaglandin E2;7-[3-hydroxy-2-(3-hydroxy-1-octenyl)-5-oxocyclopentyl]-5-heptenoic acid;Dinoprostone;PGE2;hCG-α-(59-92)-peptide (34 residues);Paraquat derivative;Paraquat hexanoate (PQ-h);Monoquat;Diquat;9,10-Dihydro-8a,10a-Diazoniaphenanthrene;MPTP;1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine;1,2-Naphthoquinone;N-Acetyl-S-(1,2-dihydroxy- 4-Naphthyl)cysteine; N-Acetyl-S-(1,4-dihydroxy-2-naphthyl)cysteine; N-Acetyl-S-(1,2-dihydroxy-1-hydroxy-1-naphthyl)cysteine; 2-Chloro-2',6'-diethylacetanilide (CDA)hapten; 2-[2-Chloro-(2',6'-diethyl)acetanilide]ethanoic acid; 2-Chloro-2',6'-diethylacetanilide (CDA)hapten; 2-[2-Chloro-(2',6'-diethyl)acetanilide]butanoic acid; 2-Chloro-2',6'-diethylacetanilide (CD A) Hapten; 5-(4-chloroacetamido-3,5-diethyl)phenoxypentanoic acid; CDA; 2-chloro-2',6'-diethylacetanilide; HDA; 2-hydroxy-2',6'-diethylacetanilide; 2,6-diethyl-aniline; hydroxyalachlor; alachlor ESA; alachlorethanesulfonic acid; isoproturone hapten; 3-(4-isopropylphenyl)-1-carboxypropyl-1-methylurea; chlorotolurone; 3-(3-chloro-p-tolyl)-1,1-dimethylurea; metoxurone; 3-(3-chloro (4-Methoxyphenyl)-1,1-dimethylurea; Metamitron; 4-amino-4,5-dihydro-3-methyl-6-phenyl-1,2,4-triazine-5-one; Mecoprop; (RS)-2-(4-chloro-o-tolyloxy)propionic acid; Propyzamide; 3,5-dichloro-N-(1,1-dimethylpropynyl)benzamide; Paraquat dichloride; MCPB; 4-(4-chloro-o-tolyloxy)butyric acid; Chlortoluron hapten; N-(3-chloro-4-methylphenyl)-N-methyl-N-carboxypropylurea; Methosulfuron;2-[3-(4-methoxy-6-methyl-1,3,5-triazine-2-yl)ureidosulfonyl]methyl benzoate; captoprilhaptens; captopril-4-(maleimidomethyl)-cyclohexanecarboxylic acid (MCC); captoprilhaptens; captopril disulfide modified; mercaptoethanol-MCC; mercaptoethanol-4-(maleimidomethyl)-cyclohexanecarboxylic acid, modified; captoprilhaptens; captopril without MCC; acleaciside A; acleaciside B; solamaldine; solasonine; solanine -S; Praprin; 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-Solasidine; O-β-D-Glucopyranosyl-1(1→3)-3-O-β-D-Galactopyranosyl-Solasodine; 12-Hydroxysolamardin; 12-Hydroxysolasonine; Isoanguibin; Sorabelin I; Sorabelin II; Xylosyl-β-Solamardine; α-Solanine α-Caconine; Dioscin; Indole derivatives; β-Indoleacetic acid; 2-Bromo-4,6-Dinitroaniline; 2-Chloro-4,6-Dinitroaniline; Tetril; 2,4,6-Trinitrophenyl-n-Methylnitramine; Nitramine; Tetralite; Tetril; 2-Amino-4,6-Dinitrotoluene; 2,4-Dinitroaniline; 3,5-Dinitroaniline; 2-Amino-4,6-Dinitrobenzoic acid; Disperse Blue 79; N-[5-[Bis[2-(Acetyloxy)ethyl]amino]-2-[(2-Bromo-4,6-Dinitrophenyl) [Azo]-4-ethoxyphenyl]acetamide; 1,3-dinitrobenzene; 2,6-dinitrotoluene; 4-amino-2,6-dinitrotoluene; 1,3,5-trinitrobenzene; nicergoline; ethylmorphine; 7,8-didehydro-4,5-epoxy-3-ethoxy-17-methylmorphinan-6-ol; dihydromorphine; dihydrocodeine; dihydromorphinone; hydromorphone; dihydrocodeinone; hydrocodone; naltrexone; N-cyclopropylmethyl-14-hydroxydihydromorphinone; dextromethorphan;(±)-3-methoxy-17-methylmorphinan; homatropin; endorphins and their modified derivatives (type: β-endorphin); methoenkephalin; DALEA; D-Ala(2)-D-Leu(5)-enkephalinamide; vincristine; 22-oxovincaleucoblastine; 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; benzenethanamine; 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 isomer; D-erythroneopterin; neopterin isomer; L-erythroneopterin; neop Therin isomer; D-threoneopterin; biopterin isomer; L-erythrobiopterin; biopterin isomer; D-erythrobiopterin; biopterin isomer; L-threobiopterin; biopterin isomer; D-threobiopterin; pterin-6-carboxylic acid; C7H5NiO3; pterin; thromboxane B2; (5Z,9α,13E,15S)-9,11,15-trihydroxythromboxa-5,13-dien-1-acid; 15-ketoprostaglandin F; 2α;Fumonicin B1;Macrofusin;FB1;Tyroleverine;TRH;Tyrotropin-releasing factor;Tyrotropin-releasing hormone;TRF;Protirelin;Lopremon;Tyroleverine-OH;TRH-OH;Diketopiperazine;Cyclo(HP);TRH analog;Methylated TRH;TRH analog;TRH elongation peptide;TRH-Gly;TRH elongation peptide;TRH-Gly-Lys-Arg;TRH elongation peptide;TRH-Gly-Lys-Arg-Ala;TRH elongation peptide;P7(modified:QHPGLRF);TRH elongation peptide;P10(modified) :SLRQHPGLRF); TRH elongation peptide; Ps5 (modified: proTRH[178-199]); TRH elongation peptide; TRH-Ps5 (modified: proTRH[172-199]); hypothalamic peptide; LHRH; cyanoginosine LA; cyanoginosine LB; cyanoginosine LR; cyanoginosine LY; cyanoginosine AY; cyanoginosine FR; cyanoginosine YR; Ne-acetyllysine-containing peptide; Gly-Lys(Ac)-e-aminocaproic acid (Aca)-Cys; benzoic acid; benzenecarboxylic acid; phenylformic acid; drasylic acid; m -Hydroxybenzoic acid; 3-Hydroxybenzoic acid; o-Methoxybenzoic acid; 2-Methoxybenzoic acid; o-Toluic acid; 2-Methylbenzoic acid; o-Chlorobenzoic acid; 2-Chlorobenzoic acid; o-Aminobenzoic acid; 2-Aminobenzoic acid; Thiosalicylic acid; 2-Mercaptobenzoic acid; o-Sulfhydrylbenzoic acid; Salicylamide; 2-Hydroxybenzamide; Saligenin; Saligenol; o-Hydroxybenzyl alcohol; Salicylic alcohol; 2-Cyanophenol; 2-Hydroxyphenylacetic acid; p-Hydroxybenzoic acid; p-Aminobenzoic acid Acids; 4-aminobenzoic acid; vitamin Bx; bacterial vitamin H1; p-toluic acid; p-methylaminobenzoic acid; p-chlorosalicylic acid; 4-chloro-2-hydroxybenzoic acid; 2,4-dihydroxybenzoic acid; β-resorcylic acid; 2,4-dihydroxybenzenecarboxylic acid; BRA; 4-aminosalicylic acid; 4-amino-2-hydroxybenzoic acid; p-aminosalicylic acid; gentisic acid; 2,5-dihydroxybenzoic acid; 5-hydroxysalicylic acid; picolinic acid; o-pyridinecarboxylic acid; 2-pyridinecarboxylic acid; picolinic acid N-oxide;3-Hydroxypicolinic acid; 2-Hydroxynicotinic acid; 7-Methylguanine; N; 2 -carboxymethyl-N 7 -Methylguanine; 2-(7-methyl-6-oxo-6,7-dihydro-1H-purine-2-ylamino)acetic acid; 7-methylxanthine; 7-methyluric acid; 7-methyladenine; guanine; 2-amino-1,7-dihydro-6H-purine-6-one; 2-aminohypoxanthine; adenine; 6-aminopurine; 6-amino-1H-purine; 6-amino-3H-purine; 6-amino-9H-purine; 7-(2-carboxyethyl)guanine; 7-CEGua; 7-ethylguanine; 2-amino-7-ethyl-1H-purine-6(7H)-one; 7-(2,3-dihydroxypropyl)guanine; 2-amino-7-(2,3-dihydro Xypropyl)-1H-purine-6(7H)-one; 7-(2-hydroxyethyl)guanine; 2-amino-7-(2-hydroxyethyl)-1H-purine-6(7H)-one; 7-(2-[(2-hydroxyethyl)amino]ethyl)-guanine; 2-amino-7-(2-(2-hydroxyethylamino)ethyl)-1H-purine-6(7H)-one; 7-carboxymethylguanine; 2-(2-amino-6-oxo-1,6-dihydropurine-7-yl)acetic acid; fluorescein; urushiol; quinone; biotin; His tag; FLAG tag; Strep tag; Myc tag; HA tag; Spot tag; or NE tag.

[0098] Representative antibodies that have a hapten-binding domain 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-triazole-1-yl)propanol) antibody (DTP antibody); 22-oxacalci 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'-carboxymethylphenyl phosphate antibody; 7-(carboxyethyl)guanine (7-CEGua) antibody (antibody group specific to 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;Amidochrol 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 (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 mercapturate antibody; (Adinphosmethyl) 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) antibody (mainly diflubenzuron antibody); berberine antibody; β-indoleacetic acid antibody; biopterin (L-erythro) antibody; brevetoxin PbTx-3 antibody; bromacil antibody; bromofos antibody; bromofosethyl antibody; butachlor antibody; captopril-MCC antibody; carbamazepine (CBZ) antibody; carbaryl antibody; carbaryl antibody (LIB-CNH32; LIB-CNH33, LIB-CNH36; LIB-CNH37; LIB-CNH45; LIB-CNA38); carbaryl antibody (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); Chlortalonyl 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; Chlortoluron antibody (antiserum); Cyanoginosine 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-hydroxyphenyl glycol) 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); Flucoflon 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;isoproturone antibody;anti-KB-6806 antiserum;(+)rupanin antibody;Lysophosphatidic acid (LPA) antibody; M3G Ab1 and Ab2; M3G Ab1 and Ab2; MBC antibody (anti-2-succinamide benzimidazole antiserum); Metanephrine antibody; (+) Methamphetamine antibody; Methiocarb antibody (LIB-MXNB31; LIB-MXNB-33; LIB-MXNH14 and LIB-MXNH-15 MAb); Metrachlor antibody; Metrachlor antibody; Metrachlor antibody (MAb 4082-25-4); Molinate antibody; Monulon 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-erythrotype) antibody;Nicarbazine antibody (Nic 6;Nic 7;Nic 8;and Nic 9);Nicergoline antibody (Nic-1;Nic-2;Nic-3 & BNA-1; BNA-3); norflurazone antibody; normetanephrine antibody; (o-DNCP) antibody; P10 antibody (TRH elongated peptide); paraoxone antibody (BD1 and CE3); paraquat antibody; paraquat antibody; parathion-methyl antibody; PCB antibody (antibody against 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); Propoxul antibody (LIB-PRNP15; LIB-PRNP21; LIB-PRNB21; LIB-PRNB33); Prostaglandin E2 antibody; p-Tyramine antibody; Pyrene antibody; Retronesin antibody; Retronesin antibody; Salicylic acid antibody; Sennoside A antibody (MAb 6G8); Sennoside B antibody (MAb: 7H12; 5G6; 5C7); Simazine antibody; Sulfonamide antibody (anti-TS); Sulcoflon antibody (S2B5 / 1 / C3); Sulfamethazine antibody (21C7);Synephrine antibody; Thiabendazole antibody (Antibody 300); Thiabendazole antibody (Antibody 430 and Antibody 448); Thyram antibody; THP antibody (7S and 19S); Thromboxane B2 antibody; Thymidine glycol monophosphate antibody (mAb 2, 6F, 6B, 6C); Tyroleverine (TRH) antibody; TNT antibody (AB1 antiserum and AB2 antiserum); Triadimephon antibody; Triazine antibody (AM1B5, 1); Triazine antibody (AM5C5, 3); Triazine antibody (AM5D1, 1, 2); Triazine antibody (AM7B2, 2, 1); Triazine antibody (SA5A1, 1, 1); Triazine serum (ametrine); Triazine serum (atrazine); Triazine serum (antisimazine); Triazine serum (antisimetrine); Trifluralin antibody; Trifluralin antibody; Vincristine antibody; Zearalenone antibody; Zeatin riboside antibody; E2 G2 and E4 C2; MAb Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7; LIB-BFNP23 Mab; MAb H-7 and H-9 (MAb for O,O-diethyl OP peptide); MoAb 33A7-1-1; MoAb 33B8-1-1; MoAb 33C3-1-1; MoAb 3C10-1-1 and MoAb 3E17-1-1; MoAb 45D6-5-1; MoAb 45E6-1-1; MoAb 45-1-1; MAb Mutant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (GlnL89Glu); MAb Mutant of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (GlnL89Glu / ValH37Ile / GluL3Val); MAb A variant of the Fab fragment K411B (isotype IgG2b with a κ light chain) derived from K4E7 (GlnL89Glu / ValH37Ile / GluL3Val); a variant of the Fab fragment K411B (isotype IgG2b with a κ light chain) derived from MAb K4E7 (GlnL89Glu / ValH37Ile); a variant of the Fab fragment K411B (isotype IgG2b with a κ light chain) derived from MAb K4E7 (GlnL89Glu / ValH37Ile); a variant of the Fab fragment K411B (isotype IgG2b with a κ light chain) derived from MAb K4E7 (GluH50Gln);Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GluH50X); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GlyH100aAla); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (GlyH100aSer); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (HisH95Phe); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (HisH95Tyr); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (PheL32Leu); MAb Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from K4E7 (TrpH33Phe,Tyr,Leu); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (Tryl96Phe); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (TryL96Phe); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (ValH37Ile); Mutants of Fab fragment K411B (isotype IgG2b with κ light chain) derived from MAb K4E7 (ValH37Ile); P6A7 MAb; PNAS2 6 / 3 56(1)-1-5-1; PNAS2 6 / 3 56(1)-1-5-2; PNAS2 6 / 3 Examples include 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] Figure 33 shows representative scFvs that bind to haptens, including 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, examples of small molecule ligands include folic acids, DUPA, NK-1R ligand, CAIX ligand, γ-glutamyl transpeptidase ligand, NKG2D ligand, or CCK2R ligand, all of which are small molecule ligands that specifically bind to cancer cells (i.e., receptors for these ligands are overexpressed in cancer compared to normal tissue). In certain embodiments, examples of the targeting moiety include fluorescein, fluorescein isothiocyanate (FITC), NHS, and / or fluorescein. In certain embodiments, the binding domain is specific to the targeting moiety. In certain embodiments, the binding domain contains an anti-fluorescein antibody E2 or a fragment of that antibody.

[0101] (iii) Intracellular signaling domain The intracellular portion of a protein contains one or more intracellular signaling domains. In certain embodiments, the intracellular signaling domains generate signals that promote the immune effector function of CAR-recombinant cells. In certain embodiments, the intracellular signaling domains generate stimulatory, co-stimulatory, or inhibitory signals in response to ligand binding to a recombinant fusion protein that induces stimulatory, co-stimulatory, or inhibitory activity. Examples of immune effector function include cytolytic activity and helper activity, which include cytokine secretion. The signals generated by the intracellular signaling domains can also induce proliferation, activation, differentiation, and other processes of immune cells.

[0102] A "signaling domain" refers to the functional portion of a protein that regulates cellular activity by transmitting information within the cell via a defined signaling pathway, such as by generating second messengers or acting as an effector in response to second messengers. A "stimulus" refers to the primary response induced when a stimulating molecule (e.g., CAR) or co-stimulatory molecule binds to its recognition ligand. This primary response triggers a signaling event, such as signal transduction via the appropriate signaling domain of the CAR or recombinant receptor protein. Stimulation can lead to the modified expression of specific molecules.

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

[0104] The primary intracellular signaling domain may contain a signaling motif known as an immune receptor-activated tyrosine motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0105] In certain embodiments, the CD3ζ(CD247) stimulating domain may contain amino acid residues derived from the cytoplasmic domain or a functional fragment of the T cell receptor ζ chain, sufficient to functionally transmit the initial signal necessary for cell activation. In certain embodiments, the CD3ζ stimulating domain may contain a human CD3ζ stimulating domain or a functional fragment thereof. In certain embodiments, the CD3ζ stimulating domain contains the sequence shown in SEQ ID NO: 121. In certain embodiments, the CD3ζ stimulating domain is encoded by SEQ ID NO: 124. In certain embodiments, if the intracellular signaling domain is derived from the CD3ζ molecule, this intracellular signaling domain retains a sufficient CD3ζ structure to generate a signal under appropriate conditions.

[0106] In certain embodiments, the intracellular signaling domain may include an intracellular costimulatory domain. In certain embodiments, the intracellular costimulatory signaling domain includes an intracellular costimulatory signaling domain derived from a molecule that carries a costimulatory signal 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 immune cells, recognized by a costimulatory ligand, and specifically binding to this costimulatory ligand, thereby mediating the induction of a costimulatory response (e.g., proliferation) by immune cells. Costimulatory molecules include cell surface molecules other than antigen receptors and their ligands that contribute to an efficient immune response. Costimulatory molecules may be presented in the TNF receptor protein family, immunoglobulin-like protein family, cytokine receptor family, integrin family, signaling lymphocyte activating molecule (SLAM protein) family, and activation-inducing NK cell receptor family.Examples of such co-stimulatory molecules include MHC class I molecules, B lymphocyte / T lymphocyte attenuators (BTLA, CD272), Toll ligand receptors, CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS (CD278), BAFFR, HVEM (LIGHTR), ICAM-1, and 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 Examples 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 co-stimulatory signaling domain includes 4-1BB (CD137, TNFRSF9). "4-1BB" refers to a member of the tumor necrosis factor receptor (TNFR) superfamily. In certain embodiments, the 4-1BB co-stimulatory domain includes the human 4-1BB co-stimulatory domain or a functional fragment thereof. In certain embodiments, the 4-1BB co-stimulatory domain includes the sequence shown in SEQ ID NO: 120. In certain embodiments, the 4-1BB co-stimulatory domain is encoded by SEQ ID NO: 123.

[0108] In certain embodiments, the intracellular co-stimulatory signaling domain includes CD28. CD28 is a T cell-specific glycoprotein involved in T cell activation, induction of cell proliferation, cytokine production, and promotion of T cell survival. In certain embodiments, the CD28 co-stimulatory domain includes the human CD28 co-stimulatory domain or a functional fragment thereof. In certain embodiments, the human CD28 co-stimulatory domain includes the sequence shown in Sequence ID No. 180. In certain embodiments, the human CD28 co-stimulatory domain is encoded by Sequence ID No. 182.

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

[0110] Examples of inhibitory immune cell molecules that can be recombinant to include 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 The fusion protein can be designed to include a transmembrane domain that, when expressed, links the extracellular portion of the fusion protein to the intracellular portion. The transmembrane domain can tether the protein to the cell membrane. A transmembrane domain may contain one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids related to the extracellular region of the protein from which the transmembrane domain originates (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more amino acids derived from the extracellular region of the protein from which the transmembrane domain originates) and / or one or more additional amino acids related to the intracellular region of the protein from which the transmembrane protein originates (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more amino acids derived from the intracellular region of the protein from which the transmembrane domain originates). In certain embodiments, the transmembrane domain may originate from the same protein from which the signaling domain, co-stimulatory domain, or hinge domain originates. In certain embodiments, the transmembrane domain is derived from a protein other than the protein from which the other domains of the fusion protein originate. In certain embodiments, the transmembrane domain may be selected or modified by amino acid substitution so as to prevent binding to the other domains of the fusion protein or to minimize interaction with the other domains of the fusion protein.

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

[0113] The transmembrane domain may be of natural origin or recombinant origin. If of natural origin, the transmembrane domain may be derived from a membrane-bound protein or a transmembrane protein. In certain embodiments, the transmembrane domain can transmit a signal to the intracellular domain once a target binds to the extracellular ligand-binding domain of the fusion protein. In certain embodiments, the transmembrane domain may include at least the transmembrane regions 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 domains are 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 domains of AM;Ly9;CD160;PSGL1;CD100;SLAMF6(NTB-A, Ly108);SLAM;BLAME;SELPLG;LTBR;PAG / Cbp;NKG2D; and / or NKG2C may be included. In certain embodiments, the transmembrane domains may include transmembrane domains derived from the CD28 chain or the CD8α chain. In certain embodiments, the transmembrane domain of CD8 includes SEQ ID NO: 119 or SEQ ID NO: 128 and / or is coded by SEQ ID NO: 122.

[0114] In certain embodiments, the majority of the transmembrane domain may consist of hydrophobic residues such as leucine and valine. In certain embodiments, the transmembrane domain may contain triplets consisting of phenylalanine, tryptophan, and valine at both ends. In certain embodiments, a CD28 or CD8 hinge is located extracellularly of the transmembrane domain.

[0115] (v) Linker In this specification, a linker may be a part of the fusion protein that serves to link two subordinate components or subdomains of the fusion protein. In certain embodiments, the linker can provide flexibility to various components of the fusion protein. In scFv, a linker linking the VH and VL of the antibody-derived binding domain has also been described. The linker may further include a spacer region and linking amino acids. In certain examples, if 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 other connected components and / or to allow for mobility relative to other connected components.

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

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

[0119] In certain embodiments, the spacer region includes 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 include all or part of a hinge region sequence derived from IgG1, IgG2, IgG3, IgG4, or IgD, or a combination of all or part of a hinge region sequence derived from IgG1, IgG2, IgG3, IgG4, or IgD and all or part of a CH2 region; all or part of a CH3 region; or a combination of all or part of a CH2 region and all or part of a CH3 region. In this specification, “wild-type immunoglobulin hinge region” refers to the amino acid sequence of a hinge located between the CH1 and CH2 domains of the heavy chain and linking them in the upper and middle parts of the natural antibody (in the case of IgG, IgA, and IgD), or the amino acid sequence of a hinge located between the CH1 and CH3 domains of the heavy chain and linking them (in the case of IgE and IgM).

[0120] Typical spacers include spacers consisting solely of an IgG4 hinge, spacers consisting of IgG4 hinges linked to the CH2 and CH3 domains, or spacers consisting of an IgG4 hinge linked to the CH3 domain. In certain embodiments, the spacer includes the IgG4 linker shown in SEQ ID NO: 78 or 80. The hinge region can be recombined to prevent undesirable structural interactions, such as dimerization with an unintended partner. Other examples of hinge regions that can be used in the fusion proteins described herein include hinge regions located in the extracellular regions of type I membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type or variants thereof. In certain embodiments, the hinge includes the CD8α hinge shown in SEQ ID NO: 129.

[0121] In certain embodiments, the spacer region includes the hinge region of the interdomain region (stalk region) of a type II C lectin, or the hinge region of the stalk region of a cluster of differentiation (CD) molecule. The "stalk region" of a type II C lectin or CD molecule refers to a portion of the extracellular domain located between the C lectin-like domain (CTLD) (e.g., similar to the CTLD of a natural killer cell receptor) and the hydrophobic portion (transmembrane domain) in the type II C lectin or CD molecule. For example, the extracellular domain of human CD94 (GenBank accession number AAC50291.1) corresponds to amino acid residues 34-179, while the CTLD corresponds to amino acid residues 61-176. The stalk region of the human CD94 molecule contains 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 further descriptions of the stalk region, see 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 contain linked 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-length human NKG2A protein (UniProt ID P26715.1) has a hydrophobic region (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 contains amino acids 119-231, and the stalk region contains amino acids 99-116, and this stalk region may have additional linked amino acids adjacent to it.Other type II type C lectins or CD molecules, or their extracellular ligand-binding domains, stalk regions, and CTLDs, are also known in the art (for example, see GenBank accession numbers: 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] Linked amino acids may be linkers that can be used to link fusion protein domain sequences when it is not necessary to create distance using spacers, and / or when it is undesirable to create distance using spacers. In certain embodiments, linked amino acids are short amino acid sequences that can be used to link intracellular signaling domains. In certain embodiments, the length of the linked amino acids is 9 amino acids or less.

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

[0124] (vi) Tags and selection markers In certain embodiments, the fusion protein may contain one or more tags and / or express one or more selection 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 (formerly known as STREP® tag or STREP tag II (IBA Institut fur Bioanalytik, Germany); see, for example, U.S. Patent Publication No. 7,981,632)), Softag 1, Softag 3, and V5. See Figure 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-tagged antibodies are commercially available from manufacturers such as Life Technologies, Pierce Antibodies, and GenScript. Flag-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, Life Technologies, and GenScript. Avi-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin-tagged antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Pierce Antibodies. HA-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, Cell Signal, and Abcam. Myc-tagged antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Cell Signal. Strep-tagged antibodies are commercially available from manufacturers such as Abcam, Iba, and Qiagen.

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

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

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

[0129] The transduction marker may contain a suitable fluorescent protein, such as 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). In a particular embodiment, the transduction marker contains BFP as shown in Figure 33. Furthermore, see 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 Furthermore, this disclosure includes stimulative, co-stimulative, and suppressive immune receptors that have been recombinant so as to have their activity induced by the presence of an intracellular hsp90-binding domain.

[0131] CD3 is an example of a stimuli receptor.

[0132] Examples of co-stimulatory immune cell molecules that can be recombinant to include 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. Examples of inhibitory immune cell molecules that can be recombinant to include 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. Specific 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 This includes EBDs linked to the intracellular signaling domains 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) Cells genetically modified to express an inducible fusion protein This disclosure includes cells genetically modified to express an inducible fusion protein. In this specification, “genetic modification” or “genetic engineering” refers to the addition of another genetic material, in the form of DNA or RNA, to the complete genetic material within a cell. The terms “genetically modified cell” and “recombinant cell” are used interchangeably. In certain embodiments, cells genetically modified to express an inducible fusion protein include effector immune cells. “Effector immune cells” include immune system cells having one or more effector functions (e.g., cytotoxic / killing activity, cytokine secretion, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC)). Effector immune cells are subtypes of immune cells.

[0134] The immune cells of this disclosure may be autologous / autologously derived cells ("self" cells) or non-autologous cells ("non-self" cells, e.g., allogeneic cells, syngeneic cells, or heterogeneous cells). "Autologous" cells refer to cells obtained from the same subject. "Allogeneic" cells refer to cells that are genetically different from the cells being compared but originate from the same species. "Syngeneic" cells refer to cells that are genetically identical to the cells being compared but originate from a different subject. "Heterogeneous" cells refer to cells that originate from a different species than the cells being compared. In certain embodiments, the recombinant cells of this disclosure may be autologous cells or allogeneic cells.

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

[0136] Most T cells have a T cell receptor (TCR) consisting of two types of peptide chains (α-TCR chain and β-TCR chain). γδ T cells refer to a small subset of T cells that have a different type of T cell receptor (TCR) composed of one γ chain and one δ chain.

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

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

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

[0140] "Effector memory T cells (TEM)" are antigen-experienced T cells that 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 the expression of CD62L and CCR7 and positive or negative for the expression of CD28 and CD45RA compared to naive cells or central memory cells. Effector T cells are positive for granzyme B and perforin compared to memory T cells or naive T cells.

[0141] Helper T cells assist other immune cells through functions such as activating cytotoxic T cells and macrophages or promoting the maturation of B cells. Helper T cells are activated by receiving the presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). When activated, helper T cells rapidly divide and secrete cytokines to regulate or support an active immune response.

[0142] Natural killer T (NKT) cells are a subset of T cells that co-express an 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 do not express 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) refer to immune cells that migrate from the blood into the tumor and can recognize and kill cancer cells. Myeloid infiltrating lymphocytes (MILs) are antigen-experienced immune cells that migrate to and remain in the bone marrow. Mucosal associated invariant T (MAIT) cells are innate-like T cells that are found in mucosal tissues, blood, and secondary lymphoid organs (SLOs) and exhibit an effector phenotype. MAIT cells present a semi-invariant T cell receptor (TCR) and are restricted by MR1, a major histocompatibility complex-related molecule.

[0145] Macrophages (and their precursor cells, monocytes) are present in all tissues in 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., dendritic cells in a pre-activated state) phagocytose peripheral antigens and other non-self components, become activated, migrate to the T cell area of lymphoid tissues, and present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD!01, CD148, CD209, and DEC-205.

[0147] Hematopoietic stem cells (HSCs) are undifferentiated hematopoietic cells that are capable of self-renewal and can differentiate into any other type of hematopoietic cell. HSCs are CD34+.

[0148] Hematopoietic progenitor cells (HPCs) originate from HSCs and can further differentiate into mature cell types. HPCs are self-renewing or (i) differentiate into myeloid progenitor cells, ultimately differentiating into monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets or dendritic cells, or (ii) differentiate into lymphoid progenitor cells, ultimately differentiating into T cells, B cells and NK cells. HPCs are CD24 lo Lin - CD117 + That is the case.

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

[0150] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell artificially created from non-pluripotent cells, typically adult somatic cells or terminally differentiated cells such as fibroblasts, hematopoietic cells, muscle cells, neurons, and epidermal cells, by introducing or exposing them to reprogramming factors.

[0151] (ix) Methods for recombining cells ex vivo and in vivo This disclosure provides a method for genetically recombining immune cells in vivo by utilizing a method for harvesting, concentrating, culturing, and recombining cells to express an activity-inducible fusion protein (e.g., CAR) ex vivo, and / or a cell-targeted delivery method.

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

[0153] Peripheral blood can be cited as a source of HSPC (US Patent Nos. 5,004,681; 7,399,633; and 7,147,626; and 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). See 27(2):S23-S29).

[0154] Methods for collecting blood samples, inhibiting coagulation, and processing them 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 to remove unwanted components, concentrate desired components, or lyse or remove cells sensitive to a particular reagent. Isolation may include one or more of a variety of cell preparation and cell separation steps, which may include separation based on one or more characteristics such as size, density, sensitivity or resistance to a particular reagent, and / or affinity to an antibody or other binding partner (e.g., immunoaffinity).

[0156] In certain embodiments, one or more cell populations concentrated, isolated and / or selected from a sample by the method provided herein are cells positive for one or more specific markers (e.g., cell surface markers). + ) or cells that highly express one or more specific markers (markers) hi ), or cells that are negative for one or more markers (marker - ) 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, for example, PERCOLL TM Monocytes can be isolated from peripheral blood mononuclear cells (PBMCs) by removing them using centrifugation with a concentration 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 monoclonal antibody cocktail against cell surface markers present on cells targeted for negative selection. For example, CD4 +When enriching cells by negative selection, a monoclonal antibody cocktail containing CD14 antibody, CD20 antibody, CD11b antibody, CD16 antibody, HLA-DR antibody, and CD8 antibody can generally be used.

[0158] After isolating and / or concentrating the cells, their number can be increased by proliferation. In certain embodiments, for example, U.S. Patent Publication No. 6,352,694; U.S. Patent Publication No. 6,534,055; U.S. Patent Publication No. 6,905,680; U.S. Patent Publication No. 6,692,964; U.S. Patent Publication No. 5,858,358; U.S. Patent Publication No. 6,887,466; U.S. Patent Publication No. 6,905,681; U.S. Patent Publication No. 7,144,575; U.S. Patent Publication No. 7,067,318; U.S. Patent Publication No. 7,172,869 Using the 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, T cells can be activated and proliferated before or after genetic recombination to express CAR.

[0159] Typically, T cells are proliferated by contacting a surface bound to a drug that stimulates signaling related to the CD3-TCR complex with a ligand that stimulates a costimulatory molecule on the T cell surface. In certain embodiments, PBMCs or isolated T cells are contacted with the stimulant and costimulator, for example, in a culture medium containing appropriate cytokines, usually with anti-CD3 and anti-CD28 antibodies bound to beads or other surfaces (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, stimulated, and proliferated using feeder cells, appropriate antibodies, and cytokines, for example, by utilizing the 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, artificial APCs (aAPCs) can be created by recombining K562 cells, U937 cells, 721.221 cells, T2 cells, or C1R cells, and these can be used to induce the stable expression and secretion of various costimulatory molecules and cytokines. aAPCs are described in WO03 / 057171 and U.S. Patent Publication 2003 / 0147869.

[0161] In certain embodiments, HSPC is, for example, 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; Vamun-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): They can be isolated and / or propagated according to the methods described in pp. 232-236; or Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006, or the references cited herein. The recovery and processing of other types of cells described herein are known to those skilled in the art.

[0162] In certain embodiments, the isolation, incubation, proliferation, and / or recombination steps are carried out in a sterile or sealed environment and / or by an automated method, for example, under the control of a computer connected to the apparatus in which each step is performed. The final formulation of recombinant cells into recombinant formulations for administration is described separately herein.

[0163] Furthermore, immune cells can be genetically recombined in vivo or ex vivo using target-directed viral vectors and / or nanoparticles. Viral vectors that can be used to deliver genes encoding fusion proteins to cells are described separately herein, and target-directed viral vectors (e.g., pseudotype viral vectors) are well known in the art.

[0164] As typical nanoparticles targeting cells, there are nanoparticles with cell-targeting ligands (e.g., CD3, CD4, CD8, CD34) bound to their surfaces. Since these nanoparticles have cell-targeting ligands bound to their surfaces, they are selectively taken up by selected types of cells. Next, a gene recombination component is delivered by the nanoparticles, and an activity-inducible fusion protein is expressed.

[0165] Typical nanoparticles include liposomes (tiny vesicles in which at least one layer of lipid bilayer surrounding an aqueous core forms a concentric sphere), liposome nanoparticles (a liposome structure used to encapsulate another smaller nanoparticle within its core), and lipid nanoparticles (a liposome-like structure without a 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. Typical materials include metals, transition metals, and metalloids (e.g., lithium, magnesium, zinc, aluminum, and silica).

[0166] Nanoparticles intended for delivery in vivo and uptake by cells may have an uncharged coating or a negatively charged coating, and the size of these nanoparticles may be 130 nm or less. The dimensions of the nanoparticles can be measured using conventional techniques such as dynamic light scattering and / or electron microscopy.

[0167] (x) Production of inducible fusion proteins 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. Nucleic acids encoding several regions of the activity-inducible fusion protein can be prepared by standard molecular cloning techniques, and a complete coding sequence can be constructed. The obtained coding region can be inserted into an expression vector 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 encoding an inducible fusion protein, its components, or a molecule co-expressed with an inducible fusion protein as described herein. The definition of this term includes a variety of sequence polymorphisms, mutations, and / or variants, such changes that do not substantially affect the function of the encoded protein. The term “gene” may include regulatory regions such as promoters, enhancers, and terminal regions, as well as the coding sequence. The gene sequence encoding the molecule may be DNA or RNA that induces the expression of the inducible fusion protein. These nucleic acid sequences may be DNA strand sequences transcribed into RNA or RNA sequences translated into protein.

[0169] "Code" refers to the property of a specific nucleotide sequence within a gene, such as complementary DNA (cDNA) or messenger RNA (mRNA), to function as a template for synthesizing another macromolecule, such as a defined amino acid sequence. Therefore, if mRNA corresponding to a particular gene is transcribed and translated to produce a protein in a cell or other biological system, that gene codes for this protein. A "protein-coding gene" includes any degenerate nucleotide sequence that codes for the same amino acid sequence or an amino acid sequence having a substantially similar form and function.

[0170] Multiple polynucleotide gene sequences encoding two or more portions of an expressed inducible fusion protein can be operably linked to one another and can also be linked to relevant regulatory sequences. For example, a functional relationship may exist between a regulatory sequence and an exogenous nucleic acid sequence, thereby leading to the expression of the exogenous nucleic acid sequence. In another example, if a first nucleic acid sequence and a second nucleic acid sequence are arranged to be functionally related, these first and second nucleic acid sequences may be operably linked. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA sequences are contiguous, and coding regions are linked within the same reading frame if a coding region is needed or useful.

[0171] In the representative nucleic acid constructs used in this disclosure, a promoter is operably ligated to the nucleic acid sequence encoding the inducible fusion protein, i.e., the nucleic acid sequence encoding the inducible fusion protein and the promoter are arranged such that transcription of mRNA from the DNA encoding the inducible fusion protein is promoted. The promoter may be genomically derived or synthetically produced. The promoter may or may not be associated with an enhancer, and the enhancer may be associated with a particular promoter in its native state or with another promoter. Various promoters used in cells (e.g., the CD4 promoter) are well known in the art. The promoter may be a constitutive promoter or an inducible promoter, for example, the induction may be associated with a particular type of cell or a particular developmental stage. Alternatively, many well known viral promoters are also suitable. Notable promoters include the Simian virus 40 (SV40) (e.g., early or late) viral promoter; the Moloney's mouse leukemia virus (MoMLV) long-chain terminal repeat (LTR) promoter; the Roussarcoma 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 to guide the inducible fusion protein to the cell membrane surface, and this signal sequence may include the endogenous signal sequence of the N-terminal portion of the inducible fusion protein. In some cases, it may be desirable to replace this endogenous signal sequence with another signal sequence. On the other hand, the selected signal sequence should be compatible with the secretory pathway of the cell expressing the inducible fusion protein so that the inducible fusion protein is presented on the cell surface in the cell expressing it. In certain embodiments disclosed herein, a GM-CSF signal peptide or a CD8 signal peptide is used.

[0173] Similarly, the terminal region may be composed of a native or endogenous transcriptional terminal region of the nucleic acid sequence encoding the C-terminal portion of the activity-inducible fusion protein. Alternatively, the terminal region may be derived from another source. In most cases, the source of the terminal region is generally considered not to have a significant impact on recombinant protein expression, and various 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 inducible fusion protein (e.g., CAR) can be deleted. For example, typically 10 amino acids or less, more generally 5 amino acids or less, can be deleted from both ends of the binding domain of an inducible fusion protein. It may also be desirable to insert several amino acids, typically 10 amino acids or less, more generally 5 amino acids or less, at the boundary of the binding domain. Deletion or insertion of amino acids may be a result obtained as needed in constructing an inducible fusion protein, and may result in effects such as easily creating a restriction site, facilitating manipulation, or improving expression levels. Furthermore, substitution of one or more amino acids with other amino acids may occur for similar reasons.

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

[0176] The introduction of a desired gene encoding an inducible fusion protein into cells may be carried out by any method known in the art, such as transfection, electroporation, microinjection, lipofection, transfection by calcium phosphate, infection with a viral or bacteriophage vector containing the gene sequence, cell fusion, gene transfer using chromosomes, gene transfer using microcells, spheroplast fusion, in vivo delivery using nanoparticles, 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), and triple-helix DNA (Chan and Glazer, 1997, J. Mol. Med. Examples include 75:267-282). In this field, various techniques are known for introducing foreign genes into cells (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 to the extent that the necessary development and physiological functions of the recipient cells are not excessively disrupted. These techniques can be used to stably transfer genes into cells so that the foreign gene is expressed in the cell, and in certain cases, preferably heritable and expressed in its offspring cells.

[0177] In certain embodiments, a gene encoding an inducible fusion protein can be incorporated into a vector and introduced into a cell. A “vector” is a nucleic acid molecule capable of transporting another nucleic acid. The vector may be, for example, a plasmid, cosmid, virus, or phage. An “expression vector” is a vector that, when placed under appropriate conditions, can induce the expression of a protein encoded by one or more genes incorporated within the expression vector.

[0178] Viral vectors can be obtained from various types of viruses. "Lentiviruses" refer to retroviruses that can infect both dividing and non-dividing cells and typically exhibit high viral titers. Some examples of lentiviruses include HIV (human immunodeficiency virus: including HIV1 and HIV2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0179] Other examples of viral vectors include those derived from Formy virus, adenovirus (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. Patent No. 5,604,090; Kay et al., 2000; Nakai et al., z1998), alphaviruses, cytomegalovirus (CMV), flaviviruses, herpesviruses (e.g., herpes simplex virus), influenza viruses, papillomaviruses (e.g., human papillomavirus and bovine papillomavirus; see, e.g., U.S. Patent No. 5,719,054), poxviruses, vaccinia viruses, and others. See Kozarsky and Wilson (1993); Rosenfeld et al. (1991); Rosenfeld et al. (1992); Mastrangeli et al. (1993); Walsh et al. (1993); and Lundstrom (1999). Another example is 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 on viral vectors for gene delivery, please refer to: 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. See 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] A genetic engineering method utilizing targeting may also be used. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-related protein) nuclease system is a recombinant nuclease system used in genetic engineering using bacterial systems. Information regarding the CRISPR-Cas system and its components can be found, for example, in 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. U.S. Patent Publication No. 8932814, U.S. Patent Publication No. 8945839, U.S. Patent Publication No. 8993233 and U.S. Patent Publication No. 8999641 and related applications thereto; 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 This is described in / 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.

[0181] In certain embodiments, zinc finger nucleases (ZFNs) are used as gene editing reagents. ZFNs are a type of site-specific nuclease that has been recombinant to bind to and cleave DNA at specific locations.For further information regarding ZFNs and ZFNs useful within the scope of the teachings of this disclosure, see, for example, U.S. Patent Publication No. 6,534,261; U.S. Patent Publication No. 6,607,882; U.S. Patent Publication No. 6,746,838; U.S. Patent Publication No. 6,794,136; U.S. Patent Publication No. 6,824,978; U.S. Patent Publication No. 6,866,997; U.S. Patent Publication No. 6,933,113; U.S. Patent Publication No. 6,979,539; U.S. Patent Publication No. 7,013, U.S. Patent Publication No. 219; U.S. Patent Publication Nos. 7,030,215; U.S. Patent Publication Nos. 7,220,719; U.S. Patent Publication Nos. 7,241,573; U.S. Patent Publication Nos. 7,241,574; U.S. Patent Publication Nos. 7,585,849; U.S. Patent Publication Nos. 7,595,376; U.S. Patent Publication Nos. 6,903,185; U.S. Patent Publication Nos. 6,479,626; U.S. Patent Publication Nos. 2003 / 0232410 and U.S. Patent Publication Nos. 2009 / 0203140, and 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 See also 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, a transcription activator-like effector nuclease (TALEN) can be used as a gene editing reagent. TALEN is a fusion protein containing a transcription activator-like effector (TALE) DNA-binding protein and a DNA cleavage domain. For further information on TALEN, please refer to U.S. Patent Publications 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 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 See 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscow, & Bogdanove, Science 326, 1501 (2009).

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

[0184] (xi) Recombinant formulations, recombinant inducement formulations and drug compositions The formulations described herein may include ex vivo genetically modified cells (i.e., recombinant formulations), or may include viral vectors or nanoparticles (recombination inducers) that can genetically modify cells in vivo to express a CAR. As described herein, the compositions of the present invention include a drug molecule that binds to an hsp90 binding domain presented on an expressed inducible fusion protein and / or causes a conformational change in the inducible fusion protein, thereby generating intracellular signaling upon ligand binding.

[0185] A "pharmaceutical" preparation or "pharmaceutical" composition comprises an active compound for administration (e.g., genetically modified cells, viral vectors, nanoparticles, or drug molecules) formulated on a pharmaceutically acceptable carrier.

[0186] "Pharmacologically acceptable" means a compound, material, and / or dosage form that is suitable for use in contact with human and animal tissues without causing excessive toxicity, in proportion to a reasonable benefit-risk ratio, within the bounds of reasonable medical judgment. In specific cases, a pharmaceutically acceptable carrier is one that has been approved by the relevant regulatory authority (e.g., the U.S. Food and Drug Administration (US FDA)).

[0187] Depending on the context and the active compound being delivered, a "pharmaceutically acceptable carrier" may include adjuvants, excipients, flow enhancers, diluents, preservatives, pigments / colorants, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that meet the aforementioned requirements. Typical pharmaceutically acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, formulations and compositions may be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the U.S. FDA's Biologics Standards Bureau and / or other relevant regulatory authorities in other countries.

[0188] Typical pharmaceutically acceptable carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, Illinois), glycerol, ethanol, and combinations thereof. In certain embodiments, human serum albumin (HSA) or other human serum components or fetal bovine serum may be added to the carrier. In certain embodiments, the carrier for infusion contains 5% HAS or buffered saline supplemented with dextrose. Other isotonic agents include polyhydric sugar alcohols, including trihydric sugar alcohols or sugar alcohols with higher valencies, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0189] The carrier may contain buffers such as citrate buffer, succinate buffer, tartaric acid buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer and / or trimethylamine salts.

[0190] Stabilizers refer to a wide range of excipients that can have various functions, from additives that can prevent cells from adhering to the walls of a container to fillers. 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] If necessary or beneficial, the formulation may contain a local anesthetic, such as lidocaine, to relieve pain at the injection site.

[0192] Typical 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 recombinant preparations is 10 2 Numbers exceeding 10 3 Numbers exceeding 10 4 Numbers exceeding 10 5 Numbers exceeding 10 6 Numbers exceeding 10 7 Numbers exceeding 10 8 Numbers exceeding 10 9 Numbers exceeding 10 10 A number greater than 10 11 The number may exceed one.

[0194] In the recombinant formulations disclosed herein, cells are typically contained in volumes of 1 L or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the density of cells administered is typically 10 4 Density exceeding 10 7 Density exceeding 10 particles / ml, or 10 8 The density is greater than particles / ml.

[0195] The therapeutically effective dose of the active ingredient (vector or nanoparticles) contained in the recombinant derivative may be in the range of 0.1–5 μg / kg or 0.5–1 μg / kg. In other examples, doses include 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 other examples, doses include 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 higher.

[0196] The therapeutically effective amount of drug molecules contained in the composition of the present invention may be in the range of 0.1 to 5 μg / kg or 0.5 to 1 μg / kg. In other examples, doses include 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1 to 5 mg / kg, or 0.5 to 1 mg / kg. In other examples, doses include 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 higher.

[0197] In certain embodiments, the recombinant formulation may comprise one or more genetically modified cells (e.g., recombinant T cells, NK cells, or stem cells) or genetically modified cells expressing one or more inducible fusion proteins. Multiple types of genetically modified cell populations can be provided in various proportions. Furthermore, the recombinant formulation may deliver nucleic acids that can recombine two or more types of cells and / or express different types of inducible fusion proteins.

[0198] Certain recombinant formulations include immune cells expressing two or more CARs. For example, immune cells (e.g., T cells) can be recombined to express different types of CARs having different types of ligand-binding domains. Different types of ligand-binding domains can bind to different epitopes on a single cancer antigen or to different cancer antigens. In this case, the multiple CARs, each binding to a different epitope or antigen, may further include different types of hsp90-binding domains so that their activation state can be individually controlled by administering different types of drug molecules (e.g., low-molecular-weight estrogen analogs).

[0199] In certain formulations, multiple types of inducible fusion proteins are expressed, and these multiple types of inducible fusion proteins can be individually activated or inactivated because each incorporates a different type of EBD. Examples of these different types of EBD include EBD (E353A) which can be activated by administration of ES8, EBD (L384M, M421G, G521R) which can be activated by administration of CMP8, and EBD ERT2 which can be activated by administration of 4-OHT. Various other combinations can be used based on the description in this disclosure.

[0200] Representative combinations of cancer antigens that are bound by immune cells expressing various CARs and various 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. Another grouping of relevant cancer antigens based on cancer type is described separately in this disclosure. Other EBD / drug molecule combinations include the combination of EBD(E353A) and ES8, the combination of EBD(L384M, M421G, G521R) and CMP8, and the combination of EBD(ERT2) with G400V, M543A, and L544A mutations and 4-OHT. This approach, which utilizes a combination of various binding domains and various hsp90 binding domains, is referred to herein as “CAR combination therapy.”

[0201] In certain cases, administration of the formulation induces the expression of CARs and co-stimulatory immune molecules (e.g., CD28, 4-1BB, OX40, ICOS), each containing a different hsp90 binding domain. In certain cases, administration of the formulation induces the expression of CARs and two types of co-stimulatory immune molecules (e.g., CD28, 4-1BB, OX40, ICOS), each containing a different hsp90 binding domain. In certain cases, administration of the formulation induces the expression of two types of CARs and two types of co-stimulatory immune molecules (e.g., CD28, 4-1BB, OX40, ICOS), each having a different binding domain and a different hsp90 binding domain, while the two co-stimulatory immune molecules contain the same hsp90 binding domain. In a specific case, administration of the formulation induces the expression of two CARs and two co-stimulatory 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, and the two co-stimulatory immune molecules each contain a different hsp90 binding domain. In this example, the hsp90 binding domain in the co-stimulatory molecule may be the same as the hsp90 binding domain in the CAR, or it may be different from the hsp90 binding domain in the CAR.

[0202] Recombinant formulations may further contain different types of immune cells, each expressing a different type of CAR. For example, individual recombinant immune cells express only one type of CAR, but the formulation may be formulated by combining them with other immune cells that have been recombinant to express a different type of CAR (e.g., a CAR containing a different ligand-binding domain that binds to a different EBD and drug molecule combination). The immune cells may be of the same type (all T cells) or may consist of a mixture of different types (e.g., T cells, NK cells, and / or HSPCs).

[0203] Recombinant inducers can be prepared to induce immune cell populations with these characteristics in vivo (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-inducible costimulatory molecules or activity-inducible inhibitory molecules).

[0204] The formulations and compositions can be prepared for administration by, for example, injection, infusion, perfusion, 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, intrafocal 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, cryopreservation of the recombinant cell preparations of this disclosure may be useful. In this specification, “cryopreservation” means preserving cells by cooling them to a sub-zero temperature, typically 77K or -196°C (the boiling point of liquid nitrogen). Cryoprotective agents are often used during sub-zero storage to mitigate or prevent damage to cells from freezing at low temperatures or warming to room temperature. The use of cryoprotective agents and the setting of an optimal cooling rate can prevent cell damage. Suitable cryoprotective agents include dimethyl sulfoxide (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 cell temperature reaches -80°C, and the cells can be permanently stored by placing them directly into liquid nitrogen (-196°C) in a long-term ultra-low temperature storage container.

[0206] (xii) How to use The methods disclosed herein include treating subjects (humans, companion animals (dogs, cats, reptiles, birds, etc.)), livestock (horses, cattle, goats, pigs, chickens, etc.) or research animals (monkeys, rats, mice, fish, etc.) with (i) recombinant formulations and / or recombinant derivative formulations and (ii) drug compositions disclosed herein. Treatment of subjects includes delivering a therapeutically effective dose. A therapeutically effective dose includes an amount that can provide an effective dose, prophylactic treatment and / or therapeutic treatment without causing excessive toxicity.

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

[0208] "Prophylactic measures" include measures taken for subjects who do not show signs or symptoms of a disease (e.g., cancer or infection) or who show only early signs or symptoms of a disease, with the aim of reducing or mitigating the risk of further progression of that disease. Thus, prophylactic measures function as measures to prevent disease. In certain embodiments, prophylactic measures inhibit, delay, or prevent the progression of a disease.

[0209] "Therapeutic treatment" includes treatments performed on subjects exhibiting symptoms or signs of a disease, with the aim of reducing or eliminating the signs or symptoms of the disease. Therapeutic treatment can suppress, control or eliminate the presence or activity of the disease, and / or suppress, control or eliminate the side effects of the disease.

[0210] The functions of an effective dose, a prophylactic treatment, or a therapeutic treatment are not mutually exclusive, and in certain embodiments, two or more treatments may be performed depending on the administered dose.

[0211] The therapeutically effective dose can be achieved with one or more doses during a course of treatment regimen (e.g., daily, every other day, every three days, once a week, every two weeks, monthly, every two months, every four months, every six months, once a year, etc.).

[0212] As described herein, the formulations and compositions of the present invention may be administered by injection, infusion, indwelling, or implantation. Recombinant derivative formulations and drug compositions may also be administered orally or by inhalation. In certain embodiments, the formulations and compositions of the present invention are administered parenterally. "Parareal administration" and "administering parenterally" refer to methods of administration other than intestinal or topical administration, usually by injection, and include intravascular injection and intravascular infusion, intravenous injection and intravenous infusion, intramuscular injection and intramuscular infusion, intra-arterial injection and intra-arterial infusion, intrathecal injection and intrathecal infusion, intra-articular injection and intra-articular infusion, intratumoral injection and intratumoral infusion, intraperitoneal injection and intraperitoneal infusion, and subcutaneous injection and subcutaneous infusion. In certain embodiments, the formulations and compositions described herein are administered to a target by direct injection into a tumor, lymph node, or disease site. In certain embodiments, drug compositions are administered orally.

[0213] In certain examples, the Disclosure provides a method for performing cellular immunotherapy in a subject with a disease or disorder, comprising the step of administering a recombinant or recombinant inducer formulation that constitutively expresses a CAR in vivo in an activated state "off". In this example, the method further comprises the step of administering a drug composition that can activate the CAR by binding to an antigen. In some embodiments, the drug composition is delivered before the administration of the recombinant or recombinant inducer formulation, simultaneously with the administration of the recombinant or recombinant inducer formulation, or at a point after the administration of the recombinant or recombinant inducer formulation.

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

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

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

[0217] In some embodiments, one or more drug compositions are administered together with a recombinant or recombinant derivative formulation, and if toxicity due to the recombinant or recombinant derivative formulation is observed, administration of the one or more drug compositions is discontinued until the toxicity decreases. After the symptoms of toxicity have subsided, administration of the 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 drug composition is administered together with a recombinant or recombinant inducer, but if a reduction in cancer cells or virus-infected cells in the subject's body is observed, administration of the drug composition can be discontinued for a certain period to allow the recombinant cells to rest. Administration of the drug composition can also be discontinued if the cancer goes into remission or the infection is eliminated.

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

[0220] Cancers that can be treated with recombinant formulations or recombinant inducers and drug compositions disclosed herein include cancers such as bladder cancer, head and neck cancer, breast cancer, colorectal 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); and lymphomas such as leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, and Burkitt lymphoma. Myeloid hematopoietic malignancies; myeloid hematopoietic malignancies such as acute myeloid leukemia, chronic myeloid leukemia, and promyelocytic leukemia; mesenchymal tissue-derived tumors such as fibrosarcoma and rhabdomyosarcoma; other tumors such as neuroblastoma and glioma; tumors of the central and peripheral nervous systems such as astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tissue-derived tumors such as fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors such as malignant melanoma, xeroderma pigmentosum, keratosaccharomyceta, seminomas, follicular thyroid cancer, and teratoma. Other representative cancers that can be treated in accordance with this disclosure include lymphoid hematopoietic malignancies, such as T-cell tumors and B-cell tumors, including T-cell diseases such as pre-lymphocytic leukemia (T-PLL), including small cell pre-T lymphocyte leukemia and cerebral-like cell pre-T lymphocyte leukemia; large granular lymphocyte leukemia (LGL); Sézary syndrome (SS); adult T-cell leukemia / lymphoma (ATLL); hepatosplenic T-cell lymphoma; peripheral / postthymic T-cell lymphoma (multimorphic and immunoblastic subtypes); angioimmunoblastic T-cell lymphoma; vascular central (nasal) T-cell lymphoma; undifferentiated (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 with a ligand-binding domain that binds to that cancer antigen can be selected. Representative cancer antigens include: bladder cancer antigen: MUC16, PD-L1, EGFR; breast cancer antigen: HER2, ERBB2, ROR1, PD-L1, EGFR, MUC16, FOLR, CEA; cholangiocarcinoma antigen: mesothelin, PD-L1, EGFR; colorectal cancer antigen: CEA, PD-L1, EGFR; glioblastoma antigen: 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 Examples include 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. Using these classifications, CAR combination therapies can be constructed as described separately herein. CAR combination therapies may include activity-inducible costimulatory molecules and / or activity-inducible inhibitory molecules.

[0222] Certain 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 cases, cancer samples obtained from subjects can be evaluated for the presence or absence of specific biomarkers or cell surface markers. For example, breast cancer cells obtained from a subject may 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 having binding domains that bind to these antigens are selected. Various combinations of such tumor antigens or cell surface molecules and CARs may be selected to constitute CAR combination therapy.

[0224] In certain embodiments, an anticancer effect can be obtained by a therapeutically effective amount of the formulation and the drug composition. Anticancer effects include a reduction in the number of malignant cells, a reduction in the number of metastases, a reduction in tumor volume, an extension of life expectancy, induction of chemotherapy sensitivity or radiosensitivity in cancer cells, inhibition of angiogenesis near cancer cells, inhibition of cancer cell proliferation, inhibition of tumor growth, prevention or reduction of metastasis, extension of the lifespan of the subject, suppression of cancer-related pain, and / or a reduction in cancer recurrence or relapse after treatment.

[0225] Infections that can be treated with the formulations and compositions of this disclosure include bacterial infections, viral infections, fungal infections, parasitic infections, and arthropod infections. In certain embodiments, the infections are chronic infections. In certain embodiments, bacterial infections include infections caused by Staphylococcus, Streptococcus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Escherichia coli, Listeria monocytogenes, Salmonella, Vibrio, Chlamydia trachomatis, Neisseria gonorrhoeae, or Treponema pallidum. In certain embodiments, viral infections include those caused by 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, fungal infections include those caused by Trichophyton or Candida species. In certain embodiments, parasitic infections include infections caused by Giardia, Toxoplasma, human pinworm, Trypanosoma cruzi, Echinococcus, cysticercosis, Toxocara, Trichomonas, and Entamoeba histolytica. In certain embodiments, arthropod infections include infections transmitted by arthropods infected with viruses or bacteria, such as California encephalitis, Chikungunya fever, Dengue fever, Eastern equine encephalitis, Poissant encephalitis, St. Louis encephalitis, West Nile fever, yellow fever, Zika fever, Lyme disease, or babesiosis.

[0226] In certain embodiments, an anti-infective effect can be obtained by a therapeutically effective amount of the formulation and the drug composition. Anti-infective effects include a reduction in the amount or level of infectious pathogens, a reduction in fatigue, a reduction in loss of appetite, suppression of weight loss, a reduction in fever, a reduction in night sweats, a reduction in chills, a reduction in pain and soreness, a reduction in diarrhea, a reduction in bloating, a reduction in abdominal pain, a reduction in rash, a reduction in cough and / or runny nose.

[0227] In certain embodiments, administration of the drug composition is discontinued to obtain an anti-adverse effect. This anti-adverse effect can reduce or eliminate negative effects caused by the administration of the formulation, such as cytokine storms (cytokine release syndrome), tumor lysis syndrome (TLS), and B-cell deficiency, which are induced by transplantation.

[0228] When administering the drug, the therapeutically effective dose (also referred to herein as “dose”) can first be estimated based on the results of in vitro assays and / or animal model studies. Using such information, a more accurate determination of the effective dose for the target subject can be made. The actual dose administered to a particular subject can be determined by a physician, veterinarian, or researcher, taking into account parameters such as the target, body weight, disease severity, disease type, disease stage, past or concomitant therapeutic interventions, the subject’s idiopathic disease, and physical and physiological factors including the route of administration.

[0229] The therapeutically effective dose of the recombinant product to be administered is 10 2 More than one cell, 10 3 More than one cell, 10 4 More than one cell, 10 5 More than one cell, 10 6 More than one cell, 10 7 More than one cell, 10 8 More than one cell, 10 9 More than one cell, 10 10 More than one cell, or 10 11 This includes a number of cells exceeding one.

[0230] Useful doses for administering recombinant derivatives or drug compositions may be, for example, in the range of 0.1 to 5 μg / kg or 0.5 to 1 μg / kg. In other examples, doses include 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 to 5 mg / kg, or 0.5 to 1 mg / kg. In other examples, doses include 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 higher.

[0231] (xiii) kit This disclosure also includes kits comprising materials useful for carrying out aspects of this disclosure. These kits may include, for example, cells (e.g., immune cells), nucleic acids encoding activity-inducible fusion proteins (e.g., CARs, costimulatory molecules and / or inhibitory molecules), transfection reagents, assay reagents, drug molecules, buffers, cellular nutrients and growth media, cell sorting molecules (e.g., Dynabeads), tubes, wells, and low-molecular-weight estrogen analogs (e.g., tamoxifen, 4-OHT, ES8, CMP8).

[0232] (xiv)hsp90 client The activity-inducible fusion proteins disclosed herein may include a binding domain derived from another client molecule of hsp90 or a domain thereof. The hsp90 client proteins described herein are classified as transcription factors, kinases, and “others,” as shown at picard.ch / downloads / hsp90 interactors.

[0233] Examples of client transcription factors for hsp90 include: 12(S)-HETE receptor; AF9 / MLLT3; steroid receptors in all vertebrates (GR, MR, ERα, ERβ, PR, AR); AGL24; ATF3; BBX; BCL-6; Bclaf1; BES1; BrZ7; BZR1; C20orf194; CAR; CEBPE; Cwt1; CXXC1; intracytoplasmic 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: Dioxin receptor (=AhR), Sim, HIF-1α, HIF-2α, HIF-3α; PCGF6; POGK; PPARα, PPARβ, PPARγ; PRDM1; PREB; PXR; REST; REV-ERBα; (of Aspergillus) RlmA; SETDB1; SIM2; SLFN11; SOC1; SOX11; Sp1; SREBF1; SREBP1; SREBP2; Stat2; Stat3 (a plural of lipid raft caveolin-1) Examples of fused receptors include Stat3, Stat5, SUP, TADA2A, TBX22, TCF25, TDP-43, TEAD2, TFDP3, THAP4, TonEBP / OREBP, TRIM32, Tup1, Twist1, Ure2, USP1, VDR, VP16, steroid (anseridiol) receptors of the aquatic fungus genus Cottonweed, WT1, ZBED4, ZBTB17, ZBTB20, ZC3H7B, ZNF215, ZNF509, and ZNF74.

[0234] Examples of client kinases for hsp90 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;EBV's BGLF4;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 (Escherichia 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 (mutants 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-binding 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;(of the Aspergillus genus) MpkA;Mps1;mTOR;MUSK;MYLK2;MYLK3;MYLK4;NEK11;NEK8;NEK9;NIK;NPM-Alk;NPR2;NTRK1;NTRK2;NTRK3;NUAK2;Nucreophosmin-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 Examples include 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; aldokereductase 1B10; ANAPC2; ANKMY2; annexin A2; ANP receptor; ANP32C / D; Apaf-1; apoB; APOBEC-3B, APOBEC-3C, APOBEC-3G; Arb1; ARD1; Argonaut 1 (Ago1); Argonaut 2 (=Ago2=GERp95); Argonaut 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; calpain1; carponin; CARM1; caspase8; β-catenin; CB2 cannabinoid receptor; Ccp1; CCDC117; type III CD38; CD79a; Cdc13; Cdc14; Cdc25a and Cdc25c; Cdk5 activator p35; CPEB1, CPEB2, CPEB3; CFTR (nascent polypeptide and mutant polypeptide); ChAT; CheZ (Escherichia coli); Chl1; Chronophin; cineole synthesis Enzyme 1; Clathrin heavy chain; CLC-1 chloride channel; CLC-2 chloride channel; Clostridium CDT toxin; Clostridium iota toxin; Clasterin; COG complex; COI1; Complement component 9 (C9); Cry toxin; 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 (Escherichia coli); DNMT1; Dsn1; DTX4; E6^E7; EBAX-1; Emc2; ENC1; eNOS, nNOS(?); Human delayed-rectifying 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 Folliclin; 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 12Glucocerebrosidase; 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; Huntintin; Importin 4 (IPO4); Importin α1; Importin α6 (KPNA5); Ino80; Inositol 1,4,5-triphosphate receptor 3; Integrin α2; Integrin α4; Integrin αL; IL-1β; IRS-2; Japanese encephalitis virus E protein; 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 cell membrane of red blood cells infected with Plasmodium); KSHV K1; KSR1; KSR2; L protein of HRSV; Lamin A / C; LAMP-2A; LANA of KS-HSV; LAP; LARP4B; Regmine; LGALS3BP; LIS1; LNX1; LOC440248; LOX1 (OLR1); LOXL2; Lpl1 (Staphylococcus aureus); LRP1 (=CD91); LRP5; LRSAM1; LSD1; LSM8; Macromolecule aminoacyl-tRNA synthetase complex; Macrophage scavenger receptor; MAP1B; MARCH9; Mdm2; MDM4; MeCatalase1; Mg 2+ Dependent phosphatidic acid phosphohydrolase; MIF; misfolding VHL; MMP2, MMP3, MMP9; μ-opioid receptor; MRE11 / Rad50 / NBS1 (MRN) complex; MRP1; Msps / XMAP215 / ch-TOG; MTA1; MTG8; MUC1; myoglobin; N-myc downstream regulatory gene 1 (NRDG1); N-WASP; Na + -K + -Cl-Cotransporter 1; NadA; NAP1; NB-LRR proteins: RPM1 and RPS2, Nod1, Nod2, NALP2, NALP3, NALP4, NALP12, IPAF, RPP4; NCC; NDRG2; NELF-E; Capsid proteins of neuronecrotic virus; Neuraminidase; Neuropeptide Y; NHE1; NHLRC1; Nibrin; NleH1 and NleH2; NMNAT2; Capsid proteins of norovirus VP1; Nox1, Nox2, Nox3, Nox5; NS1; Nsl1; nsP3 and nsP4 of chikungunyavirus; Nucleoprotein (NP) of MERS-CoV; Nup62; OGT; OsCERK1; P protein (rabies virus); P1 (capsid precursor protein P1 of picornavirus); 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; R2TP complex via Pih1; 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; Catalytic A chain of lysine; RIG-I; RNA-dep; (Takemosaic virus) RNA polymerase; RNF10; RNF111; RNF19B; RNF40; RNGTT; Rnr4; Rpb1; SCAP; SDF2; SENP3; SERCA2a; SERT(SLC6A4); SF3B3; SH3RF2; Sicily;SIR2 (SIR2RP1 of the genus Leishmania); SIRT1; SIRT2; SKP2; SKP2 complex; SLC6A14; SMYD1, SMYD2, SMYD3; snoRNP complex; SNRNP200; SOCS6; SPSB1; SPSB3; SREC-I; STING; SUR1 (subunit of ATP-sensitive potassium channel in β-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;Triose phosphate isomerase;Trithorax (and ortholog MLL);Trx1;TrxR;TSG101;Tyrosine hydroxylase;UCH-L1;UHRF1;Ulp1;uPA;Ura 2; URI complex; [of cyanobacteria] uroporphyrinogen decarboxylase (HemE); (of HSV-1) Us11; USP19; Utp21; Vaccinia core protein 4a; (of KSHV) vFLIP; vimentin; VIP1; VPS18; VPS41; WASF3; WSB2; WTAP; WWP1; XPO1; XPORT; XRCC1; ZEITLUPE; and ZMYND10.

[0236] (xv) variant Variants of sequences disclosed and cited herein are also included in this application. Indicators for determining which amino acid residues can be substituted, inserted, or deleted without loss of biological activity are computer programs well known in the art, such as DNASTAR. TMThis can be discovered using software (Madison, Wisconsin, USA). The amino acid changes in protein variants are preferably conservative, i.e., substitutions between amino acids with similar charge or substitutions between uncharged amino acids. Conservative amino acid changes include substitutions by members of the related amino acid family in the side chain.

[0237] Appropriate conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such substitutions can usually be made without altering the biological activity of the resulting molecule. Those skilled in the art will know that substituting a single amino acid in a non-essential region of a polypeptide usually does not substantially alter its biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Natural amino acids are typically classified into conserved 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 negative-charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar positive-charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6: (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu Groups 10 (nonpolar small aliphatic residues or slightly polar small aliphatic residues): 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] When making such changes, the hydrophobicity index of amino acids may be taken into consideration. The importance of the hydrophobicity index of amino acids in conferring biological functions that interact with each other on proteins is widely understood in this art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). The hydrophobicity index for each amino acid is as follows: 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 substituting a specific amino acid with another amino acid having a similar hydrophobic index or degree of hydrophobicity can yield a protein with similar biological activity, i.e., a protein with biologically equivalent functionality. When making such a change, substitutions between amino acids with hydrophobic indexes within ±2 are preferred, substitutions between amino acids with hydrophobic indexes within ±1 are particularly preferred, and substitutions between amino acids with hydrophobic indexes within ±0.5 are even more particularly preferred. Furthermore, it is well known in the art that substitutions between similar amino acids can be effectively carried out based on their 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 specific amino acids can be substituted with other amino acids that have a similar hydrophilicity value, and that such substitutions can yield biologically equivalent proteins, and in particular, immunologically equivalent proteins. When making such changes, substitutions between amino acids with hydrophilicity values ​​within ±2 are preferred, substitutions between amino acids with hydrophilicity values ​​within ±1 are particularly preferred, and substitutions between amino acids with hydrophilicity values ​​within ±0.5 are even more preferred.

[0241] As outlined above, amino acid substitutions may be made based on the relative similarity of substituents on the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, and size.

[0242] Functional variants include the addition or substitution of one or more residues that does not substantially affect the physiological function of the protein. Functional fragments include one or more deletions or cleavages that do not substantially affect the physiological function of the protein. The absence of substantial effect can be confirmed by experimentally observing comparable results in activation or binding tests. Functional variants and functional fragments of intracellular domains (e.g., intracellular signaling domains), when in the activated state described herein, transmit activation or repression signals to the same extent as the wild-type baseline intracellular domain. Functional variants and functional fragments of binding domains bind to their recognition antigens or recognition ligands at the same level as the wild-type baseline binding domain.

[0243] In certain embodiments, the VH region of the binding domain may be derived from or created based on the VH region of a known antibody, and may include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) insertions, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) deletions, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or combinations thereof, compared to the VH region of a known antibody. Insertions, deletions, or substitutions may be located at any position in the VH region, including the amino terminus, carboxyl terminus, or both ends, as long as each CDR contains no changes, or at most one, two, or three changes, and the binding domain containing the recombinant VH region can bind specifically to the target with the same affinity as the wild-type binding domain.

[0244] In certain embodiments, the VL region of the binding domain may be derived from or created based on the VL region of a known antibody, and may contain 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 combinations thereof, compared to the VL region of a known antibody. Insertions, deletions, or substitutions may be located at any position in the VL region, including the amino terminus, carboxyl terminus, or both ends, as long as each CDR contains no changes, or at most one, two, or three changes, and the binding domain containing the recombinant VL region can bind specifically to the target with the same affinity as the wild-type binding domain.

[0245] These considerations also apply to the TCR chain and other binding domains derived therefrom.

[0246] As described separately herein, gene sequence variants include codon-optimization variants, sequence polymorphisms, splice variants, and / or mutations that do not have a statistically significant effect on the function of the encoded product.

[0247] Variants of proteins, nucleic acids, 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 with the proteins, nucleic acids, or gene sequences disclosed herein.

[0248] "Sequence identity (%)" refers to the relationship between two or more sequences measured by comparing them. In this technology, "identity" also means the degree of association between protein sequences, nucleic acid sequences, or gene sequences, measured by matching between protein sequence chains, nucleic acid sequence chains, or gene sequence chains. "Identity" (often called "similarity") can be easily calculated using 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). Methods for measuring identity are preferably designed to obtain the best match between the sequences being tested. Methods for measuring identity and similarity are systematized in publicly available computer programs. Sequence alignment and identity calculations may be performed using the Megalign program (DNASTAR, Madison, Wisconsin), which is included in the LASERGENE suite of bioinformatics computing software.Multiple alignment of sequences can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989), using default parameters (gap penalty = 10, gap length penalty = 10)). Related programs include the GCG program suite (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, 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 program's default values. In this specification, "default values" means a set of numerical values ​​or parameters pre-registered in the software during software initialization.

[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. Typical stringent hybridization conditions include incubation overnight at 42°C in a solution containing 50% formamide, 5×SSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and fragment-treated 20 μg / ml denatured salmon sperm DNA, followed by washing the filter at 50°C with 0.1×SSC. Changes in hybridization stringency and signal detection are mainly achieved by adjusting the concentration of formamide (lower proportions of formamide result in lower stringency), salt conditions, or temperature. For example, moderately high stringency conditions include incubation at 37°C overnight in a solution containing 6×SSPE (20×SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml blocking salmon sperm DNA, followed by washing at 50°C with 1×SSPE and 0.1% SDS. Further lower stringency can be achieved by washing after stringent hybridization with a higher salt concentration (e.g., 5×SSC). The aforementioned conditions can be varied in various ways by adding and / or substituting other blocking reagents used to reduce the background of the hybridization experiment. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. When adding specific blocking reagents, it may be necessary to modify some of the aforementioned hybridization conditions due to compatibility issues.

[0250] (xvi) Typical Embodiments 1. An activity-inducible fusion protein containing an HSP90 binding domain. 2. The activity-inducible fusion protein according to Embodiment 1, wherein the hsp90-binding domain binds to hsp90 with an affinity lower than the affinity for binding to a drug molecule. 3. The activity-inducible fusion protein according to Embodiment 1 or 2, wherein the hsp90 binding domain includes a hormone-binding domain. 4. The activity-inducible fusion protein according to Embodiment 3, wherein the hormone-binding domain is the recombinant binding domain (EBD) of the estrogen receptor. 5. The activity-inducible fusion protein according to Embodiment 4, wherein the EBD comprises an estrogen receptor binding domain and a mutation selected from the following: 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 according to Embodiment 5, wherein the 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. 7. An activity-inducible fusion protein according to any one of Embodiments 2 to 6, wherein the drug molecule comprises a low-molecular-weight estrogen analog. 8. The activity-inducible fusion protein according to Embodiment 7, wherein the low molecular weight 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 according to Embodiment 7 or 8, wherein the low molecular weight estrogen analog comprises tamoxifen, 4-OHT, (Z)-endoxifen, ES8, or CMP8. 10. The aforementioned activity-inducible fusion protein is a chimeric antigen receptor (CAR), and when expressed in cells, it is expressed as a chimeric antigen receptor containing an extracellular portion and an intracellular portion linked via a transmembrane domain. The extracellular portion includes a ligand-binding domain, and The intracellular portion includes an intracellular signaling domain and an HSP90 binding domain. An activity-inducible fusion protein according to any one of Embodiments 1 to 9, characterized by the above. 11. The activity-inducible fusion protein according to Embodiment 10, wherein the ligand-binding domain binds to a cancer antigen or a viral antigen. 12. An inducible fusion protein according to 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-inducible fusion protein according to Embodiment 12, wherein the scFv has the sequence shown 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: 4 14. The activity-inducible fusion protein according to Embodiment 10, wherein the ligand-binding domain binds to an immune cell antigen. 15. The activity-inducible fusion protein according to Embodiment 14, 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. 16. The activity-inducible fusion protein according to Embodiment 10, wherein the ligand-binding domain binds to a hapten. 17. The activity-inducible fusion protein according to Embodiment 16, wherein the hapten comprises fluorescein, urushiol, quinone, biotin, or dinitrophenol. 18. The activity-inducible fusion protein according to Embodiment 16 or 17, 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. 19. An 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-inducible fusion protein according to Embodiment 19, wherein the spacer region includes an IgG4 hinge. 21. An activity-inducible fusion protein according to any one of Embodiments 10 to 20, wherein the intracellular portion includes a CD3ζ signaling domain. 22. An activity-inducible fusion protein according to any one of Embodiments 10 to 21, wherein the intracellular signaling domain includes a 4-1BB signaling domain. 23. An activity-inducible fusion protein according to any one of Embodiments 10 to 22, wherein the intracellular signaling domain comprises a CD3ζ signaling domain and a 4-1BB signaling domain. 24. An activity-inducible fusion protein according to any one of embodiments 10 to 23, further comprising a linked amino acid consisting of one glycine, two glycine, or three glycine molecules adjacent to the hsp90 binding domain, or not comprising a linker adjacent to the hsp90 binding domain. 25. The activity-inducible fusion protein according to Embodiment 24, wherein, in the presence of a linked amino acid consisting of 1, 2, or 3 glycine molecules, the linked amino acid is located between the 5' end of the hsp90 binding domain and the 3' end of the intracellular signaling domain. 26. An 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 one of Embodiments 10 to 25, wherein the intracellular portion does not have a linker or linked amino acids. 30. An activity-inducible fusion protein according to any one of Embodiments 10 to 29, wherein the transmembrane domain includes the transmembrane domain of CD28. 31. An activity-inducible fusion protein according to any one of embodiments 10 to 30, having the protein sequence shown in Sequence ID No. 118. 32. An activity-inducible fusion protein according to any one of Embodiments 1 to 31, wherein the hsp90 binding domain is linked to an intracellular portion of a co-stimulatory immune molecule or an inhibitory immune molecule. 33. The activity-inducible fusion protein according to Embodiment 32, wherein the co-stimulatory 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 according to 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 as described in any of Embodiments 1 to 34. 36. The nucleotide according to Embodiment 35, comprising the coding sequence shown in SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 15. 37. The nucleotide according to Embodiment 36, further comprising the coding sequence shown in Sequence ID No. 17. 38. The nucleotide according to embodiment 36 or 37, further comprising the coding sequence shown in sequence number 79. 39. A nucleotide according to any one of embodiments 36 to 39, further comprising the coding sequence shown in Sequence ID No. 124. 40. A nucleotide according to any one of embodiments 36 to 39, further comprising the coding sequence shown in Sequence ID No. 123. 41. A nucleotide according to any one of embodiments 36 to 40, further comprising the coding sequence shown in Sequence ID No. 122. 42. A nucleotide according to any one of embodiments 35 to 41, having the sequence shown in Sequence ID No. 125. 43. Cells genetically modified to express the activity-inducible fusion protein described in any of Embodiments 1 to 34. 44. The cell according to Embodiment 43, wherein the cell is genetically modified to express at least two activity-inducible fusion proteins as described in any of Embodiments 1 to 29, and each of these at least two activity-inducible fusion proteins has a different HSP90 binding domain, and each of these HSP90 binding domains binds to a different drug molecule. 45. The cell according to Embodiment 44, wherein at least two of the activity-inducible fusion proteins each further have different ligand-binding domains. 46. ​​The cell according to Embodiment 45, wherein the different ligand-binding domains each bind to a different antigen. 47. The cell according to Embodiment 46, wherein the different antigens are associated with a common cancer, a common infection, a common immune cell type, or a common hapten, or the different antigens are associated with different cancers, different infections, different immune cell types, or different haptens. 48. The cells according to Embodiment 47, wherein the common cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or a brain tumor. 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 according to Embodiment 49, wherein the T cell is a CD4+ T cell or a CD8+ T cell. 51. Cells according to any one of embodiments 43 to 48, which are induced pluripotent stem cells (iPSCs), tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), natural killer T cells (NKTs), mucosa-associated invariant T (MAIT) cells, dendritic cells, monocytes, or macrophages. 52. A system for controlling the activation state of an inducible fusion protein in vivo, It contains an activity-inducing fusion protein and a drug molecule, A system comprising the aforementioned activity-inducing fusion protein containing an HSP90-binding domain that binds to the drug molecule. 53. The system according to 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 according to embodiment 52 or 53, wherein the hsp90 binding domain includes a hormone binding domain. 55. The system according to Embodiment 54, wherein the hormone-binding domain includes a recombinant binding domain (EBD) of the estrogen receptor. 56. The system according to Embodiment 55, wherein the EBD comprises an estrogen receptor binding domain portion and a mutation 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 according to embodiment 55 or 56, wherein the EBD has the sequence shown in sequence number 4, sequence number 6, sequence number 9, sequence number 11, or sequence number 13. 58. The system according to any one of embodiments 52 to 57, wherein the drug molecule comprises a low-molecular-weight estrogen analog. 59. The system according to Embodiment 58, wherein the low molecular weight estrogen analog comprises tamoxifen, a salt of tamoxifen, a metabolite of tamoxifen, or a compound structurally similar to tamoxifen. 60. The system according to Embodiment 58 or 59, wherein the low molecular weight estrogen analog comprises tamoxifen, 4-OHT, (Z)-endoxifen, ES8, or CMP8. 61. The aforementioned activity-inducible fusion protein is a chimeric antigen receptor (CAR), and when expressed in cells, it is expressed as a chimeric antigen receptor containing an extracellular portion and an intracellular portion linked via a transmembrane domain. The extracellular portion includes a ligand-binding domain, and The intracellular portion includes an intracellular signaling domain and an HSP90 binding domain. A system according to any one of embodiments 52 to 60, characterized by the above. 62. The system according to embodiment 61, wherein the ligand-binding domain binds to a cancer antigen or a viral antigen. 63. The system according to embodiment 61 or 62, wherein the ligand-binding domain includes 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 according to embodiment 63, wherein the scFv has the sequence shown 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 according to embodiment 61, wherein the ligand-binding domain binds to an immune cell antigen. 66. The system according to 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 according to embodiment 61, wherein the ligand-binding domain binds to a hapten. 68. The system according to Embodiment 67, wherein the hapten comprises fluorescein, urushiol, quinone, biotin, or dinitrophenol. 69. The system according to 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 according to any one of embodiments 61 to 69, wherein the extracellular portion further includes a spacer region. 71. The system according to embodiment 70, wherein the spacer region includes a hinge for IgG4. 72. The system according to any one of embodiments 61 to 71, wherein the intracellular portion includes a CD3ζ signaling domain. 73. The system according to any one of embodiments 61 to 72, wherein the intracellular signaling domain includes a 4-1BB signaling domain. 74. The system according to any one of embodiments 61 to 73, wherein the intracellular signaling domain includes a CD3ζ signaling domain and a 4-1BB signaling domain. 75. The system according to any one of embodiments 61 to 74, further comprising a linked amino acid consisting of one glycine molecule, two glycine molecules, or three glycine molecules adjacent to the hsp90 binding domain, or not comprising a linker adjacent to the hsp90 binding domain. 76. The system according to Embodiment 75, wherein, if a linked amino acid consisting of 1, 2, or 3 glycine molecules is present, the linked amino acid is located between the 5' end of the hsp90 binding domain and the 3' end of the intracellular signaling domain. 77. The system according to any one of embodiments 61 to 76, wherein the hsp90 binding domain is located at the 3' end of the intracellular signaling domain. 78. The system according to any one 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 according to any one of embodiments 74 to 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 according to any one of embodiments 61 to 79, wherein the intracellular portion does not have a linker or linked amino acids. 81. The system according to any one of embodiments 61 to 80, wherein the transmembrane domain includes a transmembrane domain of CD28. 82. The system according to any one of embodiments 61 to 81, wherein the CAR has the protein sequence shown in Sequence ID No. 118. 83. The system according to any one of embodiments 52 to 82, wherein the activity-inducible fusion protein is a co-stimulatory immune molecule or an inhibitory immune molecule. 84. The system according to Embodiment 83, wherein the co-stimulatory 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 according to 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. The system according to any one of embodiments 52 to 85, wherein the system comprises at least two activity-inducible fusion proteins, each having a different HSP90 binding domain, and each of these HSP90 binding domains binds to a different drug molecule. 87. The system according to Embodiment 86, wherein the at least two activity-inducible fusion proteins each further have different ligand-binding domains. 88. The system according to embodiment 87, wherein the different ligand-binding domains each bind to a different antigen. 89. The system according to Embodiment 88, wherein the different antigens are associated with a common cancer, a common infection, a common immune cell type, or a common hapten, or the different antigens are associated with different cancers, different infections, different immune cell types, or different haptens. 90. The system according to Embodiment 89, wherein the common cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or brain tumor. 91. A system according to any one of embodiments 52 to 90, formulated for administration to a target. 92. The formulated system is (i) cells produced ex vivo expressing the activity-inducible fusion protein in the form of a recombinant formulation mounted on a pharmaceutically acceptable carrier; and / or (ii) Cell-targeting viral vectors and / or cell-targeting nanoparticles in the form of recombinant induction formulations mounted on a pharmaceutically acceptable carrier, (iii) the drug molecule in the form of a drug composition formulated on a pharmaceutically acceptable carrier and Includes, The cell-targeting viral vector and / or cell-targeting nanoparticles contain a recombinant inducer that, after administration, induces the expression of the activity-inducible fusion protein in the target cells in vivo. The system described in Embodiment 91. 93. A method for treating a subject in need of treatment, comprising the step of administering the system described in Embodiment 92 to the subject to treat the subject. 94. The method according to Embodiment 93, wherein the administration comprises administering the recombinant formulation and the drug composition. 95. The method according to Embodiment 93, wherein the administration comprises administering the recombinant inducer and the drug composition. 96. The method according to any one of embodiments 93 to 95, further comprising the step of discontinuing the administration of the drug molecule to suppress side effects caused by the administration of the system. 97. The method according to any one of embodiments 93 to 96, further comprising the step of discontinuing the administration of the drug molecule when the subject no longer requires treatment. 98. The method according to any one of embodiments 93 to 97, wherein the subject is a subject requiring treatment for cancer or a viral infection. 99. The method according to any one 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 these hsp90 binding domains binds to a different drug molecule. 100. The method according to Embodiment 99, comprising the step of administering at least two drug molecules, wherein one of the at least two drug molecules binds to the hsp90 binding domain of one activity-inducible fusion protein contained in the system, and another of the at least two drug molecules binds to the hsp90 binding domain of another activity-inducible fusion protein contained in the system. 101. The method according to Embodiment 100, further comprising the step of discontinuing the administration of at least one drug molecule. 102. The method according to Embodiment 100 or 101, further comprising the step of discontinuing the administration of all drug molecules. 103. The method according to any one of embodiments 99 to 103, wherein the at least two types of activity-inducible fusion proteins each further have different ligand-binding domains. 104. The method according to Embodiment 103, wherein the different ligand-binding domains each bind to a different antigen. 105. The method according to Embodiment 104, wherein the different antigens are associated with a common cancer, a common infection, a common immune cell type, or a common hapten, or the different antigens are associated with different cancers, different infections, different immune cell types, or different haptens. 106. The method according to Embodiment 105, wherein the common cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or a brain tumor. 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-inducible fusion protein according to any one of embodiments 1 to 34 and 107, wherein activity is induced within one hour after administration of a drug molecule bound to the HSP-binding domain. 109. An activity-inducible fusion protein according to any one of embodiments 1 to 34 and 107, wherein activity is induced within 30 minutes after administration of a drug molecule bound to the HSP-binding domain. 110. An activity-inducible fusion protein according to any one of embodiments 1 to 34 and 107, wherein activity is induced within 15 minutes after administration of a drug molecule bound to the HSP-binding domain. 111. An activity-inducible fusion protein according to any one of embodiments 1 to 34 and 107, wherein activity is induced within 5 minutes after administration of a drug molecule bound to the HSP-binding domain.

[0251] (xvii) Experimental examples shown in Figures 9-17 Further evaluation of CAR-EBD constructs was conducted to investigate the position of the EBD domain in the CAR construct and the linker connecting the EBD domain to the CAR construct. Various CAR-EBD constructs containing glycine linkers (also referred to herein as "linking amino acids") consisting of one, two, or three glycine molecules, located behind (i.e., at the 3' end of) CD3ζ were investigated (data shown). Furthermore, CAR-EBD constructs without glycine linkers, constructs with an EBD domain flanked by three glycine linkers positioned between 4-1BB and CD3ζ, and constructs with the EBD domain directly fused between 4-1BB and CD3ζ (without glycine linkers) were designed. Examples of experiments include: 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 that. EBD may be, for example, EBD(4-OHT), EBD(CMP8), or EBD(ES8).

[0252] Figures 9-17 present data for the following two types of EBD mutants. EBD(4-OHT)(SEQ ID NO: 13): Responds to 4-OHT and (Z)-endoxiphene. EBD(CMP8)(SEQ ID NO: 11): Responds to 4-OHT, (Z)-endoxiphene, and CMP8.

[0253] Figure 9 (top panel) shows a schematic diagram of Jurkat iSynPro eGFP:ffluc reporter cells. Jurkat iSynPro eGFP:ffluc reporter cells are Jurkat cell lines transduced by lentivirus to stably express an inducible synthetic promoter (iSynPro; WO2018213332A1) that regulates the expression of highly sensitive green fluorescent protein (eGFP) fused to the bioluminescent reporter firefly luciferase (ffluc). Next, clones were selected from this cell line by limiting dilution. When activated in T cells, the Jurkat iSynPro eGFP:ffluc strain induces GFP expression detectable by flow cytometry, and luciferase activity can be measured by adding D-luciferin as a substrate that catalyzes the luciferase reaction.

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

[0255] Figures 10, 14, and 15 present research results on huCD19-EBD(4-OHT) CARs with glycine linkers. Evaluation of activation curves plotting GFP expression by various huCD19-EBD(4-OHT) CARs with different glycine linkers showed that activation (on / off state) occurs in a drug-dependent manner, and that the inducibility and effect on construct leakage expression differ depending on the type of linker. For example, in this study, the Gly3 linker mutant was found to be able to enter the "on" state at lower drug concentrations and to reach maximum induction at lower drug concentrations compared to the Gly1 linker mutant and the Gly2 mutant.

[0256] The tamoxifen metabolite (Z)-endoxifen was further added to the experiment, a wider drug concentration range was established, and a control group was created in which the drug was added without tumor cells. The experiment was then repeated to investigate the activation ability in the absence of the target. Evaluation of mean fluorescence intensity (MFI) showed that both glycine linker constructs could be switched on / off by 4-OHT or (Z)-endoxifen, and similar activation curves were obtained.

[0257] Figures 11-13, 16, and 17 show the results of various B7H3-EBD CAR mutant studies. The first study aimed to confirm the functionality of the B7H3 CAR ERT2 (EBD(4-OHT)) mutant, which is responsive to 4-OHT, or the EBD(CMP8) mutant (EBD[L348M, M431G, G521R] mutant), which is responsive to both 4-OHT and CMP8. Drug concentrations of 0nM, 500nM, or 1000nM were selected. In all EBD mutants (EBD(4-OHT) or EBD(CMP8)), a clear on / off state was observed, demonstrating responsiveness to 4-OHT. Furthermore, responsiveness to CMP8 was observed only in CMP8-responsive EBD(CMP8) mutants ([L348M, M431G, G521R]), while no responsiveness to CMP8 was observed in the EBD(4-OHT) mutant (not shown).

[0258] Following this study, we conducted research using activation curves across a wide concentration range. In the B7H3-EBD(4-OHT) CAR, a clear on / off state was observed, indicating drug-dependent activation. On the other hand, in the CMP8-responsive EBD(CMP8) mutant, both 4-OHT-mediated and CMP8-mediated expression induction were observed even at a drug concentration of 0.49 nM.

[0259] (Z)-endoxifene, a metabolite of tamoxifen, was added to the experiments, and a broader drug concentration range was established to repeat the broad-concentration activation curve study. Evaluation of mean fluorescence intensity (MFI) confirmed the responsiveness of CMP8 (EBD(CMP8)[L348M, M431G, G521R]) mutants to 4-OHT or CMP8, demonstrating that (Z)-endoxifene can also induce this system. Furthermore, testing of EBD(4-OHT)-responsive mutants confirmed that this system can be induced by 4-OHT or (Z)-endoxifene, but not by CMP8.

[0260] These studies demonstrated that in all constructs, each drug could induce an on / off state.

[0261] (xviii) Conclusion The nucleic acid sequences and amino acid sequences provided herein are represented by abbreviations used for nucleotide bases and amino acid residues, as defined in Patent Law Enforcement Rule 37 CFR Rule 1.822 and also shown in Tables 1 and 3 of WIPO Standard ST.25(1998) Supplement 2. Although only one type of strand is shown for each nucleic acid sequence, complementary strands are also included in the embodiments where appropriate.

[0262] In this specification, the terms “specific binding affinity,” “specific binding,” “specific binding,” or “specific target” refer to the binding of one molecule to another molecule with a binding affinity higher than background binding. The binding domain (for example, the binding domain of a CAR containing the binding domain) is, for example, 10 5 M -1 When a binding domain binds to or associates with a target molecule with an affinity or Ka (i.e., the equilibrium binding constant of a particular binding interaction expressed in units of 1 / M) greater than or equal to that affinity, the binding domain "specifically binds" to that target molecule. In a particular embodiment, the binding domain (or CAR) is 10 6 M -1 The above 10 7 M -1 The above 10 8 M -1 The above 10 9 M -1 The above 10 10 M -1 The above 10 11 M -1 The above 10 12 M -1 or 10 13 M -1 The target binds with a Ka of the above value. The "high affinity" binding domain has 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 This refers to a binding domain having a Ka value of or greater than that.

[0263] Alternatively, affinity is the equilibrium dissociation constant (Kd) (unit: M) of a particular binding interaction (e.g., 10 -5 M~10 -13The binding domain and affinity of CAR proteins according to this disclosure can be easily measured using prior art such as competitive ELISA (enzyme-linked immunosorbent assay); binding (association); displacement assays using labeled ligands; measurement using surface plasmon resonance spectrometers such as the Biacore T100, commercially available from Biacore, Inc. (Piscataway, New Jersey); and optical biosensor technologies such as the EPIC system, commercially available from Corning Corporation, and the EnSpire, commercially available from PerkinElmer Corporation (see also, 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 the specific binding is twice, five times, ten times, twenty times, fifty times, one hundred times, one thousand times or more than that of the background binding.

[0265] In this specification, "derived from" indicates the relationship between the first molecule and the second molecule. The term "derived from" usually refers to the structural similarity between the first molecule and the second molecule, and does not imply, nor does it include, any limitation of the processes or sources for obtaining the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, this intracellular signaling domain retains a sufficient CD3ζ structure to exert the necessary function, i.e., the ability to generate a signal, under appropriate conditions. This does not imply, nor does it include, any limitation of any specific process for obtaining the intracellular signaling domain, for example, that preparation begins with the CD3ζ sequence and involves deleting undesirable sequences or introducing mutations to create the intracellular signaling domain.

[0266] As those skilled in the art will understand, each embodiment disclosed herein includes, substantially consists of, or comprises the specific components, processes, materials, or ingredients described herein. Therefore, the terms “includes” or “contains” should be interpreted as “includes, substantially consists of, or comprises.” The “includes” transition means, but is not limited to, the inclusion of components, processes, materials, or ingredients not described herein, even in large quantities. The “consists of” transition excludes all components, processes, materials, or ingredients not described herein. The “substantially consists of” transition limits the scope of the embodiment to the described components, processes, materials, or ingredients, and components, or components, that do not substantially affect the embodiment. A substantial effect means an effect that statistically significantly reduces the ability of cells expressing an inducible fusion protein to activate (e.g., antigen binding to a CAR; ligand binding to a co-stimulatory or inhibitory molecule) in the presence of the drug molecule and physiological conditions involved.

[0267] Unless otherwise stated, in this specification and in the claims, all numerical values ​​representing the quantity or properties of materials, such as molecular weight and reaction conditions, are construed in all cases as being modified with the term "approximately." Therefore, unless otherwise stated, the numerical parameters described herein and in the appended claims are approximations that will vary depending on the desired properties to be obtained by the present invention. Without limiting the scope of the claim range and equivalence principle, each numerical parameter should be interpreted at least in light of the reported significant figures and with normal rounding. To be more precise, the term “approximately” when used with a given number or range has a meaning that can be reasonably interpreted by a person skilled in the art, namely, a range of ±20% of the given number; a range of ±19% of the given number; a range of ±18% of the given number; a range of ±17% of the given number; a range of ±16% of the given number; a range of ±15% of the given number; a range of ±14% of the given number; a range of ±13% of the given number; a range of ±12% of the given number; a range of ±11% of the given number; a range of ±10% of the given number; a range of ±9% of the given number; a range of ±8% of the given number; a range of ±7% of the given number; a range of ±6% of the given number; a range of ±5% of the given number; a range of ±4% of the given number; a range of ±3% of the given number; a range of ±2% of the given number; or a range of ±1% of the given number, indicating that the number or range is somewhat greater or less than the given number or range.

[0268] While the numerical ranges and parameters representing the broad scope of this invention are approximations and approximate ranges, the numerical values ​​described in the specific examples are reported as accurately as possible. However, all numerical values ​​inherently contain certain errors that inevitably arise due to the standard deviation associated with each test measurement.

[0269] In the description of this invention (particularly in the description of the following claims), “a,” “an,” “the,” and similar demonstrative pronouns are to be interpreted as encompassing both singular and plural unless otherwise stated or the context explicitly indicates otherwise. The numerical ranges described herein are intended to be a simplified way of referring individually to each numerical value within that range. Unless otherwise stated, each numerical value is described herein as if it were described individually. Unless otherwise stated or the context explicitly indicates otherwise, any method described herein may be carried out in any suitable order. Any use of any examples provided herein, or any language indicating examples (e.g., “etc.”), is for the sole purpose of illustrating the invention in detail and does not limit the scope of the invention as described in the claims. Terms described herein should not be interpreted as referring to non-claimed components essential for carrying out the invention.

[0270] The grouping of other components of the present invention disclosed herein or the grouping of various embodiments of the present invention should not be construed as limiting the present invention. Members of each group may be described individually in this specification or in the claims, or they may be described in this specification or in the claims in combination with other members of the groups described herein or other components. For convenience and / or patentability reasons, it is anticipated that one or more members of one group may be added to another group, or one or more members may be removed from a group. In the event of such addition...

Claims

1. A single-chain activity-inducible fusion protein, In cells, it is expressed as a protein containing a transmembrane domain and an intracellular portion. The transmembrane domain is characterized by tethering the protein to the cell membrane of the cell, The intracellular portion comprises (i) an intracellular signaling domain of a co-stimulatory immune molecule or (ii) an inhibitory immune molecule, and an HSP90 binding domain. The aforementioned hsp90 binding domain, In the absence of a drug capable of binding to the hsp90-binding domain with higher affinity than hsp90, the intracellular hsp90 binds and maintains the protein in an inactive state. In the presence of an effective dose of the drug, the drug binds to and activates the protein, characterized in that A single-chain activity-inducible fusion protein.

2. The single-chain activity-inducible fusion protein according to claim 1, wherein the hsp90 binding domain comprises a recombinant estrogen-binding domain (EBD) of an estrogen receptor.

3. The single-strand activity-inducible fusion protein according to claim 2, wherein the recombinant EBD comprises an estrogen receptor binding domain and a mutation selected from the following: G521R; E353A; a combination of L384M and M421G; a combination of L384M, M421G and G521R; and a combination of G400V, M543A and L544A.

4. The single-chain activity-inducible fusion protein according to claim 2, 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.

5. The single-chain activity-inducible fusion protein according to claim 1, wherein the drug comprises tamoxifen or a salt of tamoxifen.

6. The single-chain activity-inducible fusion protein according to claim 1, wherein the drug comprises tamoxifen, 4-OHT, endoxifen, ES8, or CMP8.

7. The aforementioned single-chain activity-inducible fusion protein is a chimeric antigen receptor (CAR), and when expressed in cells, it is expressed as a chimeric antigen receptor including an extracellular component containing a ligand-binding domain, and The intracellular portion includes the intracellular signaling domain of the co-stimulatory immune molecule. The single-chain activity-inducible fusion protein according to claim 1, further characterized by the above.

8. The single-chain activity-inducible fusion protein according to claim 7, wherein the ligand-binding domain binds to a cancer antigen, viral antigen, immune cell antigen, or hapten.

9. The single-chain activity-inducible fusion protein according to claim 1, wherein the transmembrane domain comprises a CD28 transmembrane domain, and the intracellular signaling domain comprises a 4-1BB intracellular signaling domain and a CD3ζ intracellular signaling domain.

10. The single-chain activity-inducible fusion protein according to claim 1, further comprising a linked amino acid consisting of one glycine molecule, two glycine molecules, or three glycine molecules adjacent to the hsp90-binding domain, or comprising neither a linker nor a linked amino acid adjacent to the hsp90-binding domain.

11. A single-chain activity-inducible fusion protein according to claim 1, which does not contain a degron sequence.

12. The single-chain activity-inducible fusion protein according to claim 1, wherein the co-stimulatory 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.

13. The single-chain activity-inducible fusion protein according to 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.

14. A nucleotide encoding a single-chain activity-inducible fusion protein as described in Claim 1.

15. The nucleotide according to claim 14, comprising the coding sequence shown in SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO:

15.

16. The nucleotide according to claim 14, having the sequence shown in sequence number 125.

17. The drug is 1-(p-(2-(diethylamino)ethoxy)phenyl)-2-(p-methoxyphenyl)-1-phenylethanol citrate, 2-hydroxy-1,2,3-propanetricarboxylate, 2-(p-(α-phenylstyryl)phenoxy)triethylamine citrate, 2-(p-(α-(p-methoxyphenyl)styryl)phenoxy)triethylamine citrate monohydrate, 2-(p-(β-ethyl-α-phenylstyryl)phenoxy)triethyl Amine, 2-(p-(1-(p-methoxyphenyl)-2-phenylpropenyl)phenoxy)triethylamine citrate, 2-(p-(1,2-bis(p-methoxyphenyl)-1-butenyl)phenoxy)triethylamine citrate, 2-(p-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethylethylamine citrate, 2-(p-(2-(p-methoxyphenyl)-1-phenyl-1-butenyl)phenoxy)triethylamine 4-Citrate, 2-(4-(1,2-diphenylethenyl)phenoxy)-N,N-diethyl-ethaneamine, 2-(p-(p-methoxy-α-phenylphenethyl)phenoxy)triethylamine citrate, 2-(p-(p-methoxy-α-methylphenethyl)phenoxy)-triethylamine citrate, 4-chlorotamoxifen, 4-fluorotamoxifen, 4-hydroxytoremifene, 4-iodotamoxifen, 4-methyltamoxifen , 4-hydroxytamoxifen (4-OHT), α-(p-(2-(diethylamino)ethoxy)phenyl)-β-ethyl-p-methoxy-α-phenylphenethyl alcohol citrate, cis-4-hydroxytamoxifen, cis-tamoxifen, desethyltamoxifen, dihydrotamoxifen, droloxifen, droloxifen citrate, (E)-desethyltamoxifen, (E)-desethylmethyltamoxifen, (E)-N,N-dimethyl-2-(4-(1-(2-methylphenyl)-2-phenyl-1-butenyl)phenoxy)ethanamine, (E)-tamoxifen citrate, N-desmethyltamoxifen, tamoxifen citrate, toremifene, toremifene citrate, trans-4-hydroxytamoxifen, (Z)-desethylmethyltamoxifen, or (Z)-desethyltamoxifen A single-chain activity-inducible fusion protein according to claim 1, comprising:

Images