Chemically Induced Access Systems

Abstract

The present invention relates to a method for screening the efficacy and safety of candidate therapies / immunotherapy / cell therapy using a cell containing a first inducible system operable to express a first protein of interest and optionally a second protein of interest to a desired level, and optionally a second inducible system.The present invention further relates to an inducible system, in particular one or more chemically induced access systems, comprising a first plant hormone inducible access system and a second plant hormone inducible access system, and a method of using the system to control expression of a protein of interest.The present invention further relates to a method of making a cell comprising the system, and to a cell comprising the system.

Description

[Technical Field]

[0001] The present invention relates to methods for screening the efficacy and safety of candidate binding molecules / immunotherapies / cell therapies using cells containing a first inducible system, and optionally a second inducible system, operable to express a first protein of interest and optionally a second protein of interest to desired levels. The present invention further relates to inducible systems, particularly one or more chemically induced access systems, comprising a first plant hormone-inducible access system and a second plant hormone-inducible access system, and methods for using the systems to control expression of proteins of interest. The present invention further relates to methods for making cells comprising the systems, and to cells comprising the systems. [Background technology]

[0002] introduction Drug development for cancer treatment is increasingly focused on targeted therapies, whereby drugs are designed to target unique characteristics of cancer cells in the hope of avoiding side effects on healthy tissues. The burgeoning field of immuno-oncology encompasses a variety of therapeutic modalities, some of which aim to exploit antigens on the surface of cancer cells as a means to achieve selective destruction of cancer cells. Unfortunately, tumor-restricted antigens (TRAs) are largely unknown, and most are expressed at low levels in one or more healthy tissues (known as tumor-associated antigens or TAAs). The development of highly promising advanced therapeutic modalities, such as T cell-engaging antibodies and chimeric antigen receptor T (CAR-T) cell therapy, has been hindered by the lack of TRAs, where the expression of TAAs in healthy tissues can cause severe adverse effects, even death.

[0003] Currently, several in vitro risk assessment assays, such as immunogenicity and cytokine release syndrome (CRS) risk assessment assays, are considered essential during the development of such biologics and advanced therapeutic medicinal products (ATMPs). However, to date, no reliable assay exists for assessing so-called "on-target, off-tumor" effects relative to TAA expression levels. Most studies rely on evaluating multiple cell lines with varying levels of antigen expression. This has logistical (requiring the acquisition of multiple cell lines per project) and scientific limitations, as different cell lines have distinct genetic backgrounds and therefore numerous inherent differences beyond antigen expression (e.g., expression of immunomodulatory molecules, differential susceptibility to cell death, etc.), limiting data interpretation. For the evaluation of multiple antigen-targeting modalities, the use of multiple cell lines does not provide a sufficient level of control over the expression levels of each TAA.

[0004] There is a need in the industry for a portfolio of risk assessment assays, alongside CRS and immunogenicity studies, that enable drug developers to determine the threshold (or minimum) level of TAA on the cell surface that elicits a biological effect to determine the on-target but off-tumor effects of such candidates. Clinical trials are notoriously expensive, and the probability of success in oncology clinical trials is estimated to be as low as 3.4% (https: / / academic.oup.com / biostatistics / article / 20 / 2 / 273 / 4817524). Therefore, it is important for drug developers to be able to accurately assess a drug's ability to distinguish between highly TAA-expressing cancer cells and healthy cells expressing low levels of TAA, ideally at an early preclinical stage to reduce the risk of clinical failure due to off-tumor effects.

[0005] Chemically induced access (CIP) systems use membrane-permeable small-molecule inducers to control dimerization between a protein, typically a transactivator, and a DNA-binding domain when fused to an inducer-binding protein. CIPs can be adapted to bring the DNA-binding protein into proximity with the transactivator and switch on transcription of a protein of interest by positioning the DNA-binding protein's binding motif upstream of the protein's coding sequence. Such CIPs can be used to provide inducible expression of any given protein, including an antigen of interest. Despite the effectiveness of CIPs in inducible protein expression in other fields, to date, such systems have not been used in risk assessment assays for screening novel biologics and ATMPs. Furthermore, using more than one CIP system in tandem to effectively control the expression of more than one protein independently to different levels has not been achieved in this commercial context. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US6218179 [Patent Document 2] JP2005095173A [Patent Document 3] US2006 / 0099709 [Patent Document 4] WO1999 / 048916 [Patent Document 5] U.S. Patent Application Publication No. 2008-0187942 [Patent Document 6] US9060310 [Patent Document 7] US20070036810 [Non-patent literature]

[0007] [Non-Patent Document 1] https: / / academic.oup.com / biostatistics / article / 20 / 2 / 273 / 4817524 [Non-licensed document 2] L. Marignol, M. Lawler, M. Coffey & D. Hollywood (2005) Achieving hypoxia inducible gene expression in tumors, Cancer Biology & Therapy, 4:4, 365~370 [Non-licensed document 3] Madanら, PNAS 90:3928, 1993

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[0008] The present invention is directed to addressing one or more of the above-mentioned problems in the art. [Means for solving the problem]

[0009] In a first aspect of the present invention, a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise a binding domain and an effector domain; the binding domain is operable to bind to an inducer; the effector domain is selected from a transactivation domain or a DNA-binding domain; A first construct is provided in which the binding domain and effector domain of the first and second chimeric proteins are different.

[0010] In an embodiment of the first aspect of the present invention, a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise an auxin-binding domain and an effector domain; the auxin-binding domain is optionally selected from a transport inhibitor response 1 protein (TIR1) or an auxin / indole-3-acetic acid protein (AID); the effector domain is optionally selected from a transactivation domain or a DNA-binding domain selected from a Gal4 DNA-binding domain and a catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI); A first construct is provided in which the auxin binding domain and effector domain of the first and second chimeric proteins are different.

[0011] In a further embodiment of the first aspect of the invention, a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise a caffeine binding domain and an effector domain; the caffeine binding domain is optionally an anti-caffeine heavy chain antibody fragment (αCaffVHH); the effector domain is selected from a transactivation domain or optionally a DNA binding domain selected from a Gal4 DNA binding domain and a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI); A first construct is provided in which the effector domains of the first and second chimeric proteins are different.

[0012] In a further first embodiment of the first aspect of the present invention, a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise a mandipropamide (Mandi) binding domain and an effector domain; The mandipropamide binding domain is optionally linked to a modified pyrobactin receptor (PYR Mandi ), modified pyrabactin-like receptors (PYLcs Mandi ), and Abscisic Acid Insensitive 1 protein (ABI); the effector domain is selected from a transactivation domain or optionally a DNA binding domain selected from a Gal4 DNA binding domain and a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI); A first construct is provided in which the mandipropamide binding domain and the effector domain of the first and second chimeric proteins are different.

[0013] In a further first embodiment of the first aspect of the present invention, a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise a gibberellin-binding domain and an effector domain; Optionally, the gibberellin-binding domain is selected from an optionally modified gibberellin-insensitive dwarf1 protein (GID1) and a gibberellin-insensitive protein (GAI); the effector domain is selected from a transactivation domain or optionally a DNA-binding domain selected from a Gal4 DNA-binding domain and a catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI); A first construct is provided in which the gibberellin binding domain and the effector domain of the first and second chimeric proteins are different.

[0014] In a second aspect of the present invention, there is provided a second construct comprising a nucleic acid sequence encoding one or more effector domain binding sites operably linked to a nucleic acid sequence encoding a protein of interest, wherein each effector domain binding site is a dI-SceI binding site.

[0015] In a further second aspect of the present invention, there is also provided an alternative second construct comprising a nucleic acid sequence encoding one or more effector domain binding sites operably linked to a nucleic acid sequence encoding a protein of interest, wherein each effector domain binding site is a Gal4 upstream activation sequence.

[0016] Preferably, a second construct containing a dI-SceI binding site is used in conjunction with a first construct containing a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI). Preferably, a second construct containing a Gal4 upstream activating sequence is used in conjunction with a first construct containing a Gal4 DNA binding domain.

[0017] In a third aspect of the present invention, there is provided an auxin-inducible access system comprising the first construct of the first aspect and any of the second constructs of the second aspect. In a further aspect, there is provided a caffeine-inducible access system comprising any of the related first constructs defined herein above and any of the second constructs defined herein above. In a further aspect, there is provided a mandipropamid-inducible access system comprising any of the related first constructs defined herein above and any of the second constructs defined herein above. In a further aspect, there is provided a gibberellin-inducible access system comprising any of the related first constructs defined herein above and any of the second constructs defined herein above.

[0018] Preferably, any of these systems may be considered herein as an inductive system, preferably as a first inductive system.

[0019] In a fourth aspect of the invention, there is provided a vector comprising the first and / or second construct of the first or second aspect or embodiment, respectively.

[0020] In a fifth aspect of the present invention, chimeric proteins are provided comprising a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI) fused to an auxin-binding domain, optionally selected from transport inhibitor response 1 protein (TIR1) or auxin / indole-3-acetic acid protein (AID). In an alternative fifth aspect, chimeric proteins are provided comprising a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI) fused to an abscisic acid-binding domain, optionally selected from abscisic acid insensitive 1 protein (ABI1) or pyrabactin resistance-like protein (PYL1). In an alternative fifth aspect, chimeric proteins are provided comprising a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI) fused to a caffeine-binding domain, optionally selected from αCaffVHH. In an alternative fifth aspect, chimeric proteins are provided comprising a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI) fused to a caffeine-binding domain, optionally selected from PYR. Mandi , PYLcs Mandi In an alternative fifth aspect, there is provided a chimeric protein comprising a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI) optionally fused to a gibberellin binding domain selected from GID1 protein and GAI protein, which may be optionally modified.

[0021] In a further aspect of the present invention, a third chimeric protein, and Fourth chimeric protein a third construct comprising a promoter operably linked to a nucleic acid sequence encoding the third and fourth chimeric proteins each comprise an abscisic acid binding domain and an effector domain; the abscisic acid binding domain is optionally selected from ABI1 or pyrabactin resistance-like protein PYL1; the effector domain is optionally selected from a transactivation domain or a DNA binding domain selected from a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI) and a Gal4 DNA binding domain; A third construct is also provided in which the abscisic acid binding domain and effector domain of the third and fourth chimeric proteins are different.

[0022] In a further aspect of the present invention, there is also provided a fourth construct comprising a nucleic acid sequence encoding one or more effector domain binding sites operably linked to a nucleic acid sequence encoding a protein of interest, wherein each effector domain binding site is a Gal4 upstream activation sequence.

[0023] In a further aspect of the invention, there is also provided an alternative fourth construct comprising a nucleic acid sequence encoding one or more effector domain binding sites operably linked to a nucleic acid sequence encoding a protein of interest, wherein each effector domain binding site is a dI-SceI binding site.

[0024] In a further aspect of the present invention, there is also provided an abscisic acid-inducible access system comprising a third construct and a fourth construct.

[0025] Preferably, this system can be considered as an inductive system, preferably as a second inductive system.

[0026] In a further aspect of the invention, there is also provided a vector comprising the third and / or fourth construct.

[0027] In one embodiment, one or more vectors are provided which may comprise one or more of the first, second, third or fourth constructs in any combination.

[0028] Preferably, the present invention relates to any inducible system that can form the first and second inducible systems referred to herein. Below, we provide suitable examples of other inducible systems that can be used in the methods of the present invention. In a preferred embodiment, the inducible system referred to herein is a chemically induced access system, preferably selected from the above-described auxin-inducible access system, caffeine-inducible access system, mandipropamid-inducible access system, gibberellin-inducible access system, and ABA-inducible access system.

[0029] In a sixth aspect of the present invention there is provided a method of producing a cell comprising a first chemical-induced access system and / or a second chemical-induced access system as defined herein, comprising the steps of: (a) introducing a first construct into a cell and / or introducing a third construct into a cell; (b) introducing a second construct into the cell and / or introducing a fourth construct into the cell; (c) integrating the first and second constructs, and / or the third and fourth constructs into the genome of the cell. A method is provided which includes:

[0030] In one embodiment, the chemical-induced access system and the first, second, third and fourth constructs are as defined herein above.

[0031] In a sixth aspect of the present invention there is provided a method of producing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system of the third aspect defined herein, comprising the steps of: (a) introducing a first construct into a cell and / or introducing a third construct into a cell; (b) introducing a second construct into the cell and / or introducing a fourth construct into the cell; (c) integrating the first and second constructs, and / or the third and fourth constructs into the genome of the cell. A method is provided which includes:

[0032] In one embodiment, the method of the sixth aspect therefore comprises a method of producing a cell comprising both an auxin-inducible access system of the third aspect defined herein and an abscisic acid-inducible access system, the method comprising: (a) introducing the first and third constructs into a cell; (b) introducing the second and fourth constructs into the cell; (c) integrating the first, second, third, and fourth constructs into the genome of the cell. The method includes:

[0033] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are their first, second, third and fourth constructs.

[0034] In one embodiment, the first construct and the third construct may be contained on the same construct, which may be known as a guide construct. Thus, step (a) may comprise introducing the guide construct into a cell. In one embodiment, the second construct and the fourth construct may be contained on the same construct, which may be known as a delivery construct. Thus, step (b) may comprise introducing the delivery construct into a cell. Suitably, steps (a) and (b) may be in any order.

[0035] In one embodiment, step (a) may comprise introducing a viral vector comprising the first construct into the cell and / or introducing a viral vector comprising the third construct into the cell. In one embodiment, step (b) may comprise introducing a viral vector comprising the second construct into the cell and / or introducing a viral vector comprising the fourth construct into the cell. In one embodiment, the method is carried out by lentiviral integration, and therefore the viral vector may be a viral particle, preferably a lentiviral particle. In one embodiment, one viral particle may comprise the inducer construct and another viral particle may comprise the delivery construct. In one embodiment, one viral particle may comprise the first, third, second, and fourth constructs.

[0036] In a seventh aspect of the present invention there is provided a method of producing a cell comprising a first chemical-induced access system and / or a second chemical-induced access system as defined herein, comprising the steps of: (a) introducing the first construct and / or the third construct into a cell; (b) creating an integration site in the genome of the cell, the integration site comprising a first recombination site; (c) introducing into the cell an integration construct comprising the second construct and / or the fourth construct and a nucleic acid sequence encoding an integrase enzyme, wherein the second construct and / or the fourth construct further comprises a second recombination site; (d) integrating the second construct and / or the fourth construct into the genome by recombination between the first and second recombination sites using an integrase enzyme. wherein steps (a) and (b) may be performed in any order.

[0037] In a seventh aspect of the present invention there is provided a method of producing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system of the third aspect defined herein, comprising the steps of: (a) introducing the first construct and / or the third construct into a cell; (b) creating an integration site in the genome of the cell, the integration site comprising a first recombination site; (c) introducing into the cell an integration construct comprising the second construct and / or the fourth construct and a nucleic acid sequence encoding an integrase enzyme, wherein the second construct and / or the fourth construct further comprises a second recombination site; (d) integrating the second construct and / or the fourth construct into the genome by recombination between the first and second recombination sites using an integrase enzyme. wherein steps (a) and (b) may be performed in any order.

[0038] In one embodiment the method of the seventh aspect is a method of producing a cell comprising both the auxin-inducible access system and the abscisic acid-inducible access system of the third aspect defined herein, comprising the steps of: (a) introducing the first and third constructs into a cell; (b) creating an integration site in the genome of the cell, the integration site comprising a first recombination site; (c) introducing into the cell an integration construct comprising the second and fourth constructs and a nucleic acid sequence encoding an integrase enzyme, wherein the second and fourth constructs further comprise a second recombination site; (d) integrating the second construct and the fourth construct into the genome by recombination between the first and second recombination sites using an integrase enzyme. wherein steps (a) and (b) may be performed in any order.

[0039] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0040] In one embodiment, the first construct and the third construct may be contained on the same construct, which may be known as an inducer construct. Thus, step (a) may comprise introducing an inducer construct comprising the first and third constructs into a cell. In one embodiment, the second construct and the fourth construct may be contained on the same construct, which may be known as a delivery construct. Thus, step (c) may comprise introducing a delivery construct comprising the second and fourth constructs into a cell, the delivery construct further comprising a second recombination site.

[0041] In an eighth aspect of the present invention, there is provided a cell comprising (a) a first chemical-induced access system, and / or (b) a second chemical-induced access system, a first chemical-induced access system (a), (i) a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise a first inducer binding domain and an effector domain; each first inducer-binding domain operable to bind to a first inducer; the effector domain comprises a transactivation domain and a first DNA-binding domain; A first construct, wherein the effector domains of the first and second chimeric proteins are different; and (ii) a second construct comprising a nucleic acid sequence encoding one or more first DNA-binding domain binding sites operably linked to a nucleic acid sequence encoding a first protein of interest; Includes; a second chemical-induced access system (b), (i) a third chimeric protein, and Fourth chimeric protein a third construct comprising a promoter operably linked to a nucleic acid sequence encoding the third and fourth chimeric proteins each comprise a second inducer-binding domain and an effector domain; each second inducer-binding domain is operable to bind a second inducer; the effector domain comprises a transactivation domain or a second DNA-binding domain; a third construct, wherein the effector domains of the third and fourth chimeric proteins are different; and (ii) a fourth construct comprising a nucleic acid sequence encoding one or more second DNA-binding domain binding sites operably linked to a nucleic acid sequence encoding a second protein of interest. Includes; A cell is provided in which the first chemical-induced access system does not interact with the second chemical-induced access system, and one of the first or second DNA-binding domains is a dI-SceI DNA-binding domain.

[0042] Suitably, the first and second chemically induced access systems may be any of the chemically induced access systems described herein.

[0043] In one embodiment, the first and second chemical-inducible access systems may be the plant hormone or plant hormone analog-inducible access systems described herein. Preferably, in such an embodiment, the first chemical-inducible access system comprises the first and second constructs defined herein, and the second chemical-inducible access system comprises the third and fourth constructs defined herein. In one embodiment, at least one of the chemical-inducible access systems is a plant hormone-inducible access system. In one embodiment, at least one of the chemical-inducible systems is an abscisic acid-inducible access system defined herein. In one embodiment, at least one of the chemical-inducible systems is selected from the caffeine-inducible access system defined herein, the mandipropamid-inducible access system defined herein, and the gibberellin-inducible access system defined herein.

[0044] In a preferred embodiment, the first chemically induced access system is selected from a caffeine-induced access system as defined herein, a mandipropamid-induced access system as defined herein, and a gibberellin-induced access system as defined herein, and the second chemically induced access system is an abscisic acid-induced access system as defined herein.

[0045] In an alternative eighth aspect of the present invention, there is provided a cell comprising (a) a first plant hormone-inducible access system and / or (b) a second plant hormone-inducible access system, A first plant hormone-inducible access system (a) (i) a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise a first plant hormone inducer binding domain and an effector domain; each first plant hormone inducer binding domain is operable to bind to a first plant hormone inducer; the effector domain comprises a transactivation domain and a first DNA binding domain; a first construct, wherein the first plant hormone inducer binding domain and the effector domain of the first and second chimeric proteins are different; and (ii) a second construct comprising a nucleic acid sequence encoding one or more first DNA-binding domain binding sites operably linked to a nucleic acid sequence encoding a first protein of interest; Includes; a second plant hormone-inducible access system (b), (i) a third chimeric protein, and Fourth chimeric protein a third construct comprising a promoter operably linked to a nucleic acid sequence encoding the third and fourth chimeric proteins each comprise a second plant hormone inducer binding domain and an effector domain; each second plant hormone inducer binding domain is operable to bind to a second plant hormone inducer; the effector domain comprises a transactivation domain or a second DNA binding domain; a third construct, wherein the second plant hormone inducer binding domain and the effector domain of the third and fourth chimeric proteins are different; and (ii) a fourth construct comprising a nucleic acid sequence encoding one or more second DNA-binding domain binding sites operably linked to a nucleic acid sequence encoding a second protein of interest. Includes; A cell is provided in which the first plant hormone-inducible system does not interact with the second plant hormone-inducible system, and one of the first or second DNA-binding domains is a dI-SceI DNA-binding domain.

[0046] In one embodiment, the cell may contain only the first chemical-inducible access system, preferably only the first plant hormone-inducible access system. In one embodiment, the cell may contain only the second chemical-inducible access system, preferably only the second plant hormone-inducible access system. In an embodiment in which the cell contains only one system, preferably the DNA-binding domain is a dI-SceI DNA-binding domain. In one embodiment, the cell contains both the first and second chemical-inducible access systems, preferably both plant hormone-inducible access systems.

[0047] In one embodiment, the first chemical-inducible access system, preferably the first plant hormone-inducible system, and the second chemical-inducible access system, preferably the second plant hormone-inducible system, are orthogonal, and therefore, preferably, the first and second chemical-inducible access systems, preferably the first and second plant hormone-inducible systems, operate independently of each other. In one embodiment, the first inducer, preferably the first plant hormone inducer, is different from the second inducer, preferably the second plant hormone inducer, and therefore, the first inducer-binding domain, preferably the first plant hormone inducer-binding domain, is different from the second inducer-binding domain, preferably the second plant hormone inducer-binding domain. In one embodiment, the first DNA-binding domain and the second DNA-binding domain are different from each other. In one embodiment, the first and second proteins of interest are different from each other.

[0048] In one embodiment, one or more effector domain binding sites of the second and fourth constructs comprise a DNA-binding domain binding site. Preferably, one or more effector domain binding sites of the second construct comprise one or more first DNA-binding domain binding sites. Preferably, one or more effector domain binding sites of the fourth construct comprise one or more second DNA-binding domain binding sites.

[0049] Preferably, the effector domain of either the first inducible system or the second inducible system can be selected from any transactivation domain or DNA-binding domain, so long as the effector domains of the first and second chimeric proteins are different and the effector domains of the third and fourth chimeric proteins are different. Preferably, the effector domains of the first and third chimeric proteins can be transactivation domains, and preferably they can both be the same transactivation domain. Preferably, the effector domains of the second and fourth chimeric proteins can be DNA-binding domains, and preferably they are different DNA-binding domains. Preferably, one of the first or second DNA-binding domains is a dI-SceI DNA-binding domain, and preferably the other DNA-binding domain is a different DNA-binding domain. Preferably, one of the first or second DNA-binding domains is a dI-SceI DNA-binding domain, and preferably the other DNA-binding domain is selected from a LexA binding domain or a GAL4 DNA-binding domain, preferably a GAL4 DNA-binding domain. In one embodiment, the first DNA-binding domain is a dI-SceI DNA-binding domain and the second DNA-binding domain is a GAL4 DNA-binding domain.

[0050] In one embodiment, either the first or second chemical-inducible access system is a plant hormone-inducible system, selected from any inducible system in which the inducer is a plant hormone. Suitable plant hormones may be selected from, for example, auxin, abscisic acid, gibberellin, ethene, cytokinin, salicylic acid, jasmonate, brassinosteroid, peptide hormone, and caffeine. Preferably, either the first or second plant hormone-inducible system may be an auxin, abscisic acid, gibberellin, ethene, cytokinin, salicylic acid, jasmonate, brassinosteroid, peptide inducible system, or caffeine inducible system. In another embodiment, either the first or second chemical-inducible access system is a plant hormone analog-inducible system, selected from any inducible system in which the inducer is a plant hormone analog or a synthetic plant hormone. Suitable plant hormone analogs include, for example, mandipropamid. Preferably, the term "plant hormone" used herein encompasses plant hormone analogs.

[0051] In one embodiment, either the first or second plant hormone-inducible system is, in a mutually exclusive manner, an auxin-inducible access system as defined herein or an abscisic acid-inducible access system as defined herein. Preferably, the first plant hormone-inducible access system is an auxin-inducible access system as defined herein and the second plant hormone-inducible access system is an abscisic acid-inducible access system as defined herein, or vice versa. Thus, preferably, the first plant hormone inducer is auxin, the second plant hormone inducer is abscisic acid, the first plant hormone inducer-binding domain is an auxin inducer-binding domain, and the second plant hormone inducer-binding domain is an abscisic acid-binding domain.

[0052] In another embodiment, either the first or second chemical-inducible access system is a plant hormone or plant hormone analogue-inducible access system. In such an embodiment, either the first or second system is selected, in a mutually exclusive manner, from a caffeine-inducible access system, a mandipropamid-inducible access system, a gibberellin-inducible access system and an abscisic acid-inducible access system as defined herein. Preferably, in some embodiments, the first plant hormone or plant hormone analogue-inducible access system is a caffeine-inducible access system as defined herein, and the second plant hormone-inducible access system is an abscisic acid-inducible access system as defined herein, or vice versa. Preferably, in some embodiments, the first plant hormone or plant hormone analogue-inducible access system is a mandipropamid-inducible access system as defined herein, and the second plant hormone-inducible access system is an abscisic acid-inducible access system as defined herein, or vice versa. Suitably, in some embodiments, the first plant hormone or plant hormone analogue inducible access system is a gibberellin inducible access system as defined herein and the second plant hormone inducible access system is an abscisic acid inducible access system as defined herein, or vice versa.

[0053] Preferably, the or each auxin-binding domain is selected from the transport inhibitor response 1 protein (TIR1) described elsewhere herein, or the auxin / indole-3-acetic acid protein (AID) described elsewhere herein. Thus, preferably, one auxin-binding domain is TIR1, and preferably the first auxin-binding domain is TIR1 or a fragment or derivative thereof. Preferably, one auxin-binding domain is AID, and preferably the second auxin-binding domain is AID, or a fragment or derivative thereof.

[0054] Preferably, the or each caffeine binding domain is an anti-caffeine heavy chain antibody fragment (αCaffVHH) as described elsewhere herein. Thus, preferably, both the first and second caffeine binding domains are anti-caffeine heavy chain antibody fragments (αCaffVHH), or fragments or derivatives thereof.

[0055] Suitably, the or each mandipropamide binding domain is a modified pyrabactin receptor (PYR) as described elsewhere herein. Mandi ), modified pyrabactin-like receptors (PYLcs Mandi ), and Abscisic Acid Insensitive 1 protein (ABI). Preferably, one mandipropamid binding domain is ABI, and preferably, the first mandipropamid binding domain is ABI or a fragment or derivative thereof. Preferably, one mandipropamid binding domain is selected from a modified pyrabactin receptor (PYR Mandi ), or engineered pyrabactin-like receptors (PYLcs Mandi ), and preferably the second mandipropamide binding domain is a modified pyrabactin receptor (PYR Mandi ) or engineered pyrabactin-like receptors (PYLcs Mandi ) or a fragment or derivative thereof.

[0056] Preferably, the or each gibberellin-binding domain is selected from a gibberellin-insensitive dwarf1 (GID1) protein and a gibberellin-insensitive (GAI) protein, and optionally a modified GAI protein. Preferably, one gibberellin-binding domain is a gibberellin-insensitive dwarf1 protein (GID1), and preferably, a first gibberellin-binding domain is a gibberellin-insensitive dwarf1 protein (GID1) or a fragment or derivative thereof. Preferably, one gibberellin-binding domain is a gibberellin-insensitive (GAI) protein or a modified GAI protein, and preferably, a second gibberellin-binding domain is a gibberellin-insensitive (GAI) protein or a modified GAI protein, or a fragment or derivative thereof.

[0057] Preferably, the or each abscisic acid-binding domain is selected from the abscisic acid insensitive 1 protein (ABI1) described elsewhere herein, or the pyrabactin resistance-like protein (PYL1) described elsewhere herein. Preferably, one abscisic acid-binding domain is ABI1, and preferably the first abscisic acid-binding domain is ABI1 or a fragment or derivative thereof. Preferably, one abscisic acid-binding domain is PYL1, and preferably the second abscisic acid-binding domain is PYL1 or a fragment or derivative thereof.

[0058] Suitably, the or each transactivation domain is selected from Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4, and p53, NFAT, NF-κB, VP16 or VP34 in yeast, preferably VP16. Suitably, either the first or second DNA-binding domain may be, in a mutually exclusive manner, a dI-SceI DNA-binding domain or a GAL4 DNA-binding domain.

[0059] In one embodiment, a first chimeric protein comprises a VP16 transactivation domain and a TIR1 protein or a fragment or derivative thereof, and a second chimeric protein comprises a dI-SceI DNA binding domain and an AID protein or a fragment or derivative thereof, or vice versa. In one embodiment, an AIDΔ34 protein.

[0060] In one embodiment, the first chimeric protein comprises a VP16 transactivation domain and an αCaffVHH protein or a fragment or derivative thereof, and the second chimeric protein comprises a GAL4 DNA binding domain or a dI-SceI DNA binding domain and an αCaffVHH protein or a fragment or derivative thereof, or vice versa.

[0061] In one embodiment, a first chimeric protein comprises a VP16 transactivation domain and a PYR1 or PYL1 protein, or a fragment or derivative thereof, and a second chimeric protein comprises a GAL4 DNA binding domain or a dI-SceI DNA binding domain and an ABI1 protein, or a fragment or derivative thereof, or vice versa. Mandi Proteins or PYLcs Mandi Protein. In one embodiment, an ABIcs protein.

[0062] In one embodiment, a first chimeric protein comprises a VP16 transactivation domain and a GID1 protein or a fragment or derivative thereof, and a second chimeric protein comprises a GAL4 DNA binding domain or a dI-SceI DNA binding domain and a GAI protein or a fragment or derivative thereof, or vice versa. In one embodiment, the modified GAI protein.

[0063] In one embodiment, the second construct comprises 1 to 15 dI-SceI DNA binding sites, preferably 10 dI-SceI DNA binding sites, preferably in tandem, or 1 to 15 GAL4 upstream activating sequences, preferably 9 GAL4 upstream activating sequences, preferably in tandem.

[0064] In one embodiment, the third chimeric protein comprises a VP16 transactivation domain and a PYL1 protein or a fragment or derivative thereof, and the fourth chimeric protein comprises a GAL4 DNA binding domain or a dI-SceI DNA binding domain and an ABI1 protein or a fragment or derivative thereof, or vice versa. In one embodiment, a PYLcs protein. In one embodiment, an ABIcs protein.

[0065] In one embodiment, the fourth construct comprises 1 to 15 GAL4 upstream activating sequences, preferably 9 GAL4 upstream activating sequences, preferably in tandem, or 1 to 15 dI-SceI DNA binding sites, preferably 10 dI-SceI DNA binding sites, preferably in tandem.

[0066] Preferably, when a GAL4 DNA-binding domain is present in the first or third chimeric protein, the second or fourth construct must each contain 1 to 15 GAL4 upstream activating sequences, preferably 9 GAL4 upstream activating sequences, preferably in tandem. Preferably, when a dI-SceI DNA-binding domain is present in the first or third chimeric protein, the second or fourth construct must each contain 1 to 15 dI-SceI DNA-binding sites, preferably 10 dI-SceI DNA-binding sites, preferably in tandem.

[0067] It will be appreciated that, preferably, the components of the first chimeric protein and the third chimeric protein may be reversed. It will be appreciated that, preferably, the components of the second chimeric protein and the fourth chimeric protein may be reversed. It will be appreciated that, therefore, preferably, the components of the second construct and the fourth construct may also be reversed.

[0068] It is contemplated that other suitable chemically inducible access systems, and other plant hormone or plant hormone analogue inducible systems, can be used as the first and / or second plant hormone inducible access systems herein and, in combination, can be used to arrive at cells comprising a dual chemically inducible access system of the present invention that can be used to control the expression of two proteins of interest.

[0069] Suitably, any reference in any aspect or embodiment herein to an "auxin-inducible access system" may be replaced with a first chemical-inducible access system, or suitably a first plant hormone or plant hormone analogue-inducible access system, and any reference to an "abscisic acid-inducible access system" may be replaced with a second chemical-inducible access system, or suitably a second plant hormone or plant hormone analogue-inducible access system, and the corresponding components of each system as defined in the eighth aspect.

[0070] In one embodiment of the eighth aspect, there is provided a cell comprising the auxin-inducible access system and / or the abscisic acid-inducible access system of the third aspect, Auxin-induced access system (a) (i) a first chimeric protein, and Second chimeric protein a first construct comprising a promoter operably linked to a nucleic acid sequence encoding the first and second chimeric proteins each comprise an auxin-binding domain and an effector domain; the auxin-binding domain is selected from a transport inhibitor response 1 protein (TIR1) or an auxin / indole-3-acetic acid protein (AID); the effector domain is selected from a transactivation domain or a catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI); A first construct, wherein the auxin binding domain and the effector domain of the first and second chimeric proteins are different; and (ii) a second construct comprising a nucleic acid sequence encoding one or more effector domain binding sites operably linked to a nucleic acid sequence encoding the first protein of interest. wherein each effector domain binding site is a dI-SceI binding site; Abscisic acid-induced access system (b) (i) a third chimeric protein, and Fourth chimeric protein a third construct comprising a promoter operably linked to a nucleic acid sequence encoding the third and fourth chimeric proteins each comprise an abscisic acid binding domain and an effector domain; the abscisic acid binding domain is selected from abscisic acid insensitive 1 protein (ABI1) or pyrabactin resistance-like protein (PYL1); the effector domain is selected from a transactivation domain or a Gal4 DNA binding domain; The abscisic acid binding domain and the effector domain of the third and fourth chimeric proteins are different; and (ii) a fourth construct comprising a nucleic acid sequence encoding one or more effector domain binding sites operably linked to a nucleic acid sequence encoding a second protein of interest. wherein each effector domain binding site is a Gal4 upstream activation sequence.

[0071] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above.

[0072] In one embodiment, the cell may comprise only an auxin-inducible access system. In one embodiment, the cell may comprise only an abscisic acid-inducible access system. In one embodiment, the cell comprises both an auxin-inducible access system and an abscisic acid-inducible access system. In one embodiment, the cell is an inducible cell and can give rise to an inducible cell line comprising the auxin-inducible access system of the third aspect described herein and / or the abscisic acid-inducible access system.

[0073] Preferably, the chemically inducible access systems and cells containing the systems described herein can be used in a variety of methods. However, it will be understood that the methods may also utilize other inducible systems, not necessarily CIP systems. Suitable other inducible systems are described herein.

[0074] Suitably, the inducible system can be used in a variety of ways to control the expression of one or more proteins of interest in cells. Suitably, such methods are useful for screening candidate biomolecules, therapeutic agents, and / or engineered immune cells. Suitably, such methods are useful for screening candidate biomolecules, therapeutic agents, and / or engineered immune cells for a biological effect, preferably a biological effect on cells expressing the or each protein.

[0075] In a ninth aspect of the present invention there is provided a method of controlling expression of a first protein of interest and optionally a second protein of interest in a cell, comprising the steps of: (a) providing a cell comprising a first inducible system operable to express a first protein of interest, and optionally a second inducible system operable to express a second protein of interest; (b) exposing the cells to an effective concentration of a first inducer to induce a desired level of expression of a first protein of interest from a first inducible system, and optionally exposing the cells to an effective concentration of a second inducer to induce a desired level of expression of a second protein of interest from a second inducible system. A method is provided which includes:

[0076] Preferably, the first and second inducible systems are different. In a preferred embodiment, the cell comprises both the first and second inducible systems. Preferably, there is substantially no crosstalk between the first and second inducible systems.

[0077] Optionally, the method may include step (b) of culturing the cells under conditions in which the necessary components of the first and / or second inducible systems are expressed.

[0078] In some embodiments, the first and second inducible systems may be any inducible system, preferably any inducible system described herein. In one embodiment, the first and second inducible systems may be chemically induced access systems (CIP systems). In a preferred embodiment, they may be the plant hormone or plant hormone analogue inducible access systems described herein. In some embodiments, the first inducible system may be the first plant hormone inducible access system of the eighth aspect. In some embodiments, the second inducible system may be the second plant hormone inducible access system of the eighth aspect. Preferably, in such embodiments, the first inducible system comprises the first and second constructs defined herein, and the second inducible system comprises the third and fourth constructs defined herein. In one embodiment, at least one of the inducible systems is a plant hormone inducible access system. In one embodiment, at least one of the inducible systems is an abscisic acid inducible access system defined herein. In one embodiment, at least one of the inducible systems is selected from a caffeine-inducible access system as defined herein, a mandipropamid-inducible access system as defined herein, and a gibberellin-inducible access system as defined herein.

[0079] In a preferred embodiment, the first inducible system is selected from a caffeine-inducible access system as defined herein, a mandipropamid-inducible access system as defined herein, and a gibberellin-inducible access system as defined herein, and the second inducible system is an abscisic acid-inducible access system as defined herein.

[0080] In an embodiment of the ninth aspect of the present invention, there is provided a method for controlling expression of a protein of interest in a cell, comprising the steps of: (a) providing a cell comprising the first plant hormone-inducible access system and / or the second plant hormone-inducible access system of the eighth aspect; (b) culturing the cells under conditions in which the first construct and / or the third construct is expressed; (c) exposing the cells to an effective concentration of a first plant hormone inducer to induce a desired level of expression of a first protein of interest from the second construct, and / or exposing the cells to an effective concentration of a second plant hormone inducer to induce a desired level of expression of a second protein of interest from the fourth construct. A method is provided which includes:

[0081] In one embodiment of the ninth aspect, there is provided a method of controlling expression of a protein of interest in a cell, comprising: (a) providing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system as defined herein; (b) culturing the cells under conditions in which the first construct and / or the third construct is expressed; (c) exposing the cells to an effective concentration of auxin to induce the desired level of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of abscisic acid to induce the desired level of expression of the second protein of interest from the fourth construct. A method is provided which includes:

[0082] In one embodiment, the method of the ninth aspect is a method of controlling expression of a first and a second protein of interest in a cell, comprising: (a) providing a cell comprising the auxin-inducible access system and the abscisic acid-inducible access system described herein; (b) culturing the cells under conditions in which the first construct and the third construct are expressed; (c) exposing the cells to an effective concentration of auxin to induce the desired level of expression of the first protein of interest from the second construct, and exposing the cells to an effective concentration of abscisic acid to induce the desired level of expression of the second protein of interest from the fourth construct. The method includes:

[0083] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0084] In one embodiment, step (c) may comprise exposing the cells to a plurality of different concentrations of a first inducer, preferably a plant hormone inducer, preferably an auxin, to induce a plurality of different levels of expression of a first protein of interest, which may be expressible from a second construct, and / or exposing the cells to a plurality of different concentrations of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, to induce a desired level of expression of a second protein of interest, which may be expressible from a fourth construct.

[0085] In one embodiment, step (c) may comprise exposing the cells to an effective concentration of a first inducer, preferably a plant hormone inducer, preferably an auxin, and an effective concentration of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, simultaneously or at different times, as further described below.

[0086] In a tenth aspect of the invention there is provided a method of screening candidate binding molecules for a biological effect comprising the steps of: (a) providing a cell comprising a first inducible system operable to express a first protein of interest, and optionally a second inducible system operable to express a second protein of interest; (b) exposing the cells to an effective concentration of a first inducer to induce a desired level of expression of a first protein of interest, and optionally exposing the cells to an effective concentration of a second inducer to induce a desired level of expression of a second protein of interest; (c) contacting the cells with a candidate binding molecule; (d) determining whether the candidate binding molecule exerts a biological effect on cells expressing the first protein of interest and, optionally, the second protein of interest; A method is provided which includes:

[0087] Any preferred or optional features defined above in relation to the ninth aspect apply equally to the tenth aspect.

[0088] Preferably, the term "exercise" as used herein can be used synonymously with "perform", "initiate" or "cause" in reference to a biological effect.

[0089] In one embodiment of the tenth aspect of the invention there is provided a method of screening candidate binding molecules for a biological effect comprising the steps of: (a) providing a cell comprising the first plant hormone-inducible access system and / or the second plant hormone-inducible access system of the eighth aspect; (b) culturing the cells under conditions in which the first construct and / or the third construct is expressed; (c) exposing the cells to an effective concentration of a first plant hormone inducer to induce a desired level of expression of a first protein of interest from the second construct, and / or exposing the cells to an effective concentration of a second plant hormone inducer to induce a desired level of expression of a second protein of interest from the fourth construct; (d) contacting the cells with a candidate binding molecule; (e) determining whether the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0090] In one embodiment of the tenth aspect, there is provided a method of screening candidate binding molecules for a biological effect, comprising: (a) providing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system as defined herein; (b) culturing the cells under conditions in which the first construct and / or the third construct is expressed; (c) exposing the cells to an effective concentration of auxin to induce the desired level of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of abscisic acid to induce the desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with a candidate binding molecule; (e) determining whether the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0091] In one embodiment of the tenth aspect, the method comprises screening candidate binding molecules for a biological effect, comprising: (a) providing a cell comprising an auxin-inducible access system and an abscisic acid-inducible access system as defined herein; (b) culturing the cells under conditions in which the first construct and the third construct are expressed; (c) exposing the cells to an effective concentration of auxin to induce a desired level of expression of a first protein of interest from the second construct, and exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of a second protein of interest from the fourth construct; (d) contacting the cells with a candidate binding molecule; (e) determining whether the candidate binding molecule exerts a biological effect on cells expressing the first and second proteins of interest; The method includes:

[0092] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0093] In one embodiment, the biological effect comprises binding to a first and / or second protein of interest, hi one embodiment, step (d) may comprise determining whether the candidate binding molecule binds to both the first and second proteins of interest.

[0094] In one embodiment, step (c) may comprise exposing the cells to a plurality of different concentrations of a first inducer, preferably a plant hormone inducer, preferably an auxin, to induce a plurality of different levels of expression of a first protein of interest, which may be expressible from a second construct, and / or exposing the cells to a plurality of different concentrations of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, to induce a desired level of expression of a second protein of interest, which may be expressible from a fourth construct.

[0095] In one embodiment, step (c) may comprise exposing the cells to an effective concentration of a first inducer, preferably a plant hormone inducer, preferably an auxin, and an effective concentration of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, simultaneously or at different times, as further described below.

[0096] In one embodiment, step (d) may comprise determining the minimum level of expression of the first and / or second proteins of interest at which the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest.

[0097] Thus, in a further embodiment, there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which a candidate binding molecule exerts a biological effect, comprising the steps of: (a) providing a cell comprising a first inducible system operable to express a first protein of interest, and optionally a second inducible system operable to express a second protein of interest; (b) exposing the cells comprising the first inducible system, and optionally the second inducible system, to a plurality of different concentrations of a first inducer to induce a plurality of different levels of expression of the first protein of interest, and optionally exposing the cells to a plurality of different concentrations of a second inducer to induce a plurality of different levels of expression of the second protein of interest; (c) contacting the cells with a candidate binding molecule; (d) determining whether the candidate binding molecule exerts a biological effect on cells expressing the first protein of interest and optionally the second protein of interest at each level of expression of the first protein of interest and optionally the second protein of interest; and (e) determining the minimum level of expression of the first protein of interest and, optionally, the second protein of interest, at which the candidate binding molecule exerts a biological effect on cells expressing the first protein of interest and, optionally, the second protein of interest; A method is provided which includes:

[0098] In one embodiment of such aspect, there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which a candidate binding molecule exerts a biological effect, comprising: (a) providing a cell comprising the first plant hormone-inducible access system and / or the second plant hormone-inducible access system of the eighth aspect; (b) culturing cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system to a plurality of different concentrations of the first plant hormone inducer to induce a plurality of different levels of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of the second plant hormone inducer to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with a candidate binding molecule; (e) determining whether, at each level of expression of the first and / or second proteins of interest, the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest; and (f) determining the minimum level of expression of the first and / or second proteins of interest at which the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0099] In one embodiment of a further aspect, there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which a candidate binding molecule exerts a biological effect, comprising: (a) providing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system as defined herein; (b) culturing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system to a plurality of different concentrations of auxin to induce a plurality of different levels of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with a candidate binding molecule; (e) determining whether, at each level of expression of the first and / or second proteins of interest, the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest; and (f) determining the minimum level of expression of the first and / or second proteins of interest at which the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0100] In one embodiment of a further aspect, the method comprises determining the minimum levels of expression of a first and a second protein of interest in a cell at which a candidate binding molecule exerts a biological effect, the method comprising: (a) providing a cell comprising an auxin-inducible access system and an abscisic acid-inducible access system as defined herein; (b) culturing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system to a plurality of different concentrations of auxin to induce a plurality of different levels of expression of the first protein of interest from the second construct, and exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with a candidate binding molecule; (e) determining whether the candidate binding molecule exerts a biological effect on cells expressing the first and second proteins of interest at each level of expression of the first and second proteins of interest; and (f) determining the minimum level of expression of the first and / or second proteins of interest at which the candidate binding molecule exerts a biological effect on cells expressing the first and second proteins of interest; The method includes:

[0101] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0102] In one embodiment, step (b) or (c) may optionally comprise exposing the cells to a concentration of auxin and a concentration of abscisic acid, either simultaneously or at different times, as further described below.

[0103] In one embodiment, the biological effect comprises binding to the first and / or second proteins of interest. In one embodiment, step (d) or (e), as appropriate, may comprise determining whether the candidate binding molecule binds to both the first and / or second proteins of interest at each level of expression of the first and / or second proteins of interest.

[0104] In one embodiment, step (d) or (e), as appropriate, may include determining whether the candidate binding molecule exerts a biological effect on cells expressing the first and second proteins of interest at each level of expression of both the first and second proteins of interest.

[0105] In one embodiment, step (e) or (f), as appropriate, may include determining the minimum level of expression of both the first and second proteins of interest at which the candidate binding molecule exerts a biological effect on cells expressing both the first and second proteins of interest.

[0106] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest may be the level of expression at which a biological effect above background is achieved. Preferably, a biological effect at least 3 standard deviations above background is achieved, preferably a biological effect at least 4 standard deviations above background is achieved, preferably a biological effect at least 5 standard deviations above background is achieved, preferably a biological effect at least 6 standard deviations above background is achieved, preferably a biological effect at least 7 standard deviations above background is achieved, preferably a biological effect at least 8 standard deviations above background is achieved, preferably a biological effect at least 9 standard deviations above background is achieved, preferably a biological effect at least 10 standard deviations above background is achieved. Preferably, the background biological effect is the biological effect of the candidate binding molecule on control cells. Preferably, the control cell is a cell that does not contain the inducible system described herein.Preferably, the control cell does not express the or each protein of interest.Preferably, the control cell is a wild-type cell.Alternatively, the control cell may be a cell that is modified to prevent the expression of the or each protein of interest, preferably by "knocking out" the gene that encodes the or each protein of interest, and this modification can be achieved by known modification techniques such as RNA interference, RNA silencing, CRISPRi, zinc finger nuclease, TALEN, etc.

[0107] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which a candidate binding molecule exerts a biological effect on cells expressing the first and / or second proteins of interest may be the activation threshold for the first and / or second proteins of interest, which may be determined suitably using receiver operating characteristic (ROC) curve analysis, suitably using Youden's index or Youden's J statistic.

[0108] In one embodiment, the candidate binding molecule is selected from a fusion protein, an antibody (e.g., a monoclonal antibody, an antibody drug conjugate, a nanobody, a scFv, a di-scFv, a Fab, a sdAb, a F(ab)2, a glycoengineered antibody) or a binding fragment thereof, a fusion protein, an antibody-drug conjugate, an aptamer, ankyrin, a designed ankyrin repeat protein (DARPin), a peptide, a bicyclic peptide, a vaccine, a cytokine, a chemokine, a hormone, an oncolytic virus, and a bacterium; preferably, the binding molecule is an immunotherapy.

[0109] In an eleventh aspect of the invention, there is provided a method of screening candidate therapeutic agents for a biological effect, comprising the steps of: (a) providing cells comprising a first inducible system operable to express a first protein of interest, and optionally a second inducible system operable to express a second protein of interest, and immune cells; (b) exposing the cells comprising the first inducible system, and optionally the second inducible system, to an effective concentration of a first inducer to induce a desired level of expression of the first protein of interest, and optionally exposing the cells to an effective concentration of a second inducer to induce a desired level of expression of the second protein of interest; (c) contacting the immune cells with a candidate therapeutic agent; and (d) determining whether the contacted immune cells exert a biological effect on cells expressing the first protein of interest and, optionally, the second protein of interest. A method is provided which includes:

[0110] Any preferred or optional features defined above in relation to the ninth aspect apply equally to the eleventh aspect.

[0111] Optionally, the method may further comprise the step of contacting or exposing cells expressing the first protein of interest and optionally the second protein of interest to the contacted immune cells, preferably prior to step (d).

[0112] In one embodiment of the eleventh aspect of the invention there is provided a method of screening candidate therapeutic agents for a biological effect, comprising the steps of: (a) providing a cell comprising the first plant hormone-inducible access system and / or the second plant hormone-inducible access system of the eighth aspect, and an immune cell; (b) culturing cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system under conditions in which the first construct and / or the third construct are expressed; (c) exposing the cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system to an effective concentration of a first plant hormone inducer to induce a desired level of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of a second plant hormone inducer to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the immune cells with a candidate therapeutic agent; (e) determining whether the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0113] In one embodiment of the eleventh aspect, there is provided a method of screening candidate therapeutic agents for a biological effect, comprising: (a) providing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system as defined herein, and an immune cell; (b) culturing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system under conditions in which the first construct and / or the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system to an effective concentration of auxin to induce a desired level of expression of a first protein of interest from the second construct, and / or exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of a second protein of interest from the fourth construct; (d) contacting the immune cells with a candidate therapeutic agent; (e) determining whether the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0114] In one embodiment of the eleventh aspect of the invention, the method comprises screening a candidate therapeutic agent for a biological effect, the method comprising: (a) providing a cell comprising an auxin-inducible access system and an abscisic acid-inducible access system as defined herein, and an immune cell; (b) culturing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system to an effective concentration of auxin to induce a desired level of expression of a first protein of interest from the second construct, and exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of a second protein of interest from the fourth construct; (d) contacting the immune cells with a candidate therapeutic agent; (e) determining whether the contacted immune cells exert a biological effect on cells expressing the first and second proteins of interest; The method includes:

[0115] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0116] In one embodiment, step (b) or (c) may optionally comprise exposing the cells to a plurality of different concentrations of a first inducer, preferably a plant hormone inducer, preferably an auxin, to induce a plurality of different levels of expression of a first protein of interest, which may be expressible from a second construct, and / or exposing the cells to a plurality of different concentrations of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, to induce a desired level of expression of a second protein of interest, which may be expressible from a fourth construct.

[0117] In one embodiment, step (b) or (c) may optionally comprise exposing the cells to an effective concentration of a first inducer, preferably a plant hormone inducer, preferably an auxin, and an effective concentration of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, simultaneously or at different times, as further described below.

[0118] In one embodiment, step (d) or (e) may optionally include determining whether the contacted immune cells exert a biological effect on cells expressing both the first and second proteins of interest. In one embodiment, step (d) or (e) may optionally include determining whether the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest at each level of expression of the first and / or second proteins of interest.

[0119] In one embodiment, the method optionally further comprises step (e) or (f) of determining the minimum expression level of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect in the presence of the therapeutic agent.

[0120] In one embodiment, step (e) or (f) optionally includes determining the minimum expression levels of both the first and second proteins of interest at which the contacted immune cells exert a biological effect in the presence of the therapeutic agent.

[0121] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest may be the level of expression at which a biological effect greater than the background biological effect is achieved. Preferably, a biological effect at least 3 standard deviations above the background biological effect is achieved, preferably a biological effect at least 4 standard deviations above the background biological effect is achieved, preferably a biological effect at least 5 standard deviations above the background biological effect is achieved, preferably a biological effect at least 6 standard deviations above the background biological effect is achieved, preferably a biological effect at least 7 standard deviations above the background biological effect is achieved, preferably a biological effect at least 8 standard deviations above the background biological effect is achieved, preferably a biological effect at least 9 standard deviations above the background biological effect is achieved, preferably a biological effect at least 10 standard deviations above the background biological effect is achieved. Preferably, the background biological effect is the biological effect of the candidate binding molecule on control cells. Preferably, the control cell is a cell that does not contain the inducible system described herein.Preferably, the control cell does not express the or each protein of interest.Preferably, the control cell is a wild-type cell.Alternatively, the control cell may be a cell that is modified to prevent the expression of the or each protein of interest, preferably by "knocking out" the gene that encodes the or each protein of interest, and this modification can be achieved by known modification techniques such as RNA interference, RNA silencing, CRISPRi, zinc finger nuclease, TALEN, etc.

[0122] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest may be the activation threshold for the first and / or second proteins of interest, which may be determined preferably using receiver operating characteristic (ROC) curve analysis, preferably using Youden's index or Youden's J statistic.

[0123] In one embodiment, the candidate therapeutic agent is a biologic, preferably the candidate therapeutic agent is an immunotherapeutic agent, preferably selected from a fusion protein, an antibody (e.g., a monoclonal antibody, an antibody-drug conjugate, a nanobody, an scFv, a di-scFv, a Fab, an sdAb, an F(ab)2, a glycoengineered antibody) or a binding fragment thereof, a fusion protein, an antibody-drug conjugate, an aptamer, ankyrin, a designed ankyrin repeat protein (DARPin), a peptide, a bicyclic peptide, a vaccine, a cytokine, a chemokine, a hormone, an oncolytic virus, and a bacterium.

[0124] In one embodiment, the immune cells are selected from T cells, NK cells, B cells, lymphocytes, dendritic cells, and mesenchymal cells, or immortalized cells thereof, or immortalized cells thereof.

[0125] In a twelfth aspect of the present invention, there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which the immune cell exerts a biological effect in the presence of a candidate therapeutic agent, comprising the steps of: (a) providing cells comprising a first inducible system operable to express a first protein of interest, and optionally a second inducible system operable to express a second protein of interest, and immune cells; (b) exposing the cells comprising the first inducible system and optionally the second inducible system to a plurality of different concentrations of a first inducer to induce a plurality of different levels of expression of a first protein of interest, and optionally exposing the cells to a plurality of different concentrations of a second inducer to induce a plurality of different levels of expression of a second protein of interest; (c) contacting the immune cells with a candidate therapeutic agent; (d) determining whether, at each level of expression of the first protein of interest and optionally the second protein of interest, the contacted immune cells exert a biological effect on cells expressing the first protein of interest and optionally the second protein of interest; (e) determining the minimum level of expression of the first protein of interest and, optionally, the second protein of interest, at which the contacted immune cells exert a biological effect on cells expressing the first protein of interest and, optionally, the second protein of interest; A method is provided which includes:

[0126] Any preferred or optional features defined above in relation to the ninth aspect apply equally to the twelfth aspect.

[0127] Optionally, the method may further comprise the step of contacting or exposing cells expressing the first protein of interest and optionally the second protein of interest to the contacted immune cells, preferably prior to step (d).

[0128] In an embodiment of the twelfth aspect of the present invention there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which the immune cell exerts a biological effect in the presence of a candidate therapeutic agent, comprising the steps of: (a) providing a cell comprising the first plant hormone-inducible access system and / or the second plant hormone-inducible access system of the eighth aspect, and an immune cell; (b) culturing cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system under conditions in which the first construct and / or the third construct are expressed; (c) exposing the cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system to a plurality of different concentrations of the first plant hormone inducer to induce a plurality of different levels of expression of the first protein of interest from the second construct, and / or exposing the cells to a plurality of different concentrations of the second plant hormone inducer to induce a plurality of different levels of expression of the second protein of interest from the fourth construct; (d) contacting the immune cells with a candidate therapeutic agent; (e) determining whether, at each level of expression of the first and / or second proteins of interest, the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; (f) determining the minimum level of expression of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0129] In one embodiment of the twelfth aspect, there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which the immune cell exerts a biological effect in the presence of a candidate therapeutic agent, comprising: (a) providing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system as defined herein, and an immune cell; (b) culturing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system under conditions in which the first construct and / or the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system to a plurality of different concentrations of auxin to induce a plurality of different levels of expression of the first protein of interest from the second construct, and / or exposing the cells to a plurality of different concentrations of abscisic acid to induce a plurality of different levels of expression of the second protein of interest from the fourth construct; (d) contacting the immune cells with a candidate therapeutic agent; (e) determining whether, at each level of expression of the first and / or second proteins of interest, the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; (f) determining the minimum level of expression of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0130] In one embodiment of the twelfth aspect, the method comprises determining the minimum levels of expression of a first and a second protein of interest in a cell at which the immune cell exerts a biological effect in the presence of a candidate therapeutic agent, the method comprising: (a) providing a cell comprising an auxin-inducible access system and an abscisic acid-inducible access system as defined herein, and an immune cell; (b) culturing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system to a plurality of different concentrations of auxin to induce a plurality of different levels of expression of a first protein of interest from the second construct, and exposing the cells to a plurality of different concentrations of abscisic acid to induce a plurality of different levels of expression of a second protein of interest from the fourth construct; (d) contacting the immune cells with a candidate therapeutic agent; (e) determining whether, at each level of expression of the first and / or second proteins of interest, the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; (f) determining the minimum level of expression of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and second proteins of interest; The method includes:

[0131] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0132] In one embodiment, step (b) or (c) may optionally comprise exposing the cells to a predetermined concentration of a first inducer, preferably a plant hormone inducer, preferably an auxin, and a predetermined concentration of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, simultaneously or at different times, as further described below.

[0133] In one embodiment, step (d) or (e) may optionally include determining whether the contacted immune cells exert a biological effect on cells expressing the first and second proteins of interest at each level of expression of both the first and second proteins of interest.

[0134] In one embodiment, step (e) or (f) may optionally include determining the minimum level of expression of both the first and second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and second proteins of interest.

[0135] In one embodiment, the step of determining a minimum level of expression of the first and / or second protein of interest in the cell comprises determining a threshold level of expression of the first and / or second protein of interest in the cell.

[0136] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest may be the level of expression at which a biological effect greater than the background biological effect is achieved. Preferably, a biological effect at least 3 standard deviations above the background biological effect is achieved, preferably a biological effect at least 4 standard deviations above the background biological effect is achieved, preferably a biological effect at least 5 standard deviations above the background biological effect is achieved, preferably a biological effect at least 6 standard deviations above the background biological effect is achieved, preferably a biological effect at least 7 standard deviations above the background biological effect is achieved, preferably a biological effect at least 8 standard deviations above the background biological effect is achieved, preferably a biological effect at least 9 standard deviations above the background biological effect is achieved, preferably a biological effect at least 10 standard deviations above the background biological effect is achieved. Preferably, the background biological effect is the biological effect of the candidate binding molecule on control cells. Preferably, the control cell is a cell that does not contain the inducible system described herein.Preferably, the control cell does not express the or each protein of interest.Preferably, the control cell is a wild-type cell.Alternatively, the control cell may be a cell that is modified to prevent the expression of the or each protein of interest, preferably by "knocking out" the gene that encodes the or each protein of interest, and this modification can be achieved by known modification techniques such as RNA interference, RNA silencing, CRISPRi, zinc finger nuclease, TALEN, etc.

[0137] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the contacted immune cells exert a biological effect on cells expressing the first and / or second proteins of interest may be the activation threshold for the first and / or second proteins of interest, which may be determined preferably using receiver operating characteristic (ROC) curve analysis, preferably using Youden's index or Youden's J statistic.

[0138] In one embodiment, the candidate therapeutic agent is a biologic, preferably the candidate therapeutic agent is an immunotherapeutic agent, preferably selected from a fusion protein, an antibody (e.g., a monoclonal antibody, an antibody drug conjugate, a nanobody, a scFv, a di-scFv, a Fab, a sdAb, a F(ab)2, a glycoengineered antibody) or a binding fragment thereof, a fusion protein, an antibody-drug conjugate, an aptamer, ankyrin, a designed ankyrin repeat protein (DARPin), a peptide, a bicyclic peptide, a vaccine, a cytokine, a chemokine, a hormone, an oncolytic virus, and a bacterium.

[0139] In one embodiment, the immune cells are selected from T cells, NK cells, B cells, lymphocytes, dendritic cells, and mesenchymal cells, or immortalized cells thereof.

[0140] In a thirteenth aspect of the invention, there is provided a method of screening candidate engineered immune cells for a biological effect, comprising the steps of: (a) providing a cell comprising a first inducible system operable to express a first protein of interest, and optionally a second inducible system operable to express a second protein of interest; (b) exposing the cells comprising the first inducible system and optionally the second inducible system to an effective concentration of a first inducer to induce a desired level of expression of the first protein of interest, and optionally exposing the cells to an effective concentration of a second inducer to induce a desired level of expression of the second protein of interest; (c) contacting the cells with candidate engineered immune cells; (d) determining whether the candidate engineered immune cells exert a biological effect on cells expressing the first protein of interest and, optionally, the second protein of interest; A method is provided which includes:

[0141] Any preferred or optional features defined above in relation to the ninth aspect apply equally to the thirteenth aspect.

[0142] In an embodiment of the thirteenth aspect of the invention there is provided a method of screening candidate engineered immune cells for a biological effect comprising the steps of: (a) providing a cell comprising the first plant hormone-inducible access system and / or the second plant hormone-inducible access system of the eighth aspect; (b) culturing cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system under conditions in which the first construct and / or the third construct are expressed; (c) exposing the cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system to an effective concentration of a first plant hormone inducer to induce a desired level of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of a second plant hormone inducer to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with candidate engineered immune cells; (e) determining whether the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0143] In one embodiment of the thirteenth aspect, there is provided a method of screening candidate engineered immune cells for a biological effect, comprising: (a) providing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system as defined herein; (b) culturing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system under conditions in which the first construct and / or the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system to an effective concentration of auxin to induce a desired level of expression of a first protein of interest from the second construct, and / or exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of a second protein of interest from the fourth construct; (d) contacting the cells with candidate engineered immune cells; (e) determining whether the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0144] In one embodiment of the thirteenth aspect, the method comprises screening candidate engineered immune cells for a biological effect, comprising: (a) providing a cell comprising an auxin-inducible access system and an abscisic acid-inducible access system as defined herein; (b) culturing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system to an effective concentration of auxin to induce a desired level of expression of a first protein of interest from the second construct, and exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of a second protein of interest from the fourth construct; (d) contacting the cells with candidate engineered immune cells; (e) determining whether the candidate engineered immune cells exert a biological effect on cells expressing the first and second proteins of interest; (f) determining the expression level of the first and / or second proteins of interest exerting a biological effect in the contacted engineered immune cells. The method includes:

[0145] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0146] In one embodiment, step (b) or (c) may optionally comprise exposing the cells to a plurality of different concentrations of a first inducer, preferably a plant hormone inducer, preferably an auxin, to induce a plurality of different levels of expression of a first protein of interest, which may be expressible from a second construct, and / or exposing the cells to a plurality of different concentrations of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, to induce a desired level of expression of a second protein of interest, which may be expressible from a fourth construct.

[0147] In one embodiment, step (b) or (c) may optionally comprise exposing the cells to an effective concentration of a first inducer, preferably a plant hormone inducer, preferably an auxin, and an effective concentration of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, simultaneously or at different times, as further described below.

[0148] In one embodiment, step (d) or (e) optionally includes determining whether the candidate engineered immune cells exert a biological effect on cells expressing both the first and second proteins of interest. In one embodiment, exerting a biological effect may include targeting cells expressing the first and / or second proteins of interest.

[0149] In one embodiment, the method optionally further comprises step (e) or (f) determining the minimum expression level of the first and / or second protein of interest at which the contacted engineered immune cells exert a biological effect.

[0150] In one embodiment, step (e) or (f), as appropriate, may include determining the minimal expression levels of both the first and second proteins of interest at which the candidate engineered immune cells exert a biological effect.

[0151] In one embodiment, the minimum level of expression of the first and / or second protein of interest at which the candidate engineered immune cell exerts a biological effect may be the level of expression at which a biological effect above background is achieved. Preferably, a biological effect at least 3 standard deviations above background is achieved, preferably a biological effect at least 4 standard deviations above background is achieved, preferably a biological effect at least 5 standard deviations above background is achieved, preferably a biological effect at least 6 standard deviations above background is achieved, preferably a biological effect at least 7 standard deviations above background is achieved, preferably a biological effect at least 8 standard deviations above background is achieved, preferably a biological effect at least 9 standard deviations above background is achieved, preferably a biological effect at least 10 standard deviations above background is achieved. Preferably, the background biological effect is the biological effect of the candidate binding molecule on control cells. Preferably, the control cell is a cell that does not contain the inducible system described herein.Preferably, the control cell does not express the or each protein of interest.Preferably, the control cell is a wild-type cell.Alternatively, the control cell may be a cell that is modified to prevent the expression of the or each protein of interest, preferably by "knocking out" the gene that encodes the or each protein of interest, and this modification can be achieved by known modification techniques such as RNA interference, RNA silencing, CRISPRi, zinc finger nuclease, TALEN, etc.

[0152] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the candidate engineered immune cells exert a biological effect may be the activation threshold for the first and / or second proteins of interest. Suitably, this may be determined using receiver operating characteristic (ROC) curve analysis, suitably using Youden's index or Youden's J statistic.

[0153] In one embodiment, the candidate engineered immune cells are selected from cells expressing a CAR or a T cell receptor (TCR), preferably selected from CAR T cells, TCR T cells, CAR NK cells, CAR macrophages, and CAR B cells.

[0154] In a fourteenth aspect of the present invention there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which a candidate engineered immune cell exerts a biological effect, comprising the steps of: (a) providing a cell comprising a first inducible system operable to express a first protein of interest, and optionally a second inducible system operable to express a second protein of interest; (b) exposing the cells comprising the first inducible system, and optionally the second inducible system, to a plurality of different concentrations of a first inducer to induce a plurality of different levels of expression of the first protein of interest, and optionally exposing the cells to a plurality of different concentrations of a second inducer to induce a plurality of different levels of expression of the second protein of interest; (c) contacting the cells with candidate engineered immune cells; (d) determining whether, at each level of expression of the first protein of interest and optionally the second protein of interest, the candidate engineered immune cells exert a biological effect on cells expressing the first protein of interest and optionally the second protein of interest; and (e) determining the minimum level of expression of the first protein of interest and optionally the second protein of interest at which the candidate engineered immune cells exert a biological effect on cells expressing the first protein of interest and optionally the second protein of interest; A method is provided which includes:

[0155] Any preferred or optional features defined above in relation to the ninth aspect apply equally to the fourteenth aspect.

[0156] In a fourteenth aspect of the present invention there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which a candidate engineered immune cell exerts a biological effect, comprising the steps of: (a) providing a cell comprising the first plant hormone-inducible access system and / or the second plant hormone-inducible access system of the eighth aspect; (b) culturing cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the first plant hormone-inducible access system and / or the second plant hormone-inducible access system to a plurality of different concentrations of the first plant hormone inducer to induce a plurality of different levels of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of the second plant hormone inducer to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with candidate engineered immune cells; (e) determining whether, at each level of expression of the first and / or second proteins of interest, the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; and (f) determining the minimum level of expression of the first and / or second proteins of interest at which the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes:

[0157]

[0033] In one embodiment of the fourteenth aspect, there is provided a method for determining the minimum level of expression of at least one protein of interest in a cell at which a candidate engineered immune cell exerts a biological effect, comprising: (a) providing a cell comprising an auxin-inducible access system and / or an abscisic acid-inducible access system as defined herein; (b) culturing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and / or the abscisic acid-inducible access system to a plurality of different concentrations of auxin to induce a plurality of different levels of expression of the first protein of interest from the second construct, and / or exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with candidate engineered immune cells; (e) determining whether, at each level of expression of the first and / or second proteins of interest, the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; (f) determining the minimum level of expression of the first and / or second proteins of interest at which the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest; A method is provided which includes

[0158] In one embodiment of the fourteenth aspect, the method comprises determining the minimum levels of expression of a first and a second protein of interest in a cell at which a candidate engineered immune cell exerts a biological effect, the method comprising: (a) providing a cell comprising an auxin-inducible access system and an abscisic acid-inducible access system as defined herein; (b) culturing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system under conditions in which the first construct and the third construct are expressed; (c) exposing the cells containing the auxin-inducible access system and the abscisic acid-inducible access system to a plurality of different concentrations of auxin to induce a plurality of different levels of expression of the first protein of interest from the second construct, and exposing the cells to an effective concentration of abscisic acid to induce a desired level of expression of the second protein of interest from the fourth construct; (d) contacting the cells with candidate engineered immune cells; (e) determining whether, at each level of expression of the first and second proteins of interest, the candidate engineered immune cells exert a biological effect on cells expressing the first and second proteins of interest; (f) determining the minimum level of expression of the first and / or second proteins of interest at which the candidate engineered immune cells exert a biological effect on cells expressing the first and second proteins of interest; The method includes:

[0159] In one embodiment, the auxin-inducible access system and the abscisic acid-inducible access system are as defined herein above, as are the first, second, third and fourth constructs.

[0160] In one embodiment, step (b) or (c) may optionally comprise exposing the cells to a predetermined concentration of a first inducer, preferably a plant hormone inducer, preferably an auxin, and a predetermined concentration of a second inducer, preferably a plant hormone inducer, preferably abscisic acid, simultaneously or at different times, as further described below.

[0161] In one embodiment, step (d) or (e), as appropriate, may include determining whether the candidate engineered immune cells exert a biological effect on cells expressing the first and second proteins of interest at each level of expression of both the first and second proteins of interest.

[0162] In one embodiment, step (e) or (f), as appropriate, may comprise determining the minimum level of expression of both the first and second proteins of interest at which the candidate engineered immune cells exert a biological effect on cells expressing both the first and second proteins of interest.

[0163] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest may be the level of expression at which a biological effect above background is achieved. Preferably, a biological effect at least 3 standard deviations above background is achieved, preferably a biological effect at least 4 standard deviations above background is achieved, preferably a biological effect at least 5 standard deviations above background is achieved, preferably a biological effect at least 6 standard deviations above background is achieved, preferably a biological effect at least 7 standard deviations above background is achieved, preferably a biological effect at least 8 standard deviations above background is achieved, preferably a biological effect at least 9 standard deviations above background is achieved, preferably a biological effect at least 10 standard deviations above background is achieved. Preferably, the background biological effect is the biological effect of the candidate binding molecule on control cells. Preferably, the control cell is a cell that does not contain the inducible system described herein.Preferably, the control cell does not express the or each protein of interest.Preferably, the control cell is a wild-type cell.Alternatively, the control cell may be a cell that is modified to prevent the expression of the or each protein of interest, preferably by "knocking out" the gene that encodes the or each protein of interest, and this modification can be achieved by known modification techniques such as RNA interference, RNA silencing, CRISPRi, zinc finger nuclease, TALEN, etc.

[0164] In one embodiment, the minimum level of expression of the first and / or second proteins of interest at which the candidate engineered immune cells exert a biological effect on cells expressing the first and / or second proteins of interest may be the activation threshold for the first and / or second proteins of interest. Suitably, this may be determined using receiver operating characteristic (ROC) curve analysis, suitably using Youden's index or Youden's J statistic.

[0165] In one embodiment, the candidate engineered immune cells are selected from cells expressing a CAR or a T cell receptor (TCR), preferably selected from CAR T cells, TCR T cells, CAR NK cells, CAR macrophages, and CAR B cells.

[0166] In a fifteenth aspect of the present invention, there is provided a method of inducing a first chemical-inducible access system, such as an auxin-inducible access system, comprising the steps of: (a) providing a cell comprising a first chemical-inducible access system, such as the auxin-inducible access system of the eighth aspect; (b) culturing the cells under conditions in which the first and second chimeric proteins are expressed from the first construct; (c) exposing the cells to an effective concentration of a first inducer, such as an auxin, to cause the first and second chimeric proteins to associate. A method is provided which includes:

[0167] In such embodiments, the first chemical-inducible access system, eg, auxin-inducible access system, the first and second constructs are as defined herein above.

[0168] In a further embodiment of the invention, there is provided a method of inducing a second chemically inducible access system, e.g., an abscisic acid inducible access system, comprising: (a) providing a cell comprising a second chemical-inducible access system, such as the abscisic acid-inducible access system of the eighth aspect; (b) culturing the cells under conditions in which the third and fourth chimeric proteins from the third construct are expressed; (c) exposing the cells to an effective concentration of a second inducer, e.g., abscisic acid, to allow the third and fourth chimeric proteins to associate. A method is provided which includes:

[0169] In such embodiments, the second chemically inducible access system, eg, an abscisic acid inducible access system, the third and fourth constructs are as defined herein above.

[0170] In a further embodiment of the invention, there is provided a method of inducing a first and / or second plant hormone-inducible access system, comprising: (a) providing a cell comprising the first and / or second plant hormone-inducible access system of the eighth aspect; (b) culturing the cells under conditions in which the first and second chimeric proteins from the first construct and / or the third and fourth chimeric proteins from the third construct are expressed; (c) exposing the cells to an effective concentration of a first plant hormone inducer to cause the first and second chimeric proteins to associate, and / or exposing the cells to an effective concentration of a second plant hormone inducer to cause the third and fourth chimeric proteins to associate. A method is provided which includes:

[0171] In such an embodiment, the cell may be a cell of the eighth aspect.

[0172] In a further embodiment of the present invention there is provided a method for inducing an auxin-inducible access system and an abscisic acid-inducible access system, comprising the steps of: (a) providing a cell comprising an auxin-inducible access system and an abscisic acid-inducible access system as defined herein; (b) culturing the cells under conditions in which the first and second chimeric proteins from the first construct and the third and fourth chimeric proteins from the third construct are expressed; (c) exposing the cells to an effective concentration of auxin to cause the first and second chimeric proteins to associate, and exposing the cells to an effective concentration of abscisic acid to cause the third and fourth chimeric proteins to associate. A method is provided which includes:

[0173] In such an embodiment, the auxin-inducible access system, the abscisic acid-inducible access system, the first, second, third and fourth constructs are as defined herein above.

[0174] In one embodiment, the method of inducing the access system may comprise exposing the cells to a plurality of different concentrations of a first plant hormone inducer, preferably auxin, to induce a plurality of different levels of expression of a first protein of interest from a second construct, and / or exposing the cells to a plurality of different concentrations of a second plant hormone inducer, preferably abscisic acid, to induce a desired level of expression of a second protein of interest from a fourth construct.

[0175] In one embodiment, step (c) may comprise exposing the cells to an effective concentration of auxin and an effective concentration of abscisic acid, either simultaneously or at different times, as further described below.

[0176] In such an embodiment, the cell may be a cell of the eighth aspect.

[0177] In a sixteenth aspect of the present invention there is provided a method for determining whether a first compound, such as an auxin compound, is present in a sample, the method comprising: (a) providing a cell comprising a first chemical-inducible access system, such as the auxin-inducible access system of the eighth aspect, wherein the protein of interest of the second construct is a reporter; (b) culturing the cells under conditions in which the first and second chimeric proteins are expressed from the first construct; (c) contacting the sample with cells; and (d) assessing expression of the reporter to determine whether a compound, such as an auxin compound, is present in the sample. A method is provided which includes:

[0178] In such embodiments, the first chemical-inducible access system, eg, auxin-inducible access system, the first and second constructs are as defined herein above.

[0179] In a seventeenth aspect, there is provided a method for determining whether a second compound, such as an abscisic acid compound, is present in a sample, the method comprising: (a) providing a cell containing a second chemical-inducible access system, e.g., an abscisic acid-inducible access system, wherein the protein of interest of the fourth construct is a reporter; (b) culturing the cells under conditions in which the third and fourth chimeric proteins from the third construct are expressed; (c) contacting the sample with cells; and (d) assessing expression of the reporter to determine whether a second compound, such as an abscisic acid compound, is present in the sample. A method is provided which includes:

[0180] In such embodiments, the second chemically inducible access system, eg, an abscisic acid inducible access system, the third and fourth constructs are as defined herein above.

[0181] In a further embodiment, there is provided a method of determining whether a first compound and / or a second compound is present in a sample, the method comprising: (a) providing a cell comprising a first inducible system operable to express a first protein of interest and / or a second inducible system operable to express a second protein of interest, wherein the first protein of interest is a first reporter and the second protein of interest is a second reporter; (b) contacting the sample with cells; and (c) assessing expression of the first and / or second reporter to determine whether the first and / or second compound is present in the sample. A method is provided which includes:

[0182] Suitable inducible systems are defined herein. In one embodiment, the first and second inducible systems may be chemically induced access systems (CIP systems). In a preferred embodiment, they may be plant hormone or plant hormone analog inducible systems described herein. In some embodiments, the first inducible system may be the first plant hormone inducible access system of the eighth aspect. In some embodiments, the second inducible system may be the second plant hormone inducible access system of the eighth aspect. Suitably, in such embodiments, the first inducible system comprises the first and second constructs defined herein, and the second inducible system comprises the third and fourth constructs defined herein. In one embodiment, at least one of the inducible systems is a plant hormone inducible access system. In one embodiment, at least one of the inducible systems is an abscisic acid inducible access system defined herein. In one embodiment, at least one of the inducible systems is selected from a caffeine inducible access system defined herein, a mandipropamide inducible access system defined herein, and a gibberellin inducible access system defined herein.

[0183] In a preferred embodiment, the first inducible system is selected from a caffeine-inducible access system as defined herein, a mandipropamid-inducible access system as defined herein, and a gibberellin-inducible access system as defined herein, and the second inducible system is an abscisic acid-inducible access system as defined herein.

[0184] In a further embodiment, there is provided a method of determining whether a first compound and / or a second compound is present in a sample, the method comprising: (a) providing a cell comprising a first plant hormone-inducible access system and a second plant hormone-inducible access system, wherein the protein of interest of the second construct is a first reporter and the protein of interest of the fourth construct is a second reporter; (b) culturing the cells under conditions in which the first and second chimeric proteins from the first construct and the third and fourth chimeric proteins from the third construct are expressed; (c) contacting the sample with cells; and (d) assessing expression of the first and / or second reporter to determine whether the first and / or second compound is present in the sample. A method is provided which includes:

[0185] In such an embodiment, the cell may be a cell of the eighth aspect.

[0186] In a further embodiment, there is provided a method for determining whether an auxin compound and / or an abscisic acid compound is present in a sample, comprising the steps of: (a) providing a cell containing an auxin-inducible access system and an abscisic acid-inducible access system, wherein the protein of interest of the second construct is a first reporter and the protein of interest of the fourth construct is a second reporter; (b) culturing the cells under conditions in which the first and second chimeric proteins from the first construct and the third and fourth chimeric proteins from the third construct are expressed; (c) contacting the sample with cells; and (d) assessing expression of the first and / or second reporter to determine whether auxin and / or abscisic acid compounds are present in the sample. A method is provided which includes:

[0187] In such an embodiment, the auxin-inducible access system, the abscisic acid-inducible access system, the first, second, third and fourth constructs are as defined herein above.

[0188] In one embodiment, step (d) may comprise assessing expression of both the first and second reporters to determine whether auxin and abscisic acid compounds are present in the sample.

[0189] In an eighteenth aspect of the present invention there is provided a kit comprising a first inducible access system of the third aspect, such as the auxin-inducible access system of the third aspect, a vector of the fourth aspect, or a cell comprising said system; and a first inducer compound, such as an auxin compound, as defined above.

[0190] In a nineteenth aspect of the present invention there is provided a kit comprising a second inducible access system as defined herein above, such as an abscisic acid inducible access system, a vector comprising said system, or a cell comprising said system as defined herein above, and a second inducer compound, such as an abscisic acid compound.

[0191] In one embodiment, the kit may include components for the detection of both the first compound and the second compound, for example components for both the detection of auxin and the detection of abscisic acid.

[0192] Preferably, any reference to a first construct in the above embodiments is a reference to the first construct according to the first embodiment. Preferably, any reference to a second construct in the above embodiments is a reference to the second construct according to the second embodiment. Preferably, any reference to an auxin-inducible access system in the above embodiments is a reference to an auxin-inducible access system according to the third embodiment. Preferably, any reference to a third construct in the above embodiments is a reference to the third construct as defined herein above. Preferably, any reference to a fourth construct in the above embodiments is a reference to the fourth construct as defined herein above. Preferably, any reference to an abscisic acid-inducible access system in the above embodiments is a reference to the abscisic acid-inducible access system as defined herein above.

[0193] Suitably, any reference in the above aspects to an auxin-inducible access system and / or an abscisic acid-inducible access system may encompass one of these two systems or both of these two systems. Suitably, such reference may encompass an auxin-inducible access system, optionally in combination with an abscisic acid-inducible access system, and any corresponding constructs thereof, optionally in combination.

[0194] It will be appreciated by those skilled in the art that the cells and methods described herein may suitably contain third, fourth, fifth, sixth, or multiple inducible systems operable to express multiple proteins of interest at different, controllable levels, using the same constructs, steps, and techniques described herein for the first and second inducible systems. [Brief explanation of the drawings]

[0195] [Figure 1]Figure 1 shows a schematic diagram of an exemplary auxin (indole-3-acetic acid (IAA))-controlled, chemically induced access (CIP) for titratable expression of a first protein of interest (TAOI expression), which may be a first target antigen of interest. Panel A1) shows an IAA activator cassette containing a simplex virus VP16 transactivation domain (VP16AD) fused to TIR1 and a catalytically inactive I-SceI homing endonuclease (dI-SceI) fused to AIDΔ34. Panel A2) shows that the expression construct employs the 18-bp recognition sequence of I-SceI, downstream of which the target antigen of interest (TAOI) is located. Panel B) shows that dI-SceI associates with its recognition sequence but is unable to activate transcription due to the lack of a transactivator domain. In the presence of IAA, AIDΔ34 associates with TIR1, bringing the VP16 transactivator domain into close proximity with the upstream region of TAOI, activating transcription. [Figure 2] Figure 1 shows the cytotoxicity of IAA to HEK293 and CHO-K1 cells analyzed by flow cytometry. HEK293 and CHO-K1 cells were treated with the indicated concentrations of IAA for 24 hours, then stained with eFluor780 fixable viability dye and analyzed by flow cytometry. Viability is reported as the percentage of cells that do not take up the dye. [Figure 3] Figure 1 shows graphs demonstrating the effect of IAA on T cell proliferation and activation. PBMCs from two donors were stimulated with human T activator CD3 / CD28 beads in the presence of a range of IAA concentrations. Proliferation of CD4+ (Panel A) and CD8+ (Panel B) T cells was assessed by flow cytometry using a proliferation dye. IFNγ production was assessed by ELISA (Panel C). n=3, graphs report mean ± SD. [Figure 4]Figure 1 shows the expression of the IAA activator cassette in HEK293 (A) and CHO-K1 (B) cells as determined using an anti-VP16 detection antibody. Stained parental HEK293 or CHO-K1 cells are shown in dark gray, and cells transduced with the IAA activator cassette are shown in light gray. [Figure 5] Figure 1 shows IAA treatment of IAA activator HEK293 (A) and CHO-K1 (B) cells transduced with EGFP reporter constructs containing various numbers of i-SceI binding site(s) as indicated. Analysis was performed by flow cytometry after 24 hours of incubation with the indicated concentrations of IAA. [Figure 6] Figure 1 shows induction of EGFP expression in IAA HEK293 (A) and CHO-K1 (B) cells containing the IAA activator cassette and 10x and 5x i-SceI reporter constructs seeded overnight in 96-well plates. IAA was added at the indicated concentrations, and cells were incubated for an additional 24 hours. EGFP expression was detected by flow cytometry analysis. [Figure 7] Figure 1 shows IAA dose-dependent induction of EGFP expression. CHO-K1 (A) and HEK293 (B) cells containing the IAA activator cassette and 10x i-SceI reporter construct were seeded overnight in 96-well plates. IAA was added at the indicated concentrations, and the cells were incubated for an additional 24 hours. EGFP expression was detected by flow cytometry analysis. [Figure 8]This is a schematic diagram showing abscisic acid (ABA)-regulated chemically induced access (CIP) for titratable expression of a second protein of interest (TAO2 expression), which may be a second target antigen of interest. Panel A1) shows the ABA activator cassette containing the yeast Gal4 DNA-binding domain (Gal4DBD) fused to ABIcs and the herpes simplex virus VP16 transactivation domain (VP16AD) fused to PYLcs. Expression of these two fusion proteins is operably linked by the inclusion of the Thosea asigna 2A (T2A) self-cleaving peptide. Panel A2) shows that the expression construct employs nine repeats of the Gal4 upstream activating sequence (UAS) under whose control the target antigen of interest (TAOI) is placed. Panel B) shows that the Gal4 DNA-binding domain associates with the Gal4 UAS but is unable to activate transcription due to the lack of a transactivator domain. In the presence of ABA, PYLcs associate with ABIcs, bringing the VP16 transactivator domain into close proximity with the upstream region of the target antigen (gene) of interest, activating transcription. [Figure 9] Figure 1 shows the cytotoxicity of ABA to HEK293 and CHO-K1 cells analyzed by flow cytometry. HEK293 and CHO-K1 cells were treated with the indicated concentrations of ABA for 24 hours, then stained with eFluor780 fixable viability dye and analyzed by flow cytometry. Viability is reported as the percentage of cells that do not take up the dye. [Figure 10] Figure 1 shows graphs demonstrating the effect of ABA on T cell proliferation and activation. PBMCs from two donors were stimulated with the human T activator CD3 / CD28 in the presence of a range of concentrations of IAA. Proliferation of CD4+ (Panel A) and CD8+ (Panel B) T cells was assessed by flow cytometry using a proliferation dye. IFNγ production was assessed by ELISA (Panel C). n=3, graphs report mean ± SD. [Figure 11]Figure 1 shows the expression of the ABA activator cassette in HEK293 (A) and CHO-K1 (B) cells as determined using an anti-VP16 detection antibody. Stained parental HEK293 or CHO-K1 cells are shown in dark gray, and cells transduced with the ABA activator cassette are shown in light gray. [Figure 12] Figure 1 shows ABA treatment of ABA-activator CHO-K1 and HEK293 cells transduced with tagBFP (A) and CD19 (B) reporter constructs. Cells were treated with 1000 μM ABA for 24 h and then analyzed by flow cytometry for tagBFP and CD19 expression. [Figure 13] Figure 1 shows ABA dose-dependent induction of CD19 expression. CHO-K1 cells containing an ABA activator cassette and a 9xGal4 UAS CD19 reporter construct were seeded overnight in a 96-well plate. ABA was added at the indicated concentrations, and the cells were incubated for an additional 48 hours. CD19 expression was detected by flow cytometry analysis, and the number of CD19 receptors per cell was quantified using a Quantibrite PE bead fluorescence quantification kit (BD Biosciences). CD19 receptors per cell of Ramos, Raji, and B cells isolated from PBMCs are shown for comparison. [Figure 14] FIG. 1 is a schematic diagram showing (A) the constructs used in the examples to prepare an auxin-inducible CIP system and (B) the constructs used in the examples to prepare an abscisic acid-inducible CIP system. [Figure 15] Figure 1 shows quantification of IAA dose-dependent expression of CD19. CHO-K1 cells containing an IAA activator cassette and 10x i-SceI CD19 reporter construct were seeded overnight in a 96-well plate. IAA was added at the indicated concentrations, and the cells were further incubated for 72 hours. CD19 expression was assessed by flow cytometry and quantified as the number of receptors per cell. CD19 expression levels of CD19-positive B cells found in peripheral blood mononuclear cells (PBMCs) and Ramos cells were calculated by comparison in the same experiment. [Figure 16] Figure 1 shows validation of the orthogonality of the ABA and IAA CIP systems. CHO-K1 ABA activator CD19 reporter cells and CHO-K1 IAA activator CD19 reporter cells were treated separately with (A) 1000 μM ABA and (B) 1000 μM IAA, respectively, for 24 h. After incubation, cells were analyzed for CD19 expression by flow cytometry. [Figure 17] This figure shows independent T cell activation by CD19 expressing CHO-K1 ABA or IAA CIP systems, assessed using Jurkat NFAT T cell activation reporter cells. CHO-K1 IAA or ABA activator CD19 reporter cells were plated with a range of concentrations of IAA or ABA (7.18 μM to 500 μM) to induce CD19 expression for 24 hours. After 24 hours, IAA and ABA were removed from the system, and Jurkat NFAT luciferase reporter cells were added along with 10 ng / mL and 25 ng / mL of the CD19 x CD3 bispecific antibody Blincyto. NFAT activation was determined by luminescence after 24 hours (48 hours from the start of the assay) and plotted against CD19 expression, quantified by staining with PE anti-CD19, on a curve generated using the Quanti-Brite PE receptor quantification kit. n=3, graphs report mean ± SD. [Figure 18] FIG. 1 shows that CHO-K1 cells containing the ABA-inducible CD22 and IAA-inducible CD19 CIP system were treated with a range of concentrations of IAA or ABA, and CD22 and CD19 expression was measured by flow cytometry. [Figure 19]Figure 1 shows HER2 expression induced by ABA to screen the biological activity of the anti-HER2 ADCs trastuzumab emtansine or trastuzumab deruxtecan. A) HER2 expression in CHO-K1 ABA HER2 cells treated with a range of ABA concentrations. B) Target cell cytolysis in HER2-expressing CHO-K1 ABA HER2 cells treated with the anti-HER2 ADCs trastuzumab emtansine or trastuzumab deruxtecan. n=3, graphs report mean±SD. [Figure 20] Figure 1 shows a T cell-dependent cytotoxicity (TDCC) assay using CHO-K1 ABA-activator CD19 cells and CD19xCD3 bispecific T cell engager (TCE). CD19 expression was induced with a range of concentrations of ABA, and then CD8+ T cells isolated from PBMCs of four donors were added at a 3:1 ratio in the presence of 225 pM CD19xCD3 bispecific antibody TCE. A) Target cell death after 48 hours. B) CD8+ T cell proliferation measured by Ki-67 staining. C) IFN-γ production by CD8+ T cells after 48 hours. For all panels, n=3, and graphs report the mean ± SD. [Figure 21]Figure 1 shows a comparison of the cytotoxicity of two CD19xCD3 bispecific T cell engagers (TCEs) across a range of CD19 receptor expression levels achieved by treating CHO-K1 ABA CD19 cells with a range of ABA concentrations. T cells were isolated from donor PBMCs (n=10) and used with TCE1 or TCE2. A and C). T cell-mediated cytotoxicity (A) and IFN-gamma production (C). Each data point represents a single T cell donor. B and D) Estimates of the minimum number of receptors at which a biological effect in the form of cytotoxicity (B) and IFN-gamma production (D) was observed for each bispecific antibody TCE. This was defined as the minimum number of receptors at which an effect higher than the mean background effect plus three standard deviations was observed, where background was the biological effect observed when using wild-type CHO as target cells, assuming a target CD19 receptor count of 0. Data are presented as medians, bars, and confidence intervals. The Wilcoxon matched-pairs signed-rank test was used to compare the two treatments. E and F) Receiver operating characteristic (ROC) curves for both TCE1 and TCE2 to determine activation thresholds for a cohort of 10 donors for cytolysis (E) and IFN-gamma production (F). Youden's index for each molecule and readout was used to determine the receptor activation thresholds shown on the graphs. The same threshold of background + 3 standard deviations cutoff was used to classify results as positive for each level of receptor expression and each donor. [Figure 22]Figure 1 shows CD19 CAR-T cell activity against target CHO-K1 ABA-activated CD19 cells. A) Schematic of the CD19 CAR construct used in this experiment, consisting of FMC63 anti-CD19 scFv (CD19VL and CD19VH), CD8 stalk and transmembrane domains, and 41BB and CD3ζ intracellular domains. B) Expression of the CD19 CAR construct after transduction of T cells from three donors, detected via flow cytometry in CD3-positive T cells using an anti-FMC63 scFv antibody. C) Cytolytic activity of CD19 CAR-T cells against CHO-K1 ABA CD19 cells expressing a range of CD19 levels via treatment with a range of ABA concentrations. D) IFN-γ release by effector CD19 CAR-T cells after 24 hours of incubation with target cells. For C and D, n=3, graphs report mean ± SD. [Figure 23] Figure 1 shows the activity of dual CD19 / CD22 CAR Jurkat NFAT luciferase reporter cells against target CHO-K1 cells expressing the ABA-inducible CD22 and IAA-inducible CD19 CIP systems. A) Schematic of the CD19 / CD22-targeting CAR used in this experiment, consisting of FMC63 anti-CD19 scFv (CD19VL, CD19VH), m971 anti-CD22 scFv (CD22VL, CD22VH), CD8 stalk and transmembrane domains, and 41BB and CD3ζ intracellular domains. B) Luciferase activity, measured in relative luminescence units (RLU), of CD19 / CD22 CAR Jurkat NFAT luciferase reporter (effector) cells after addition to target CHO-K1 ABA CD22 IAA CD19 cells induced with a range of concentrations of either IAA, ABA, or both. [Figure 24]

[0023] Figure 1 shows the activity of dual CD19 / CD22 CAR-T cells generated from primary T cells derived from PBMCs of three donors against target CHO-K1 cells containing the ABA-inducible CD22 and IAA-inducible CD19 CIP system. A) CD19 / CD22 CAR expression detected in CD3-positive T cells transduced from three donors. B) CD19 and CD22 expression in CHO-K1 IAA CD19 ABA CD22 target cells after induction with a range of concentrations of IAA or ABA, separately or together. C) Cytolysis of target CHO-K1 IAA CD19 ABA CD22 cells induced by CD19 / CD22 CAR-positive T cells. For C, n=3, error bars report mean±SD. [Figure 25] Figure 1 shows the evaluation of the effect of HER2 expression induced by treating CHO-K1 ABA HER2 cells with a range of concentrations of ABA on the antibody-dependent cellular cytotoxicity (ADCC) activity of trastuzumab, pertuzumab, or the combination of the two. PBMCs from three healthy donors were recruited at an effector-to-target ratio of 10:1 (each represented by a data point), and co-cultures were treated with 20 μg / mL of individual or combined drugs. Target cell cytolysis was assessed using the xCELLIgence RTCA system. Data are presented as mean ± SD. [Figure 26]Figure 1: Caffeine-controlled inducible expression system. A1) A caffeine activator cassette containing the yeast Gal4 DNA-binding domain (Gal4DBD) and the herpes simplex virus VP16 transactivation domain (VP16AD), ​​each fused to an αCaffVHH nanobody isolated from the llama (Lama glama). Expression of these two fusion proteins is operably linked by the inclusion of the Thosea asigna 2A (T2A) self-cleaving peptide. A2) A target antigen of interest (TAOI) under the control of nine repeats of the Gal4 upstream activation sequence (UAS). B) The Gal4 DNA-binding domain associates with the Gal4 UAS but is unable to activate transcription due to the lack of a transactivator domain. In the presence of caffeine, the αCaffVHH nanobody dimerizes, bringing the VP16 transactivator domain into close proximity with the upstream region of the TAOI, activating transcription of the TAOI. [Figure 27] Figure 1: Inducible expression system controlled by Mandi. A) Chemical structure of Mandipropramid (Mandi). B1 and D1) The Mandi activator cassette contains VP16AD fused to PYRMandi (B1) or PYLcsMandi (D1) and Gal4DBD fused to ABI. Expression of these two fusion proteins is operably linked by the inclusion of a T2A self-cleaving peptide. B2 and D2) Target antigen of interest (TAOI) under the control of nine repeats of Gal4 UAS. C and E) Gal4DBD associates with Gal4 UAS but cannot activate transcription due to the lack of a transactivator domain. In the presence of Mandi, PYRMandi (C) and PYLcsMandi (E) associate with ABI, bringing the VP16 transactivator domain into close proximity with the upstream region of TAOI, activating transcription of TAOI. [Figure 28]Figure 1: Gibberellin-controlled inducible expression system. A1) The gibberellin activator cassette contains VP16AD fused to GID1 and Gal4DBD fused to GAI. Expression of these two fusion proteins is operably linked by the inclusion of a T2A self-cleaving peptide. A2) A target antigen of interest (TAOI) under the control of nine repeats of the Gal4 UAS. B) Gal4DBD associates with the Gal4 UAS but cannot activate transcription because it lacks a transactivator domain. In the presence of gibberellin, GID1 associates with GAI, bringing the VP16 transactivator domain into close proximity with the upstream region of TAOI, activating transcription of TAOI. DETAILED DESCRIPTION OF THE INVENTION

[0196] The inventors have surprisingly overcome problems in the art associated with in vitro risk assessment of candidate therapeutic agents, such as immunotherapeutics, by establishing an inducible platform that allows for the independent control of expression of one or two different target antigens in a titratable manner, allowing for the expression of any target antigen of interest to any desired level.

[0197] The inventors have discovered that expression of a first target antigen of interest and, optionally, a second target antigen of interest (TAOI1 and TAOI2) can be precisely controlled via an inducible system, specifically a chemically induced access (CIP) system, and that such a system may be derived from a plant hormone signaling system, specifically the auxin plant signaling pathway, the abscisic acid plant signaling pathway, the caffeine plant signaling pathway, or the gibberellin plant signaling pathway. The inventors have discovered that using at least one inducible system, it is possible to develop engineered cell lines with simultaneously inducible and titratable expression of up to two different antigens of interest, TAOI1 and / or TAOI2. Advantageously, the use of plant hormone inducible systems means that these systems can be induced using plant hormones or their analogs that are small, non-toxic molecules without undesirable effects on animal cells. Chemically induced access (CIP) systems derived from plant hormone signaling systems can also be based on plant hormone analogs, such as mandipropamide (Mandi).

[0198] In its preferred embodiment, the present invention provides a modified CIP system comprising a first auxin-inducible access system, a caffeine-inducible access system, a mandipropamid-inducible access system, or a gibberellin-inducible access system, and / or a second abscisic acid-inducible access system, resulting in precisely titratable induction of TAOI1 and / or TAOI2 with a large dynamic range and compatibility with both immortalized cell lines and primary human cells.

[0199] However, the system's advantages apply equally to methods using either inducible system. Taken together, these features make cells containing an inducible system, such as the modified CIP system, suitable for in vitro characterization of candidate targeted therapies using cell-based bioassays and for complex cocultures of primary human immune cells and immortalized cell lines. The system provides an in vitro risk assessment assay that allows drug developers to determine the threshold (or minimum) level of cell surface antigen that elicits biological effects and thereby determines the efficacy and safety of candidate therapeutics, particularly on-target and off-tumor effects. This assessment can also be performed simultaneously for up to two different antigens, thereby better reflecting the in vivo tumor environment and enabling the development of candidate therapies that can target more than one antigen. This information is useful for informing candidate drug selection and allows safer therapeutics to be introduced into the clinic, thereby increasing the chances of success.

[0200] The invention will now be further described with reference to the following heading sections: Any of the features described in any of the sections may be applied to any of the aspects of the invention in any practical combination.

[0201] Inductive System As described above, the methods of the present invention can utilize any inducible system operable to control the expression of a protein of interest in cells, such that the cells can be used to screen biomolecules, candidate therapeutic agents, or engineered immune cells for their activity against the protein. Such inducible systems can be used individually or in pairs to express two or more different antigens, optionally at different levels, in the same cell for such screening methods. In a preferred embodiment, the inducible system used in the methods can be a chemical-inducible access system based on a plant hormone signaling pathway.

[0202] Suitably, inducible systems that can be used in the methods of the present invention include systems induced by the presence of an inducer, the absence of a repressor, or any other suitable physical or chemical change.

[0203] By way of non-limiting example, inducible systems for use in embodiments of the present invention may be a forskolin-inducible system, a hypoxia-inducible system, a tetracycline-regulatable (e.g., inducible or repressible) system, an alcohol-inducible system, a steroid-inducible system, a mifepristone (RU486)-inducible system, an ecdysone-inducible system, a rapamycin-inducible system, a metallothionein-inducible system, a hormone-inducible system, a plant hormone or analog-inducible system, a coumarate-inducible system, a temperature-inducible system, a pH-inducible system, and a metal-inducible system.

[0204] As discussed further below, a variety of suitable inductive systems have been described in the art, and others are discussed herein. Those skilled in the art will be able to select one or more suitable inductive systems for use in various embodiments of the present invention.

[0205] Hypoxia Induction System In some embodiments, the inducible system is a hypoxia inducible system.

[0206] In some embodiments, the hypoxia-inducible system comprises a hypoxia-inducible promoter operably linked to a sequence encoding a protein of interest. In some embodiments, the hypoxia-inducible promoter comprises at least one hypoxia response element (HRE) capable of binding to and activating hypoxia-inducible factor (HIF).

[0207] HIF is a family of transcription factors activated by a decrease in cellular oxygen levels. Under normal oxygen conditions, HIF undergoes hydroxylation and subsequent degradation. Hypoxic conditions stabilize HIF and prevent its degradation. This allows HIF to translocate to the nucleus, bind to HREs, and activate HRE-responsive genes.

[0208] A hypoxia-inducible promoter typically comprises an HRE that is capable of binding to and activating HIF operably linked to a minimal promoter. The particular promoter associated with the HRE can be selected depending on the circumstances, but a minimal promoter is typically preferred, particularly when it is desired to minimize background expression levels.

[0209] HREs are generally composed of a multimer of short conserved sequences called HIF-binding sites (HBSs). As the name suggests, HIF binds to the HBSs, activating the HRE and driving transcription of genes encoding proteins of interest. Preferably, an HRE contains multiple HBSs. Preferably, the spacing between adjacent core consensus sequences in adjacent HBSs is 3 to 50 nucleotides.

[0210] A suitable description of the hypoxia induction system can be found, for example, in US20110158947A1, which is incorporated herein by reference. Thus, in some embodiments, the hypoxia-inducible promoter is an adenosine A2B receptor (A2BR) promoter, a plasminogen activator receptor (uPAR) VEGF receptor (VEGFR1 and VEGFR2) promoter, a platelet-derived endothelial cell growth factor / thymidine phosphorylase (PDECGF / TP) promoter, a nitric oxide synthase (NOS) promoter, a phosphoglycerate kinase-1 (PGK-1) promoter, a pyruvate kinase M (PK-M) promoter, a glucose transporter 1 (GLUT1) promoter, a hypoxia-inducible factor (HIF-1) promoter, an early growth response 1 (Egr-1) promoter, a nuclear factor kB (NFkB) promoter, a hepatocyte growth factor activator (HGFA) promoter, a vascular endothelial growth factor (VEGF ... VEGF promoter, CXCL8 promoter, CCL11 promoter, transforming growth factor-beta (TGF-β) promoter, procollagen promoter, integrin-linked kinase (ILK) promoter, K1PDC1 promoter, erythropoietin (EPO) promoter, serine / threonine kinase-15 (STK15) promoter, histone demethylase Jumonji domain-containing 1A (JMJD1A) promoter, endothelin-2 (EDN2) promoter, choline kinase (Chk) promoter, sphingosine kinase 1 promoter, carcinoembryonic antigen (CEA, ceacam5) promoter, monocyte chemotactic protein-1 (MCP-1 / CCL2) promoter, MCP-5 (Ccl1 2) promoter, can be selected from the group consisting of prostate-specific antigen (PSA) promoter, c-Met promoter, matrix metalloproteinase class III beta-tubulin (TUBB3) promoter, glutamine:fructose-6-phosphate amidotransferase (GFAT) promoter, protein phosphatase 1 nuclear-targeting subunit beta-secretase (BACE1) promoter, and plasminogen activator inhibitor-1 (PAI-1) promoter.

[0211] Other naturally occurring hypoxia-inducible promoters are described in WO2016 / 146819, which is incorporated herein by reference. See, e.g., Table 4.

[0212] Hypoxia response elements are described in L. Marignol, M. Lawler, M. Coffey & D. Hollywood (2005) Achieving hypoxia inducible gene expression in tumors, Cancer Biology & Therapy, 4:4, 365-370; US6218179; Madan et al., PNAS 90:3928, 1993; JP2005095173A, US2006 / 0099709; and WO1999 / 048916. A mouse hypoxia response element is disclosed in US5942434.

[0213] Forskolin-inducible system In some embodiments, the inducible system is a forskolin-inducible system.

[0214] In some embodiments, the forskolin-inducible system comprises a forskolin-inducible promoter operably linked to a sequence encoding a protein of interest, hi some embodiments, the forskolin-inducible promoter comprises a forskolin-inducible cis-regulatory element (CRE) capable of being bound by CREB and / or AP1.

[0215] Although the CRE / promoter is referred to as forskolin-inducible, it can also be induced by other agents. The mechanism of induction by forskolin is via activation of adenylyl cyclase and the resulting increase in intracellular cAMP. Therefore, the CRE / promoter can also be induced by other activators of adenylyl cyclase or factors that increase intracellular cAMP.

[0216] Preferably, the CRE comprises at least two, more preferably at least three transcription factor binding sites (TFBS) for CREB and / or AP1.

[0217] Preferably, the forskolin-inducible promoter comprises a CRE, as discussed above, linked to a minimal promoter or proximal promoter, preferably linked to a minimal promoter.

[0218] The minimal promoter can be any suitable minimal promoter. A variety of minimal promoters are known in the art. Suitable minimal promoters include, but are not limited to, the CMV minimal promoter (CMV-MP), the YB-TATA minimal promoter (YB-TABA), the HSV thymidine kinase minimal promoter (MinTK), the SV40 minimal promoter (SV40-MP), or G6PC-MP (which is a non-TATA box MP derived from liver).

[0219] cAMP response elements are also described in US8986937, which is incorporated herein by reference. Exemplary naturally occurring cAMP-inducible promoters described therein include the PEPCK promoter (Roesler et al. (1998) The Journal of Biological Chemistry, 273, 14950-14957); a promoter containing a cAMP response element (CRE) located at position 294 relative to the translation start site of the human cyclin D2 promoter (Muniz et al. (2006) Biology of Reproduction 75(2): 279-288); and a promoter containing the cAMP response element (CRE) of the lactate dehydrogenase A subunit promoter (Welfeld et al. (1989) J. Biol. Chem. 264(12):6941-7). Exemplary cAMP-inducible promoters include the 236-nucleotide glycoprotein hormone alpha subunit promoter (AF401991) containing a cyclic AMP (cAMP) regulatory element (CRE), as described in U.S. Patent Application Publication No. 2008-0187942, published August 7, 2008, and incorporated herein by reference. Such elements can be used in forskolin-inducible promoters, as described above.

[0220] US9060310, incorporated herein by reference, describes additional cAMP response elements, such as various CRE palindromes and hairpins of SEQ ID NOs: 2, 3, 8, 9, 10, and 11. Such cAMP response elements can be used in forskolin-inducible promoters as described above.

[0221] US20070036810 and Mayr B, Montminy M., Nat Rev Nat Rev Mol Cell Biol 2001 Aug;2(8):599-609 (both incorporated by reference) disclose cAMP response elements containing the palindromic sequence TGACGTCA or asymmetric variations containing a CRE half-site with the core sequence TGAC. Such elements can be used in forskolin-inducible promoters as described above.

[0222] Temperature-Induced System The inducible system may be a temperature inducible system, for example, induced by a decrease in temperature.

[0223] In some embodiments, the temperature-inducible system includes a temperature-sensitive promoter operably linked to a sequence encoding a protein of interest. Preferably, the promoter is a synthetic cold-shock-responsive promoter derived from the S1006a gene (calcyclin) in CHO cells. The temperature sensitivity of the S1006a gene (calcyclin) promoter was identified by (Thaisuchat et al., 2011) and is incorporated herein by reference. In some embodiments, the inducible promoter is one of the synthetic cold-shock-responsive promoters shown in Figure 2 of (Thaisuchat et al., 2011). These promoters are induced by a decrease in temperature, as shown in Figure 3 of (Thaisuchat et al., 2011). Most of these synthetic promoter constructs exhibit expression similar to the known promoter SV40 at 37°C and are induced two- to three-fold when the temperature is decreased to 33°C. In some preferred embodiments, the inducible promoter is sps5 from Figure 2 of (Thaisuchat et al., 2011). In some preferred embodiments, the inducible promoter is sps8 from Figure 2 of (Thaisuchat et al., 2011).

[0224] pH-inducible system The inducible system may be a pH-inducible system, for example, induced by a decrease or increase in pH to which a cell containing the inducible system is exposed.

[0225] Preferably, the inducible system may comprise a pH-sensitive promoter operably linked to a sequence encoding the protein of interest. Preferably, the pH-sensitive promoter is a promoter that can be induced by a decrease in pH, i.e., a promoter that can be induced under acidic conditions. Suitable acid-inducible promoters are described in (Hou et al., 2016), which is incorporated herein by reference.

[0226] In some embodiments, the inducible promoter is a synthetic promoter derived from the YGP1 gene or CCW14 gene that is inducible under acidic conditions. The inducibility of the YGP1 gene or CCW14 gene under acidic conditions was studied and improved by modifying transcription factor binding sites in (Rajkumar et al., 2016), which is incorporated herein by reference. In some embodiments, the inducible promoter is one of the synthetic promoters inducible under acidic conditions shown in Figures 1A, 2A, 3A, and 4A of (Rajkumar et al., 2016). These promoters are induced by a decrease in pH, as shown in Figures 1B, 2B, 3B, and 4B of (Rajkumar et al., 2016). Most of these synthetic promoters are induced up to 10-15-fold when the pH is reduced from 6 to 3. In some preferred embodiments, the inducible promoter is YGP1pr from Figure 1 of (Rajkumar et al., 2016). In another preferred embodiment, the inducible promoter is YGP1pr from Figure 1 of (Rajkumar et al., 2016).

[0227] Osmolality induction The inducible system may be an osmolality-induced system.

[0228] Preferably, the osmolality-induced system may comprise an osmolality-sensitive promoter operably linked to a sequence encoding a protein of interest. Preferably, the osmolality-induced promoter is described in Zhang et al., https: / / doi.org / 10.1007 / s11033-012-1566-3, incorporated herein by reference.

[0229] Carbon source induction The inducible system may be a carbon source inducible system, preferably induced by the addition of a specific carbon source, such as a non-sugar carbon source. Alternatively, the inducible promoter may be induced by the withdrawal or absence of the carbon source.

[0230] Preferably, the carbon source-inducible system comprises a carbon source-inducible promoter operably linked to a sequence encoding the protein of interest. Suitable promoters induced by the presence or absence of various carbon sources are described in (Weinhandl et al., 2014), which is incorporated herein by reference.

[0231] Alcohol (e.g., ethanol) induction The inducible system may be an alcohol-inducible system, preferably induced by the addition of ethanol. Preferably, the alcohol-inducible system comprises an alcohol-inducible promoter operably linked to a sequence encoding a protein of interest. Suitable promoters induced by ethanol are described in Matsuzawa et al., https: / / doi.org / 10.1007 / s00253-013-4812-2, which is incorporated herein by reference.

[0232] Amino acid derivatization The inducible system may be an amino acid inducible system, preferably induced by the addition of one or more amino acids. Preferably, the amino acid inducible system may include an amino acid inducible promoter operably linked to a sequence encoding a protein of interest. Preferably, the amino acid may be an aromatic amino acid. Preferably, the amino acid may be GABA (gamma-aminobutyric acid), which is also a neurotransmitter. Suitable promoters inducible by aromatic amino acids and GABA are described in Kim et al., https: / / doi.org / 10.1007 / s00253-014-6303-5, incorporated herein by reference.

[0233] Ecdysone induction The inducible system may be a steroid hormone-inducible system, preferably one that is induced by a steroid hormone. Preferably, the steroid hormone-inducible system may comprise a steroid hormone-inducible promoter operably linked to a sequence encoding a protein of interest. Preferably, the steroid hormone may be ecdysone. A mammalian ecdysone-inducible system was created by No, Yao, and Evans (No, Yao, and Evans, 1996) and is incorporated herein by reference. Expression of a modified ecdysone receptor in mammalian cells allows for induction of expression from an ecdysone-responsive promoter upon addition of ecdysone, as shown in Figure 2 of (No, Yao, and Evans, 1996). This system exhibited lower basal activity and higher inducibility than the tetracycline-inducible system, as shown in Figure 6 of (No, Yao, and Evans, 1996). A suitable commercially available inducible system is available from Agilent technologies and is described in (Agilent technologies, 2015), which is incorporated herein by reference.

[0234] Tetracycline-regulated induction The inducible system may be a tetracycline-inducible system. Suitably, the tetracycline-inducible system may comprise a promoter that is induced by the presence or absence of tetracycline or a derivative thereof operably linked to a sequence encoding a protein of interest.

[0235] In some embodiments, the promoter can be induced by the presence or absence of tetracycline or its derivatives. A suitable promoter induced by the absence of tetracycline or its derivatives is a promoter in a tet-OFF system. In the tet-OFF system, a tetracycline-controlled transactivator (tTA) enables transcriptional activation of a tTA-dependent promoter in the absence of tetracycline or its derivatives. tTA and tTA-dependent promoters were first developed by (Gossen and Bujard, 1992), which is incorporated herein by reference. tTA was developed by fusing the Tn10-encoded tetracycline resistance operon (tet repressor) of Escherichia coli with an activating cyclin-controlled transactivator (tTA), and a tTA-dependent promoter was developed by combining a tet operator sequence with a minimal promoter from the human cytomegalovirus promoter IE (hCMV-IE) (Gossen and Bujard, 1992). Upon addition of tetracycline or a derivative thereof, tTA is unable to bind to its target sequence within the tTA-dependent promoter, resulting in abolishment of expression from the tTA-dependent promoter. This is shown in Figure 1A and described on page 96 of (Jaisser, 2000), which is incorporated herein by reference. The mechanism of the conformational change brought about by the binding of tetracycline or a derivative thereof to tTA is described in Orth et al., https: / / doi.org / 10.1038 / 73324, which is incorporated herein by reference.

[0236] A preferred promoter induced by the presence of tetracycline or its derivatives is a promoter in the tet-ON system. In the tet-ON system, the reverse tetracycline-controlled transactivator (rtTA) enables transcriptional activation of a tTA-dependent promoter in the presence of tetracycline or its derivatives, as described in Gossen et al., DOI:10.1126 / science.7792603, which is incorporated herein by reference. In the absence of tetracycline or its derivatives, tTA can no longer bind to its target sequence in the tTA-dependent promoter, resulting in a loss of expression from the tTA-dependent promoter. This is shown in Figure 1B and described on page 96 of (Jaisser, 2000). Preferably, an improved variant of the reverse tetracycline-controlled transactivator (rtTA) can be used.

[0237] Suitable improved variants are listed in Table 1 of (Urlinger et al., 2000), which is incorporated herein by reference. Variants rtTA-S2 and rtTA-M2 were shown to have lower basal activity in Figure 3 of (Urlinger et al., 2000), indicating minimal background expression from a tTA-dependent promoter in the absence of tetracycline or its derivatives. In addition, rtTA-M2 exhibited increased sensitivity to tetracycline and its derivatives, as shown in Figure 3 of (Urlinger et al., 2000). It functions at a concentration ten times lower than rtTA. In some preferred embodiments, the improved variant of rtTA is rtTA-M2 from (Urlinger et al., 2000).

[0238] Alternative improved variants are listed in Table 1 of (Zhou et al., 2006). Most of these variants were shown to have higher transcriptional activity and doxycycline sensitivity than rtTA, as shown in Figure 3 of (Zhou et al., 2006). The best-performing variant was 7-fold more active and 100-fold more sensitive to doxycycline. In some preferred embodiments, the improved variant of rtTA is V14, V15, or V16 from (Zhou et al., 2006).

[0239] A suitable commercially available tetracycline inducible system is the T-Rex system from (Life-Technologies, Inc., 2014).

[0240] Kumart induction In some embodiments, the inducible system may be a coumarate-inducible system, preferably one in which the promoter is inducible by the presence or absence of coumarate. Preferably, the coumarate-inducible system may comprise a promoter inducible by the presence or absence of coumarate operably linked to a sequence encoding a protein of interest.

[0241] In the coumarate switch system from (Mullick et al., 2006), incorporated herein by reference, the repressor CymR blocks transcription from a promoter containing a CuO sequence placed downstream of the promoter. Upon addition of coumarate, the CymR repressor is unable to bind to CuO, allowing transcription from the CuO-containing promoter to proceed. This is shown in Figure 1B and Figure 2 from (Mullick et al., 2006).

[0242] In an alternative coumarate switch system, a chimeric transactivator (cTA) created from a fusion of CymR and the activation domain of VP16 does not prevent transcription from a promoter containing a CuO sequence upstream of the promoter in the presence of coumarate. In the absence of coumarate, the chimeric transactivator (cTA) binds to the CuO sequence and prevents transcription. This is shown in Figure 1C and Figure 3 from (Mullick et al., 2006).

[0243] In the third configuration, the reverse chimeric transactivator (rcTA) prevents transcription from promoters containing a CuO sequence upstream of the promoter in the absence of coumarate. In the presence of coumarate, rcTA binds to the CuO sequence, allowing transcription from promoters containing the CuO sequence to proceed. This is shown in Figure 1D and Figure 7 from (Mullick et al., 2006).

[0244] A suitable commercially available coumarate inducible system can be found from SBI Biosciences (SBI, 2020), which is incorporated herein by reference.

[0245] 4-hydroxytamoxifen (OHT) induction The inducible system may preferably be a 4-hydroxytamoxifen (OHT) inducible system, comprising a promoter that can be induced by 4-hydroxytamoxifen (OHT). Preferably, the inducible system may comprise a promoter that is induced by the presence of 4-hydroxytamoxifen (OHT) operably linked to a sequence encoding a protein of interest. A suitable 4-hydroxytamoxifen inducible promoter is described by Feil et al., https: / / doi.org / 10.1006 / bbrc.1997.7124, which is incorporated herein by reference.

[0246] Gas induction The inducible system may preferably be a gas-inducible system comprising a gas-inducible promoter, such as a promoter that may be acetaldehyde-inducible. Preferably, the inducible system comprises a promoter that is induced by the presence of a gas operably linked to a sequence encoding a protein of interest. Suitable gas-inducible promoters are described in Weber et al., https: / / doi.org / 10.1038 / nbt1021, incorporated herein by reference.

[0247] Riboswitch, ribozyme and aptazyme induction The inducible system may be a riboswitch, ribozyme, or aptazyme inducible system, and preferably is induced by the presence or absence of a ribozyme. The ribozyme, in turn, is induced by a ligand. Preferably, the inducible system comprises a promoter that is induced by the presence of a ribozyme operably linked to a sequence encoding a protein of interest.

[0248] The inducible promoter may be induced in the absence of a metabolite. In some embodiments, the metabolite may be glucosamine-6-phosphate responsive. A suitable ribozyme that acts as a glucosamine-6-phosphate responsive gene repressor is described by Winkler et al., https: / / doi.org / 10.1038 / nature02362, which is incorporated herein by reference.

[0249] Protein expression can also be downregulated by ligand-induced aptazymes. Protein expression can be downregulated by aptazymes, which downregulate protein expression through self-cleavage induced by small ribozyme molecules, causing mRNA degradation (Zhong et al., 2016), which is incorporated herein by reference. A suitable aptazyme is shown in Figure 4A of (Zhong et al., 2016). These aptazymes reduce the relative expression of the target gene, as shown in Figure 4 of (Zhong et al., 2016).

[0250] On the other hand, protein expression can also be upregulated by small molecule-dependent ribozymes.Ribozymes can be tetracycline-dependent.Suitable tetracycline-dependent ribozymes can be turned on by preventing ribozyme cleavage, otherwise cleave mRNA in the absence of ligand, as described in Beilstein et al., https: / / doi.org / 10.1021 / sb500270h, which is incorporated herein by reference.

[0251] Protein expression can also be regulated by guanine-dependent aptazymes, as described by Nomura et al., https: / / doi.org / 10.1039 / C2CC33140C, incorporated herein by reference.

[0252] Additionally, RNA constructs combining drug-inducible allosteric ribozymes with microRNA precursor analogs that allow for chemical induction of RNAi in mammalian cells are described in Kumar et al., https: / / doi.org / 10.1021 / ja905596t, incorporated herein by reference.

[0253] Metallothionein induction The inducible system may preferably be a metallothionein-inducible system comprising a metallothionein-inducible promoter as described in the literature. Preferably, the inducible system comprises a promoter that is induced by the presence of metallothionein operably linked to a sequence encoding a protein of interest. See, for example, Shinichiro Takahashi, "Positive and Negative Regulators of the Metallothionein Gene," Molecular Medicine Reports, March 9, 2015, pp. 795-799, which is incorporated herein by reference.

[0254] Rapamycin induction The inducible system may be a rapamycin-inducible system. Preferably, the inducible system comprises a promoter induced by the presence of rapamycin operably linked to a sequence encoding a protein of interest. The inducible promoter can be induced by a small molecule drug, such as rapamycin. A humanized system for pharmacological control of gene expression using rapamycin is described in Rivera et al., Nature Medicine 2, 1028-1032 (1996) https: / / doi.org / 10.1038 / nm0996-1028, incorporated herein by reference.

[0255] rheoswitch The inducible system may be a rheoswitch. Preferably, the system may include a promoter that can be induced by a small synthetic molecule. In some embodiments, these small synthetic molecules may be diacylhydrazine ligands. A suitable system for such inducible up- and down-regulation of gene expression is described in Cress et al., https: / / cancerres.aacrjournals.org / content / 66 / 8_Supplement / 27.2, incorporated herein by reference.

[0256] CRISPR induction Gene expression can be induced by CRISPR-based transcriptional regulators. Nuclease-deficient Cas9 can be directed to a sequence of interest by designing its associated single guide RNA (sgRNA), and gene expression can be modulated by tethering an effector domain to the sgRNA-Cas9 complex, as shown in Figure 1A of (Ferry, Lyutova, and Fulga, 2017), incorporated herein by reference. Preferably, the inducible system can be an inducible CRISPR-TR platform. A suitable multifunctional inducible CRISPR-TR platform based on minimal manipulation of sgRNAs is described in (Ferry, Lyutova, and Fulga, 2017).

[0257] CRISPR-based transcriptional regulation can in turn be induced by drugs. A suitable drug-inducible CRISPR-based transcriptional regulator system is shown in (Zhang et al., 2019).

[0258] Chemically induced access induction The inducible system may be a chemically induced access system. A suitable such small molecule-based system for controlling protein abundance or activity is described in Liang et al., 10.1126 / scisignal.2001449, incorporated herein by reference.

[0259] Gene expression may also be chemically induced by induced proximity caused by chemical molecules that combine two protein-binding surfaces, as shown in (Belshaw et al., 1996), which is incorporated herein by reference. Transcriptional activation of a gene of interest by chemically induced proximity by molecules that combine two protein-binding surfaces is shown in Figure 3 of (Belshaw et al., 1996).

[0260] As discussed above, in a preferred embodiment of the present invention, the or each inducible system is a chemical-inducible access system, in particular a plant hormone or plant hormone analogue-inducible access system.

[0261] Therefore, suitably, in a preferred embodiment of the present invention, the inducible system used in the method of the present invention is a chemical-inducible access system, more preferably a plant hormone or plant hormone analogue-inducible access system.

[0262] Therefore, suitably in a preferred embodiment of the method of the present invention, the cell provided may comprise a cell of the eighth aspect of the present invention. Suitably, the cell provided comprises a first and optionally a second chemical-induced access system.

[0263] In one embodiment, the first and second chemical-inducible access systems may be the plant hormone or plant hormone analog-inducible access systems described herein. Preferably, in such an embodiment, the first chemical-inducible access system comprises the first and second constructs defined herein, and the second chemical-inducible access system comprises the third and fourth constructs defined herein. In one embodiment, at least one of the chemical-inducible access systems is a plant hormone-inducible access system. In one embodiment, at least one of the chemical-inducible systems is an abscisic acid-inducible access system defined herein. In one embodiment, at least one of the chemical-inducible systems is selected from the caffeine-inducible access system defined herein, the mandipropamid-inducible access system defined herein, and the gibberellin-inducible access system defined herein.

[0264] In a preferred embodiment, the first chemically induced access system is selected from a caffeine-induced access system as defined herein, a mandipropamid-induced access system as defined herein, and a gibberellin-induced access system as defined herein, and the second chemically induced access system is an abscisic acid-induced access system as defined herein.

[0265] Preferred plant hormone or plant hormone analogue inducible access systems for use in the present invention are described in detail below.

[0266] Components of chemical-induced access systems Effector domain The first construct of the first inducible system according to the invention comprises two effector domains, one in the first chimeric protein and one in the second chimeric protein, and the third construct of the second inducible system also comprises two effector domains, one in the third chimeric protein and one in the fourth chimeric protein.

[0267] Preferably, the effector domain of either the first inducible system or the second inducible system can be selected from any transactivation domain or DNA-binding domain, so long as the effector domains of the first and second chimeric proteins are different and the effector domains of the third and fourth chimeric proteins are different. Preferably, the effector domains of the first and third chimeric proteins can be transactivation domains, and preferably they can both be the same transactivation domain. Preferably, the effector domains of the second and fourth chimeric proteins can be DNA-binding domains, and preferably they are different DNA-binding domains.

[0268] Preferably, the transactivation domains of the first and third chimeric proteins may be any transactivation domain, preferably selected from Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4, and p53, NFAT, NF-κB, VP16, and VP34 in yeast, as further described below.

[0269] Preferably, the DNA binding domains of the second and fourth chimeric proteins may be any DNA binding domain, preferably selected from LexA, dI-SceI, and Gal4, as further described below.

[0270] Preferably, the effector domain of each first construct is selected from a transactivation domain or a DNA-binding domain. Preferably, any DNA-binding domain or transactivation domain can be used in the first construct. Therefore, preferably, any transactivation domain can be used in the first chimeric protein. Therefore, preferably, any DNA-binding domain can be used in the second chimeric protein. Preferably, for example, the transactivation domain of the first construct, preferably the first chimeric protein, can be selected from Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4, and p53, NFAT, NF-κB, VP16, or VP34 in yeast. Preferably, for example, the DNA-binding domain of the first construct, preferably the second chimeric protein, can be selected from LexA, dI-SceI, and Gal4. In one embodiment, the transactivation domain of the first construct, preferably the first chimeric protein, is VP16. In one embodiment, the DNA binding domain of the first construct, preferably the second chimeric protein, is a catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI).

[0271] Preferably, the effector domain of each third construct is selected from a transactivation domain or a DNA-binding domain. Preferably, any DNA-binding domain or transactivation domain can be used in the third construct. Therefore, preferably, any transactivation domain can be used in the third chimeric protein. Therefore, preferably, any DNA-binding domain can be used in the fourth chimeric protein. Preferably, for example, the transactivation domain of the third construct, preferably the third chimeric protein, can be selected from Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4, and p53, NFAT, NF-κB, VP16, or VP34 in yeast. Preferably, for example, the DNA-binding domain of the third construct, preferably the fourth chimeric protein, can be selected from LexA, dI-SceI, and Gal4. In one embodiment, the transactivation domain of the third construct, preferably the third chimeric protein, is VP16. In one embodiment, the DNA binding domain of the third construct, preferably the fourth chimeric protein, is a Gal4 DNA binding domain.

[0272] In some embodiments, the effector domain is a transactivation domain, as described above.

[0273] Preferably, the transactivation domain of the first or third construct is brought into close proximity with a dI-SceI DNA binding domain. Preferably, the proximity of a transactivation domain according to the invention to a dI-SceI DNA binding domain induces transcription from the dI-SceI binding site.

[0274] Preferably, the transactivation domain of the first or third construct may be associated with a dI-SceI DNA binding domain. Preferably, the transactivation domain may be bound to a dI-SceI DNA binding domain.

[0275] Preferably, the transactivation domain of the first or third construct is brought into close proximity with a Gal4 DNA binding domain. Preferably, the proximity of the transactivation domain of the present invention to the Gal4 DNA binding domain induces transcription from a Gal4 upstream activation sequence.

[0276] Suitably, the transactivation domain of the first or third construct may be associated with a Gal4 DNA binding domain. Suitably, the transactivation domain may be linked to a Gal4 upstream activation sequence.

[0277] It will be appreciated by those skilled in the art that the transactivation domain according to the present invention may be any transactivation domain. By way of example only, the transactivation domain may be Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4, and p53, NFAT, NF-κB, VP16, or VP34 in yeast.

[0278] Preferably, the transactivation domain is VP16.

[0279] More preferably, the transactivation domain is herpes simplex virus VP16.

[0280] Preferably, the VP16 transactivation domain comprises the sequence set forth in SEQ ID NO: 11 or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0281] Preferably, the VP16 transactivation domain consists of the sequence set forth in SEQ ID NO:11.

[0282] In some embodiments, the effector domain is a DNA binding domain, as described above.

[0283] It will be appreciated by those skilled in the art that the DNA binding domain according to the present invention may be any DNA binding domain. By way of example only, the DNA binding domain may be LexA, dI-SceI or Gal4.

[0284] Preferably, the DNA binding domain is either dI-SceI or Gal4, as described herein below.

[0285] In the context of the present invention, modified endonucleases are provided in which the endonucleases have been modified to be catalytically inactive while retaining the ability to bind DNA.

[0286] In some embodiments, the effector domain is a modified I-SceI endonuclease DNA binding domain. Suitably in the first or third construct embodiments.

[0287] In a preferred embodiment, the modified endonuclease is an I-SceI endonuclease that has been modified so that it is catalytically inactive while retaining its ability to bind DNA. An I-SceI endonuclease that is catalytically inactive while retaining DNA-binding function is sometimes referred to herein as a "dead" I-SceI (dI-SceI).

[0288] It will be appreciated that the endonuclease (e.g., I-SceI) can be modified by any means or at any location that effectively removes the function of the endonuclease but retains DNA binding, such that the modified endonuclease is catalytically inactive, leaving only the DNA binding function. Suitably, the modification may comprise one or more mutations.

[0289] An engineered mutant or variant shall be understood as a protein or nucleic acid sequence that shares sequence identity with the original sequence (or with a specific fragment of the original sequence), but which contains at least one alteration (e.g., substitution, addition, or deletion) compared to the original sequence.

[0290] In a preferred embodiment, the dI-SceI has at least one mutation in its active site.

[0291] Suitably, the mutation may be a substitution, addition or deletion.

[0292] Preferably, the mutation is a substitution. Preferably, the mutation is a non-conservative substitution. "Non-conservative" means that the substitution does not retain the characteristics of the original amino acid residue at the recited position.

[0293] In a preferred embodiment, the modified I-SceI comprises at least one modification (e.g., substitution, addition, or deletion) compared to the original sequence. Suitably, the modified I-SceI comprises at least 2 modifications, at least 3 modifications, at least 4 modifications, at least 5 modifications, at least 6 modifications, at least 7 modifications, at least 8 modifications, at least 9 modifications, at least 10 modifications, at least 11 modifications, at least 12 modifications, at least 13 modifications, at least 14 modifications, at least 15 modifications, at least 16 modifications, at least 17 modifications, at least 18 modifications, at least 19 modifications, at least 20 modifications, at least 21 modifications, at least 22 modifications, at least 23 modifications, at least 24 modifications, at least 25 modifications, at least 26 modifications, at least 27 modifications, at least 28 modifications, at least 29 modifications, or at least 30 modifications.

[0294] Preferably, the modified I-SceI comprises at least 35 modifications, at least 40 modifications, at least 45 modifications, at least 50 modifications, at least 55 modifications, at least 60 modifications, at least 75 modifications, at least 80 modifications, at least 85 modifications, at least 90 modifications, at least 95 modifications, or at least 100 modifications compared to the original sequence.

[0295] Preferably, the modified I-SceI comprises at least 120 modifications, at least 140 modifications, at least 160 modifications, at least 180 modifications, at least 200 modifications, at least 220 modifications, at least 240 modifications, at least 260 modifications, at least 280 modifications or at least 300 modifications compared to the original sequence.

[0296] In a preferred embodiment, dI-SceI preferably comprises at least two mutations in its active site, preferably at least two non-conservative substitutions in its active site, preferably at least two alanine substitutions in its active site.

[0297] Preferably, dI-Scel comprises substitutions at positions 44 and / or 145 of SEQ ID NO: 12. Preferably, dI-Scel comprises substitutions at positions 44 and 145 of SEQ ID NO: 12. Preferably, dI-Scel comprises substitutions Asp44Ser and / or Asp145Ala in SEQ ID NO: 12. Preferably, dI-Scel comprises substitutions Asp44Ser and Asp145Ala in SEQ ID NO: 12.

[0298] Advantageously, the DNA recognition region of dI-Scel remains unchanged (compared to the original sequence), thus retaining its DNA binding function.

[0299] Suitably, the dI-SceI DNA binding domain may comprise the sequence set forth in SEQ ID NO: 12, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, and comprising substitutions at positions 44 and / or 145, or corresponding positions, as described above.

[0300] "Position corresponding thereto," as used herein, means the same position as the referenced position, but in an orthologous or homologous sequence, e.g., the same position in the same protein from a different organism. Corresponding positions can be determined by one skilled in the art by alignment of a reference sequence with an orthologous or homologous sequence. Suitable alignment tools are available and well known in the art, such as BLAST.

[0301] Preferably, the dI-SceI DNA binding domain consists of the sequence set forth in SEQ ID NO:13.

[0302] Alternatively, the DNA binding domain may be LexA, preferably LexA from E. coli.

[0303] Preferably, the LexA DNA binding domain may comprise the sequence set forth in SEQ ID NO: 21 or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0304] Preferably, the LexA DNA binding domain consists of SEQ ID NO:21.

[0305] Alternatively, the DNA binding domain may be Gal4, preferably Gal4 from S. cerevisiae.

[0306] Preferably, the Gal4 DNA binding domain may comprise the sequence set forth in SEQ ID NO: 22 or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0307] Preferably, the Gal4 DNA binding domain consists of SEQ ID NO:22.

[0308] Auxin-binding domain The first or similarly third nucleic acid construct according to the present invention may comprise two auxin-binding domains as plant hormone inducer-binding domains, one in the first or third chimeric protein and one in the second or fourth chimeric protein. Preferably, the auxin-binding domains are selected from the F-box transport inhibitor response 1 (TIR1) protein and the transcriptional corepressor Auxin / Indole-3-acetic acid (AUX / IAA) protein (referred to as AID), or a fragment or derivative thereof.

[0309] "Fragment or derivative thereof," as used herein, means a modified form of a protein, such as a truncated form of the protein, or a mutated form of the protein, suitable types of truncations and mutations are described elsewhere herein. Reference herein to any protein component of a system may also refer to a fragment or derivative of said protein, some of which are specifically described herein.

[0310] According to the present invention, the F-box transport inhibitor response 1 (TIR1) protein and the transcriptional corepressor auxin / indole-3-acetic acid (AUX / IAA) protein (termed AID) heterodimerize in the presence of auxin.

[0311] In one embodiment, the auxin binding domain is the F-box transport inhibitor response 1 protein (TIR1).

[0312] TIR1 is a member of the F-box family of proteins, which are known to be protein motifs of approximately 50 amino acids that function to mediate protein-protein interactions.

[0313] Preferably, the F-box protein is TIR1. Advantageously, the TIR1 according to the present invention is capable of expressing the TIR1 gene in any plant, such as Amborella trichopoda, Arabidopsis thaliana, Brachypodium distachyon, Brassica rapa, Brassica napus, papaya, Cicer arietinum, citrus, cucumber, Eucalyptus grandis, Glycine max, Gossypium raimondii, Marchantia polymorpha, Medicago truncatula, rice, Oryza sativa, Petunia The origin may be from Phalaenopsis hybrida, Phalaenopsis equestris, Physcomitrella patens, Norway spruce (Picea abies), cottonwood (Populus trichocarpa), peach (Prunus persica), castor bean (Ricinus communis), Selaginella moellendorffii, tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), sorghum (Sorghum bicolor), wheat (Triticum aestivum), einkorn (Triticum urartu), bladderwort (Utricularia gibba), grape (Vitis vinifera), or corn (Zea mays).

[0314] In a preferred embodiment, the TIR1 protein is rice TIR1 (Oryza sativa TIR1; osTIR1).

[0315] Preferably, the rice TIR1 (osTIR1) is modified. Hence, preferably, the osTIR1 is a modified mutant or variant of a reference osTIR1.

[0316] An engineered mutant or variant shall be understood as a protein or nucleic acid sequence that shares sequence identity with the original sequence (or with a specific fragment of the reference sequence), but contains at least one alteration (e.g., substitution, addition, or deletion) compared to the reference sequence.

[0317] Suitably, the modification may comprise one or more mutations. Suitably, the mutations may be substitutions, additions or deletions.

[0318] The term "reference sequence" as used herein means the entire native or wild-type sequence of the same protein or nucleic acid from the same organism, or a fragment thereof. Preferably, the reference sequence is unmodified.

[0319] In a preferred embodiment, the modified TIR1 comprises at least one alteration (e.g., a substitution, addition, or deletion) compared to a reference sequence. Preferably, the modified variant of TIR1 comprises at least two alterations, at least three alterations, at least four alterations, at least five alterations, at least six alterations, at least seven alterations, at least eight alterations, at least nine alterations, at least ten alterations, at least eleven alterations, at least 12 alterations, at least 13 alterations, at least 14 alterations, at least 15 alterations, at least 16 alterations, at least 17 alterations, at least 18 alterations, at least 19 alterations, at least 20 alterations, at least 21 alterations, at least 22 alterations, at least 23 alterations, at least 24 alterations, at least 25 alterations, at least 26 alterations, at least 27 alterations, at least 28 alterations, at least 29 alterations, or at least 30 alterations compared to the reference sequence.

[0320] Preferably, the modified TIR1 comprises at least 35 modifications, at least 40 modifications, at least 45 modifications, at least 50 modifications, at least 55 modifications, at least 60 modifications, at least 75 modifications, at least 80 modifications, at least 85 modifications, at least 90 modifications, at least 95 modifications, or at least 100 modifications compared to the reference sequence.

[0321] Preferably, the modified TIR1 comprises at least 120 modifications, at least 140 modifications, at least 160 modifications, at least 180 modifications, at least 200 modifications, at least 220 modifications, at least 240 modifications, at least 260 modifications, at least 280 modifications or at least 300 modifications compared to the reference sequence.

[0322] In a preferred embodiment, the modified TIR1 comprises at least two mutations. Preferably, the modified TIR1 comprises at least two substitutions. Preferably, the modified TIR1 comprises a substitution at position 7 and / or 10 of SEQ ID NO: 14. Preferably, the modified TIR1 comprises a substitution at position 7 and 10 of SEQ ID NO: 14. Preferably, the modified TIR1 comprises the substitutions E7K and / or E10K in SEQ ID NO: 14. Preferably, the modified TIR1 comprises the substitutions E7K and E10K in SEQ ID NO: 14.

[0323] Suitably, TIR1 comprises the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, and may comprise a substitution at or corresponding to positions 7 and / or 10 as described above.

[0324] Preferably, TIR1 consists of the sequence set forth in SEQ ID NO:15.

[0325] Optionally, the modified TIR1 may further comprise a mutation at position 74 of SEQ ID NO: 14. Therefore, optionally, the modified TIR1 may comprise mutations at positions 7 and / or 10 and / or 74 of SEQ ID NO: 14. Therefore, optionally, the modified TIR1 may comprise mutations at positions 7, 10 and 74 of SEQ ID NO: 14. Suitably, the modification at position 74 may be a substitution. Suitably, the substitution is F74G. Optionally, the modified TIR1 comprises the substitutions E7K and / or E10K and / or F74G in SEQ ID NO: 14. Suitably, the modified TIR1 comprises the substitutions E7K, E10K and F74G in SEQ ID NO: 14.

[0326] Suitably, TIR1 comprises the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, and may comprise a substitution at or corresponding to positions 7 and / or 10 and / or 74 as described above.

[0327] Preferably, TIR1 consists of the sequence set forth in SEQ ID NO: 22. Preferably, when the auxin is 5-Ph-IAA, TIR1 is used with an additional modification at position 74. Advantageously, constructs using this modified TIR1 do not show detectable leaky degradation and require lower ligand concentrations.

[0328] As previously discussed, the transcriptional corepressor auxin / indole-3-acetic acid (AUX / IAA) protein (termed AID) heterodimerizes with TIR1 in the presence of auxin.

[0329] In one embodiment, the auxin binding protein is therefore an AID protein.

[0330] Suitably, the AID protein may originate from any of the following plant species: Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, Brassica napus, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Scutellaria baicalensis, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia crus-galli, grape, or maize.

[0331] Suitably, the AID is a truncated version of the AID protein.

[0332] In the context of the present invention, a truncated version of AID is a truncated version of a reference sequence (ie, not the full-length sequence), but shares complete sequence identity with the corresponding portion of the reference sequence.

[0333] In one embodiment, the AID protein is a truncation of the sequence set forth in SEQ ID NO: 16. Suitably, the truncation may comprise up to 100 amino acids, up to 90 amino acids, up to 80 amino acids, up to 70 amino acids, up to 60 amino acids, up to 50 amino acids, up to 40 amino acids, up to 30 amino acids. Suitably, the truncation is from the N-terminus or C-terminus of the amino acid sequence.

[0334] In a preferred embodiment of the present invention, the truncated version of AID is a 133 amino acid truncated version in which C-terminal domains 3 and 4 are removed, also known as Delta34 variant (AIDΔ34). Preferably, AIDΔ34 is a truncated version of the reference sequence. Preferably, AIDΔ34 is a C-terminal truncation of the reference sequence.

[0335] Suitably, AIDΔ34 comprises the sequence set forth in SEQ ID NO: 17 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto.

[0336] Preferably, AIDΔ34 consists of the sequence set forth in SEQ ID NO:17.

[0337] In an alternative embodiment of the invention, the truncated version of AID has a 63 amino acid truncation at the N-terminus and a 97 amino acid truncation at the C-terminus, which is alternatively known as an mAID variant. Preferably, the mAID is a truncated version of the reference sequence.

[0338] Suitably, the mAID comprises the sequence set forth in SEQ ID NO: 23 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto.

[0339] Suitably, the mAID consists of the sequence set forth in SEQ ID NO:23.

[0340] Abscisic acid binding domain The first or similarly third nucleic acid construct according to the present invention may comprise two abscisic acid-binding domains as plant hormone inducer-binding domains, one in the third or first chimeric protein and one in the fourth or second chimeric protein. Preferably, the abscisic acid-binding domains are selected from abscisic acid insensitive 1 protein (ABI1) or pyrabactin resistance-like protein (PYL1), or fragments or derivatives thereof.

[0341] According to the present invention, the ABI1 and PYL1 proteins heterodimerize in the presence of abscisic acid.

[0342] In one embodiment, the abscisic acid binding domain is ABI1.

[0343] ABI1 is a member of the protein serine / threonine phosphatase 2C class (PP2C).

[0344] Preferably, the PP2C protein is ABI1. Preferably, ABI1 according to the present invention may originate from any plant, for example, Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, rapeseed, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Scutellaria baicalensis, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia crus- tularensis, grape, or maize.

[0345] In a preferred embodiment, the ABI1 protein is Arabidopsis ABI1.

[0346] Preferably, the ABI1 protein is modified, and therefore preferably the ABI1 protein is a modified mutant or variant of a reference ABI1 protein.

[0347] An engineered mutant or variant shall be understood as a protein or nucleic acid sequence that shares sequence identity with the original sequence (or with a specific fragment of the reference sequence), but contains at least one alteration (e.g., substitution, addition, or deletion) compared to the reference sequence.

[0348] Suitably, the modification may comprise one or more mutations. Suitably, the mutations may be substitutions, additions or deletions.

[0349] Suitable such modifications are defined herein above with respect to the auxin-binding domain.

[0350] The term "reference sequence" as used herein means the entire native or wild-type sequence of the same protein or nucleic acid from the same organism, or a fragment thereof. Preferably, the reference sequence is unmodified.

[0351] Preferably, the ABI1 protein has been modified by deletion, preferably by truncation, which leaves only the complementary surface of the ABI1 protein operable to interact with the PYL1 protein, preferably the ABI1 protein has been modified to consist of the complementary surface of the ABI1 protein.

[0352] In one embodiment, the ABI1 protein comprises a truncation at its N-terminus and at its C-terminus. In one embodiment, the ABI1 protein comprises a truncation of amino acids 1 to 125 from its N-terminus, preferably 125 amino acids. In one embodiment, the ABI1 protein comprises a truncation of amino acid residues 424 to 434 from its C-terminus, preferably 10 amino acids. Preferably, the ABI1 protein comprises amino acids 126 to 423 of SEQ ID NO: 24.

[0353] In one embodiment, the ABI1 protein consists of amino acids 126 to 423 of SEQ ID NO: 24, also known as "ABI1cs." In one embodiment, the ABIcs protein consists of SEQ ID NO: 25.

[0354] As previously discussed, the PYL1 protein heterodimerizes with the ABI1 protein in the presence of abscisic acid.

[0355] In one embodiment, the abscisic acid binding protein is therefore a PYL1 protein.

[0356] Suitably, the PYL1 protein may originate from any of the following plant species: Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, Brassica napus, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Scutellaria baicalensis, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia crus-galli, grape, or maize.

[0357] In a preferred embodiment, the PYL1 protein is Arabidopsis PYL1.

[0358] Preferably, the PYL1 protein is modified, and therefore preferably the PYL1 protein is a modified mutant or variant of a reference PYL1 protein.

[0359] An engineered mutant or variant shall be understood as a protein or nucleic acid sequence that shares sequence identity with the original sequence (or with a specific fragment of the reference sequence), but contains at least one alteration (e.g., substitution, addition, or deletion) compared to the reference sequence.

[0360] Suitably, the modification may comprise one or more mutations. Suitably, the mutations may be substitutions, additions or deletions.

[0361] Suitable such modifications are defined herein above with respect to the auxin-binding domain.

[0362] The term "reference sequence" as used herein means the entire native or wild-type sequence of the same protein or nucleic acid from the same organism, or a fragment thereof. Preferably, the reference sequence is unmodified.

[0363] Preferably, the PYL1 protein has been modified by deletion, preferably by truncation, which leaves only the complementary surface of the PYL1 protein operable to interact with the ABI1 protein, preferably the PYL1 protein has been modified to consist of the complementary surface of the PYL1 protein.

[0364] In one embodiment, the PYL1 protein comprises a truncation at its N-terminus and at its C-terminus. In one embodiment, the PYL1 protein comprises a truncation of amino acids 1 to 32, preferably 32 amino acids, from its N-terminus. In one embodiment, the PYL1 protein comprises a truncation of amino acid residues 210 to 221, preferably 12 amino acids, from its C-terminus. Preferably, the PYL1 protein comprises amino acids 33 to 209 of SEQ ID NO: 26.

[0365] In one embodiment, the PYL1 protein consists of amino acids 33-209 of SEQ ID NO: 26, also known as "PYLcs." In one embodiment, the PYLcs protein consists of SEQ ID NO: 27.

[0366] Caffeine-binding domain The first or similarly third nucleic acid construct according to the present invention may comprise two caffeine binding domains as inducer binding domains, one in the first or third chimeric protein and one in the second or fourth chimeric protein.

[0367] Suitably, the caffeine binding domain may be an anti-caffeine antibody, or a fragment or derivative thereof.

[0368] "Fragment or derivative thereof", as used herein, means modified forms of a protein, such as truncated forms of the protein, Fab domains, scFvs, minibodies, camelid heavy chain antibodies, VHH domains, single variable domains, or mutated forms of the protein, suitable types of truncations and mutations are described elsewhere herein. Reference herein to any protein component of the system may also refer to fragments or derivatives of said protein, some of which are specifically described herein.

[0369] In one embodiment, the caffeine binding domain is an anti-caffeine antibody heavy chain fragment, preferably an anti-caffeine antibody VHH domain (αCaffVHH).

[0370] According to the present invention, two αCaffVHH domains homodimerize in the presence of caffeine.

[0371] Suitably, any two identical anti-caffeine antibodies or functional binding fragments or derivatives thereof capable of homodimerizing in the presence of caffeine can also be used as caffeine-binding domains.

[0372] Advantageously, the caffeine binding domain according to the present invention may originate from any camelid, and in a preferred embodiment, from a llama.

[0373] Suitably, the caffeine binding domain is an anti-caffeine antibody heavy chain fragment, preferably an anti-caffeine antibody VHH domain of camel, suitably llama origin (αCaffVHH). In one embodiment, the caffeine binding domain may comprise the sequence set forth in SEQ ID NO: 31.

[0374] Suitably, the αCaff VHH domain may comprise the sequence set forth in SEQ ID NO: 31, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Suitably, the caffeine binding domain may consist of SEQ ID NO: 31. Suitably, the αCaff VHH domain may consist of SEQ ID NO: 31.

[0375] Mandipropamide (Mandi) binding domain The first or similarly third nucleic acid construct according to the present invention may comprise two mandipropamide (Mandi) binding domains as inducer binding domains, one in the third or first chimeric protein and one in the fourth or second chimeric protein.

[0376] Preferably, the mandipropamid binding domain is a protein selected from the group consisting of abscisic acid insensitive 1 protein (ABI1), PYR Mandi Arabidopsis ABA receptor PYR1 sextuplicate mutant 10 ), PYLcs Mandi (The mutation is PYR Mandi The PYR1-like PYL1cs complementary surface region sextuple mutant transferred from Arabidopsis thaliana (or a fragment or derivative thereof).

[0377] "Fragment or derivative thereof," as used herein, means a modified form of a protein, such as a truncated form of the protein, or a mutated form of the protein, suitable types of truncations and mutations are described elsewhere herein. Reference herein to any protein component of a system may also refer to a fragment or derivative of said protein, some of which are specifically described herein.

[0378] According to the present invention, ABI1 and PYR Mandi Proteins, as well as ABI1 and PYLcs MandiThe proteins heterodimerize in the presence of Mandi.

[0379] In one embodiment, the mandipropamide binding domain is ABI1.

[0380] ABI1 is a member of the protein serine / threonine phosphatase 2C class (PP2C).

[0381] Preferably, the PP2C protein is ABI1. Preferably, ABI1 according to the present invention may originate from any plant, for example, Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, rapeseed, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Scutellaria baicalensis, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia crus- tularensis, grape, or maize.

[0382] In a preferred embodiment, the ABI1 protein is Arabidopsis thaliana ABI1. Suitably, it comprises SEQ ID NO: 24. Suitably, it consists of SEQ ID NO: 24.

[0383] Preferably, the ABI1 protein is modified, and therefore preferably the ABI1 protein is a modified mutant or variant of a reference ABI1 protein.

[0384] An engineered mutant or variant shall be understood as a protein or nucleic acid sequence that shares sequence identity with the original sequence (or with a specific fragment of the reference sequence), but contains at least one alteration (e.g., substitution, addition, or deletion) compared to the reference sequence.

[0385] Suitably, the modification may comprise one or more mutations. Suitably, the mutations may be substitutions, additions or deletions.

[0386] Suitable such modifications are defined herein above with respect to the auxin-binding domain.

[0387] The term "reference sequence" as used herein means the entire native or wild-type sequence of the same protein or nucleic acid from the same organism, or a fragment thereof. Preferably, the reference sequence is unmodified.

[0388] Preferably, the ABI1 protein has been modified by deletion, preferably by truncation, which leaves only the complementary surface of the ABI1 protein operable to interact with the PYL1 protein, preferably the ABI1 protein has been modified to consist of the complementary surface of the ABI1 protein.

[0389] In one embodiment, the ABI1 protein comprises a truncation at its N-terminus and at its C-terminus. In one embodiment, the ABI1 protein comprises a truncation of amino acids 1 to 125 from its N-terminus, preferably 125 amino acids. In one embodiment, the ABI1 protein comprises a truncation of amino acid residues 424 to 434 from its C-terminus, preferably 10 amino acids. Preferably, the ABI1 protein comprises amino acids 126 to 423 of SEQ ID NO: 24.

[0390] In one embodiment, the ABI1 protein consists of amino acids 126 to 423 of SEQ ID NO: 24, also known as "ABI1cs." In one embodiment, the ABIcs protein consists of SEQ ID NO: 25.

[0391] As previously discussed, PYR Mandi The protein heterodimerizes with the ABI1 protein in the presence of Mandi.

[0392] In one embodiment, the mandipropamide binding protein is therefore PYR Mandi It is a protein.

[0393] Preferably, PYR Mandi The protein may originate from any of the following plant species: Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, rapeseed, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Japanese cedar, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia crus-galli, grape, or maize.

[0394] In a preferred embodiment, PYR Mandi The protein is Arabidopsis PYR Mandi Preferably, the PYR protein comprises SEQ ID NO: 33, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Mandi The protein consists of SEQ ID NO:33.

[0395] As discussed previously, PYLcs Mandi The protein heterodimerizes with the ABI1 protein in the presence of mandipropamide.

[0396] In one embodiment, the mandipropamide binding protein is therefore PYL1cs Mandi It is a protein.

[0397] Preferably, PYL1cs MandiThe protein may originate from any of the following plant species: Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, rapeseed, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Japanese cedar, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia crus-galli, grape, or maize.

[0398] In a preferred embodiment, PYL1cs Mandi The protein is PYL1cs from Arabidopsis thaliana. Mandi Preferably, the PYL1cs sequence comprises SEQ ID NO: 35, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Mandi The protein consists of SEQ ID NO:35.

[0399] Gibberellin-binding domain The first or similarly third nucleic acid construct according to the present invention may contain two gibberellin-binding domains as plant hormone inducer-binding domains, one in the first or third chimeric protein and one in the second or fourth chimeric protein.

[0400] Preferably, the gibberellin-binding domain is selected from the gibberellin-insensitive dwarf1 (GID1) protein or the gibberellin-insensitive (GAI) protein or a fragment or derivative thereof.

[0401] "Fragment or derivative thereof," as used herein, means a modified form of a protein, such as a truncated form of the protein, or a mutated form of the protein, suitable types of truncations and mutations are described elsewhere herein. Reference herein to any protein component of a system may also refer to a fragment or derivative of said protein, some of which are specifically described herein.

[0402] According to the present invention, the gibberellin-insensitive dwarf1 (GID1) protein and the gibberellin-insensitive (GAI) protein heterodimerize in the presence of gibberellin.

[0403] In some embodiments, the GAI protein may be modified. Preferably, the GAI protein may be modified by truncation at either the C- or N-terminus, preferably the C-terminus. Preferably, the modified GAI protein consists of amino acids 1-92 (or nucleotides 1-276) of the full-length GAI protein. Preferably, references herein to a "modified GAI protein" or a "GAI protein that may be modified" or a "GAI protein fragment" refer to this truncated form.

[0404] Therefore, preferably, also according to the present invention, the gibberellin-insensitive dwarf1 (GID1) protein and the gibberellin-insensitive (GAI) protein, i.e., amino acids 1 to 92 of the modified GAI protein, heterodimerize in the presence of gibberellin.

[0405] In one embodiment, the gibberellin-binding domain is a gibberellin-insensitive dwarf1 (GID1) protein.

[0406] Preferably, GID1 according to the present invention may originate from any plant, such as Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, rapeseed, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Scutellaria baicalensis, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia fasciata, grape, or maize.

[0407] In a preferred embodiment, the GID1 protein is Arabidopsis GID1. Suitably, GID1 may comprise the sequence set forth in SEQ ID NO: 37, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In one embodiment, the GID1 protein consists of SEQ ID NO: 37.

[0408] In one embodiment, the gibberellin-binding protein is a gibberellin-insensitive (GAI) protein.

[0409] In a preferred embodiment, the GAI protein is an Arabidopsis GAI protein. Suitably, GAI may comprise the sequence set forth in SEQ ID NO: 48, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In one embodiment, the GAI protein consists of SEQ ID NO: 48.

[0410] Suitably, the GAI protein may originate from any of the following plant species: Amborella trichopoda, Arabidopsis thaliana, Brassica rapa, rapeseed, papaya, chickpea, Citrus, cucumber, Eucalyptus grandis, soybean, Gossypium raimondii, Marchantia polymorpha, Medicago sativa, rice, petunia, Phalaenopsis equestris, Physcomitrella patens, Norway spruce, cottonwood, peach, castor bean, Scutellaria baicalensis, tomato, eggplant, potato, sorghum, wheat, einkorn, Utricularia crus-galli, grape, or maize.

[0411] In one embodiment, the gibberellin-binding protein is a modified GAI protein, preferably nucleotides 1 to 276 (amino acids 1 to 92) of SEQ ID NO: 48. In one embodiment, the modified GAI protein consists of amino acids 1 to 92 of SEQ ID NO: 48.

[0412] Suitably, the modified GAI protein, i.e., nucleotides 1-276 (amino acids 1-92) of the full-length GAI protein, comprises the sequence set forth in SEQ ID NO: 39, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In one embodiment, the modified GAI protein consists of SEQ ID NO: 39.

[0413] auxin Auxins are a class of plant hormones that control growth and development in plants. Auxins are known to regulate transcription by promoting the degradation of the AID family of transcriptional repressors. In the context of the present invention, auxins act as plant hormone inducers for the chemically induced access system of the present invention. Preferably, auxins induce heterodimerization of the auxin-binding protein TIR1 and AID.

[0414] Suitably, the auxin may be any auxin, or a precursor or mimetic thereof.

[0415] Suitably, the auxin may be indole-3-acetic acid, 4-chloroindole-3-acetic acid, phenylacetic acid, 5-phenyl-indole-3-acetic acid (5-Ph-IAA), indole-3-butyric acid, or indole-3-propionic acid. Suitably, the auxin may be a synthetic auxin, such as 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D).

[0416] In a preferred embodiment, the auxin used in the present invention is indole-3-acetic acid (IAA).

[0417] Preferably, the IAA may be IAA1 or IAA2.

[0418] abscisic acid Abscisic acid (ABA) is an isoprenoid plant hormone that functions in plant developmental processes, such as regulating seed and bud dormancy and stomatal closure. It is also important for responses to environmental stress. In the context of the present invention, abscisic acid acts as a plant hormone inducer for the chemically induced access system of the present invention. Preferably, abscisic acid induces heterodimerization of the abscisic acid-binding proteins ABI1 and PYL1.

[0419] Suitably, the term abscisic acid may also include abscisic acid precursors such as zeaxanthin, xanthin or abscisic aldehyde, or mimetics such as pyrabactin. Suitably, the abscisic acid may be synthetic.

[0420] In a preferred embodiment, abscisic acid (ABA) is used in the present invention.

[0421] Caffeine Caffeine is a xanthine alkaloid that acts as a natural insecticide, serving as a toxic substance that prevents herbivores and insects from consuming plant leaves, seeds, and other parts. Caffeine also plays a role in enhancing plant reproductive success by attracting pollinators such as bees and butterflies. Caffeine can be considered a plant hormone. Caffeine acts as an inducer for the chemically induced access system of the present invention. Preferably, caffeine induces homodimerization of the caffeine-binding protein αCaffVHH.

[0422] In a preferred embodiment, caffeine is used in the present invention.

[0423] Mandipropamide (Mandi) Mandipropamid is a fungicide widely used in agriculture. Mandipropamid acts as an inducer for the chemically induced access system of the present invention. Preferably, in a manner similar to that of ABA, mandipropamid binds to the mandipropamid-binding proteins ABI1 and PYR. Mandi Heterodimerization of ABI1 and PYLcs Mandi Therefore, mandipropamide can preferably function when used in conjunction with the ABA-inducible access system described herein. Preferably, mandipropamide is a plant hormone analog.

[0424] In a preferred embodiment, mandipropamide is used in the present invention.

[0425] Gibberellin Gibberellins are plant hormones that play an important role in regulating various aspects of plant growth and development, such as seed germination, stem elongation, leaf expansion, flowering, and fruit development, as well as a variety of physiological processes in plants. Gibberellins act as plant hormone inducers for the chemically induced access system of the present invention. Preferably, gibberellins induce heterodimerization of the gibberellin-binding proteins GID1 and GAI, as well as heterodimerization of GID1 and a modified GAI protein (i.e., nucleotides 1-276 (amino acids 1-92) of the full-length GAI protein).

[0426] Suitably, the term gibberellin may include any compound of the gibberellin family, or a precursor or mimetic thereof.

[0427] Preferably, the gibberellin may be selected from, for example, any one of GA1, GA3, GA4, and GA7.

[0428] In a preferred embodiment, gibberellin GA3 is used in the present invention.

[0429] Protein of interest In the context of the present invention, a protein of interest may be any protein that is expressed or displayed inside, within, or on the surface of a cell. Preferably, in some embodiments, the protein of interest is expressed or displayed on the surface of a cell.

[0430] Preferably, the protein of interest may be an antigen. Preferably, an antigen may be considered to be any protein that is typically expressed or presented on the surface of a cell. Such antigens may be referred to herein as target antigens of interest, i.e., "TAO1" or "TAO12."

[0431] Suitably, the antigen may be any immunostimulatory antigen. Suitably, the antigen may be any antigen to which a therapeutic agent or drug will be bound. Such therapeutic agents or drugs are defined herein.

[0432] Suitably, the therapeutic agent or drug, e.g., immunotherapeutic agent, that binds to an antigen of interest may be selected from a fusion protein, an antibody (e.g., a monoclonal antibody, a bispecific antibody, a multispecific antibody, an antibody drug conjugate, a nanobody, a scFv, a di-scFv, a Fab, a sdAb, a F(ab)2, a glycoengineered antibody) or a binding fragment thereof, an antibody-like molecule, a fusion protein, an aptamer, ankyrin, an ankyrin repeat protein (DARPin), a peptide, a bicyclic peptide, a small molecule, a vaccine, a T cell, a natural killer (NK) cell, a cell expressing a CAR, e.g., a CAR T cell or a CAR NK cell, an oncolytic virus, a cytokine, a chemokine, a hormone, a bacterium, a tumor-infiltrating lymphocyte, a dendritic cell, a macrophage, or a mesenchymal cell.

[0433] Preferably, the therapeutic agent or drug is a biologic. Preferably, the therapeutic agent or drug is an immunotherapeutic. Preferably, the immunotherapy binds to an antigen of interest. In some embodiments, the immunotherapeutic may be an antibody or a binding fragment thereof.

[0434] In other embodiments, the immunotherapeutic agent that binds to an antigen of interest may be an immune cell, such as a T cell, a NK cell, a B cell, a tumor-infiltrating lymphocyte, a dendritic cell, a macrophage, a mesenchymal cell, or an immortalized version thereof. Preferably, the immunotherapeutic agent that is an immune cell may also be an engineered immune cell. Preferably, the engineered immune cell may express a CAR. Therefore, preferably, the engineered immune cell may be selected from a CAR T cell, a TCR T cell, a CAR B cell, a CAR-macrophage, or a CAR NK cell.

[0435] In a preferred embodiment, the antigen of interest may be any antigen that is a therapeutic target. By way of example, the antigen may be CD19, BCMA, CD123, mesothelin, GD2, CD20, CD33, CD47, HER2, CD22, CD13, PSMA, EGFR vIII, EGFR, CD38, EpCAM, PSCA, CEA, HIV, glypican-3, FLT3, NKG2D, claudin 18.2, DLL3, CS1, MUC16, CD3, PD-L1, 4-1BB, PD-1, LAG3, CTLA-4, MUC1, 5T4, CD40, CD155, OX-40, NY-ESO, ROR1, TROP2, VEGFRI, VEGFRII, CLL, CD30, CD70, CD133, TIM-3, L1CAM, ICOS, DLL4, FRalpha, WT1, IL13Ralpha, Lewis-Y, or cMET.

[0436] In one embodiment, the first antigen of interest may be CD19 and the second antigen of interest may be CD22.

[0437] In one embodiment, the first antigen of interest may be CD38 and the second antigen of interest may be BCMA.

[0438] In one embodiment, the first antigen of interest may be PD-L1 and the second antigen of interest may be HER2.

[0439] In one embodiment, the first antigen of interest may be HER2 and the second antigen of interest may be HER3.

[0440] In one embodiment, the first antigen of interest may be CD13 and the second antigen of interest may be TIM3.

[0441] In one embodiment, the first antigen of interest may be CD155 and the second antigen of interest may be PD-L1.

[0442] In one embodiment, the first antigen of interest may be CD19 and the second antigen of interest may be CD20.

[0443] In one embodiment, the first antigen of interest may be EGFR and the second antigen of interest may be MET.

[0444] In one embodiment, the first antigen of interest may be PD-1 and the second antigen of interest may be ICOS.

[0445] Therefore, preferably the immunotherapeutic agent is anti-CD19, anti-BCMA, anti-CD123, anti-mesothelin, anti-GD2, anti-CD20, anti-CD33, anti-HER2, anti-CD22, anti-CD30, anti-PSMA, anti-EGFR vIII, anti-EGFR, anti-CD38, anti-EpCAM, anti-PSCA, anti-CEA, anti-HIV, anti-glypican-3, anti-FLT3, anti-NKG2D, anti-claudin 18.2, anti-DLL3, anti-CS1, anti-MUC16, anti-CD3, anti-PD-L1, anti-4-1BB, anti-PD-1, anti-LAG3, anti-CTLA-4, anti-MUC1, anti-5T4, anti-CD40, anti-OX-40, anti-NY-ESO, anti-ROR1, anti-TROP2, anti-VEGFRII, anti-CLL, anti-CD30, anti-CD70, anti-CD133, anti-TIM-3, anti-L1CAM, anti-ICOS, anti-DLL4, anti-FRalpha, anti-WT1, anti-IL13Ralpha, anti-Lewis-Y or anti-cMET antibody, or a binding fragment thereof.

[0446] In some embodiments, therefore, the immunotherapeutic agent may be one or more antibodies directed against one or both of the paired first and second antigens listed above.

[0447] Preferably, the antigen is an antigen associated with a disease or disorder.

[0448] Suitably, the antigen may be associated with any disease or disorder, for example an infectious disease, an autoimmune disease, an inflammatory disease, a cancer, a genetic or inherited disease.

[0449] Suitable infectious diseases may include viral, bacterial, fungal, or protozoal infections.

[0450] Suitable viral infections include COVID-19, SARS, MERS, influenza, the common cold, respiratory syncytial virus infection, adenovirus infection, parainfluenza virus infection, norovirus infection, rotavirus infection, astrovirus infection, measles, mumps, rubella, chickenpox, herpes zoster, roseola, smallpox, disease V, chikungunya virus infection, HPV infection, hepatitis A, B, C, D or E, warts, herpes, molluscum contagiosum, Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, polio, viral meningitis, viral encephalitis, rabies, Zika virus infection, West Nile virus infection, HIV / AIDS, hantavirus infection, and HPS.

[0451] Suitable bacterial infections include urinary tract infections, cystitis, impetigo, bacterial food poisoning, campylobacteriosis, C. difficile infection, bacterial cellulitis, MRSA, CRPA, VRSA, sepsis, erysipelas, necrotizing fasciitis, bacterial folliculitis, gonorrhea, chlamydia, syphilis, Mycoplasma genitalium, bacterial vaginosis, pelvic inflammatory disease, tuberculosis, whooping cough, Haemophilus influenzae disease, pneumonia, bacterial meningitis, Lyme disease, cholera, botulism, tetanus, anthrax, cryptosporidiosis, diphtheria, E. coli infection, legionellosis, leptospirosis, listeriosis, salmonellosis, shigellosis, staphylococcal infection, streptococcal infection, TSS, typhoid, and Yersenia infection.

[0452] Suitable autoimmune diseases include asthma, psoriasis, MS, rheumatoid arthritis, reactive arthritis, lupus, inflammatory bowel syndrome / disease, type 1 diabetes, Guillain-Barre syndrome, demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, ulcerative colitis, antiphospholipid syndrome, Kawasaki disease, hair loss, vitiligo, scleroderma, Sjogren's syndrome, Crohn's disease, celiac disease, Addison's disease, and narcolepsy.

[0453] Suitable cardiovascular diseases include angina, heart attack, heart failure, coronary heart disease, stroke, transient ischemic attack, peripheral artery disease, aortic disease, atherosclerosis, high blood pressure, cerebrovascular disease, renal artery stenosis, aneurysm, cardiomyopathy, pulmonary heart disease, arrhythmias, rhythm disorders, endocarditis, cardiac hypertrophy, myocarditis, valvular heart disease, congenital heart disease, and rheumatic heart disease.

[0454] Suitable inflammatory diseases include any of the infectious or autoimmune diseases described above, including arthritis, asthma, tuberculosis, periodontitis, chronic ulcers, sinusitis, hepatitis, glomerulonephritis, inflammatory bowel syndrome / disease, preperfusion injury, transplant rejection, sickle cell disease, allergies, cardiovascular disease, psoriasis, cytokine-mediated pruritus, COPD, diabetes, bronchitis, Crohn's disease, atherosclerosis, dermatitis, arteritis, and lupus.

[0455] Suitable cancers include breast cancer, liver cancer, lung cancer, pancreatic cancer, brain cancer, prostate cancer, bowel cancer, rectal cancer, bone cancer, leukemia, bladder cancer, cervical cancer, endometrial cancer, eye cancer, retinoblastoma, Ewing's sarcoma, gallbladder cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, mesothelioma, myeloma, lymphoma, ovarian cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, nasal and paranasal sinus cancer, skin cancer, sarcoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, penile cancer, and vulvar cancer.

[0456] Suitably, the antigen may be associated with a disease in any system of the body, for example, a neurological disease, a cardiovascular disease, a blood disease, a skin disease, a gastrointestinal disease, a muscle disease, a bone disease, a respiratory disease, a reproductive system disease, a urinary system disease, or an endocrine disease.

[0457] Preferably, the antigen is a cancer-associated antigen. Preferably, the antigen is a tumor-associated antigen, preferably an antigen associated with a cancerous tumor. Thus, preferably, the antigen may be a tumor-associated antigen (TAA) or a tumor-restricted antigen (TRA).

[0458] In one embodiment, the antigen is a tumor-associated antigen (TAA). As explained above, TAAs are not only associated with tumor cells, but also with healthy cells. Advantageously, the present invention provides a means for assessing the binding of candidate therapeutic agents, which may be any of those listed above, to a TAA, thereby assessing their binding and biological activity in both tumor and healthy cells. In some embodiments, the first and second proteins of interest may be a first and second TAA, respectively.

[0459] First construct The first construct according to the present invention comprises a promoter operably linked to nucleic acid sequences encoding a first chimeric protein and a second chimeric protein, and therefore preferably is a first nucleic acid construct.

[0460] Preferably, the promoter may be a mammalian cell promoter. In a preferred embodiment of the present invention, the mammalian cell promoter is selected from MND, CAG EF1-α, CMV, MSCV, SV40, mouse PGK, human PGK, or UBC. In one embodiment, the promoter is MND.

[0461] Preferably, the promoter of the first construct of the present invention is operably linked to a nucleic acid sequence encoding a first chimeric protein and is operably linked to a nucleic acid sequence encoding a second chimeric protein.

[0462] "Operably linked," as used herein, means that the indicated elements are functionally related to each other and generally physically related as well. Thus, the term "operably linked," as used herein, refers to nucleotide sequences in a single nucleic acid molecule that are functionally associated. Thus, a first nucleotide sequence operably linked to a second nucleotide sequence refers to a situation in which the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a nucleotide sequence if it affects the transcription or expression of that nucleotide sequence. Those skilled in the art will understand that control sequences (e.g., promoters) need not necessarily be contiguous with a nucleotide sequence to which they are operably linked, so long as the control sequence functions to direct its expression. Thus, for example, there may be intervening untranslated but transcribed sequences between the promoter and the nucleotide sequence, and the promoter can still be considered "operably linked" to the nucleotide sequence.

[0463] "Operably linked" means integrated as part of the same nucleic acid molecule, preferably positioned and oriented to allow transcription to be initiated from a promoter. DNA operably linked to a promoter is under the transcription initiation control of the promoter or is functionally linked to it.

[0464] Preferably, therefore, the first construct is a bicistronic construct, in that it comprises a first and a second nucleic acid sequence, each of which can be considered a cistron.

[0465] In some embodiments, the first construct according to the present invention may further comprise a cleavable linker.

[0466] Preferably, a cleavable linker links the nucleic acid sequence encoding the first chimeric protein to the nucleic acid sequence encoding the second chimeric protein. Preferably, the cleavable linker is positioned between the nucleic acid sequence encoding the first chimeric protein and the nucleic acid sequence encoding the second chimeric protein.

[0467] Preferably, the cleavable linker is a nucleic acid sequence encoding a self-cleaving peptide.

[0468] Preferably, a self-cleaving peptide links the first chimeric protein and the second chimeric protein, and preferably, when the first construct is transcribed, the linker is automatically cleaved such that the first chimeric protein and the second chimeric protein are separated.

[0469] Preferably, the cleavable linker may be a 2A self-cleaving peptide. Preferably, the 2A self-cleaving peptide may be a teschovirus-1 2A (P2A) self-cleaving peptide, a foot-and-mouth disease (F2A) self-cleaving peptide, an equine rhinitis (E2A) self-cleaving peptide, or a thosea asigna (T2A) self-cleaving peptide. Preferably, the self-cleaving peptide may be derived from any particular virus in the groups listed above. In one embodiment, the cleavable linker is a porcine teschovirus P2A self-cleaving peptide.

[0470] In an alternative embodiment, the first nucleic acid construct according to the present invention may further comprise an IRES.

[0471] Preferably, an IRES is located between the nucleic acid sequence encoding the first chimeric protein and the nucleic acid sequence encoding the second chimeric protein. Preferably, the IRES ensures that the first nucleic acid sequence is translated separately from the second nucleic acid sequence to form separate first and second chimeric proteins.

[0472] Suitably, the IRES sequence may be selected from any suitable viral or cellular IRES, such as that of the encephalomyocarditis virus (EMCV).

[0473] In one embodiment, the IRES comprises the sequence set forth in SEQ ID NO: 4. In one embodiment, the IRES consists of the sequence set forth in SEQ ID NO: 4.

[0474] Preferably, the first chimeric protein and the second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention each comprise an auxin-binding domain and an effector domain, and thus the first construct comprises nucleic acid sequences encoding two auxin-binding domains and two effector domains.

[0475] Suitably, the auxin-binding domain of either the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the invention may be the transport inhibitor response 1 protein (TIR1), or a fragment or derivative thereof. Thus, the first construct comprises a nucleic acid sequence encoding the TIR1 protein, or a fragment or derivative thereof.

[0476] Suitably, the auxin-binding domain of the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the invention may be an auxin / indole-3-acetic acid protein (AID). Thus, the first construct comprises a nucleic acid sequence encoding an AID protein, or a fragment or derivative thereof.

[0477] Alternatively, the first chimeric protein and the second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention each comprise a caffeine-binding domain and an effector domain, and thus the first construct comprises nucleic acid sequences encoding two caffeine-binding domains and two effector domains.

[0478] Preferably, the caffeine-binding domains of both the first and second chimeric proteins encoded by the nucleic acid sequences of the first construct of the present invention may be anti-caffeine heavy chain antibody fragments (αCaffVHH) or fragments or derivatives thereof. Thus, the first construct comprises nucleic acid sequences encoding two αCaffVHH proteins or fragments or derivatives thereof.

[0479] Alternatively, the first chimeric protein and the second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention each comprise a mandipropamide-binding domain and an effector domain, and thus the first construct comprises a nucleic acid sequence encoding two mandipropamide-binding domains and two effector domains.

[0480] Preferably, the mandipropamide-binding domain of either the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention is PYR Mandi or PYLcs Mandi The first construct may be a PYR protein, or a fragment or derivative thereof. Mandi , or PYLcs Mandi It includes nucleic acid sequences that encode proteins, or fragments or derivatives thereof.

[0481] Suitably, the mandipropamide-binding domain of the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the invention may be ABI1. Thus, the first construct comprises a nucleic acid sequence encoding an ABI1 protein, or a fragment or derivative thereof.

[0482] Alternatively, the first chimeric protein and the second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention each contain a gibberellin-binding domain and an effector domain, and thus the first construct contains nucleic acid sequences encoding two gibberellin-binding domains and two effector domains.

[0483] Preferably, the gibberellin-binding domain of either the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention may be the GID1 protein, or a fragment or derivative thereof. Thus, the first construct comprises a nucleic acid sequence encoding the GID1 protein, or a fragment or derivative thereof.

[0484] Preferably, the gibberellin-binding domain of the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention may be a GAI protein or a modified GAI protein. Thus, the first construct comprises a nucleic acid sequence encoding a GAI protein, or a fragment or derivative thereof.

[0485] Preferably, the effector domain of either the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention may be a transactivation domain, and thus the first construct comprises a nucleic acid sequence encoding a transactivation domain.

[0486] Preferably, the effector domain of either the first or second chimeric protein encoded by the nucleic acid sequence of the first construct of the present invention may be a catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI). Thus, the first construct comprises a nucleic acid sequence encoding a dI-SceI DNA-binding domain.

[0487] In the context of the present invention, the auxin binding domain and the effector domain of the first and second chimeric proteins are different.

[0488] Therefore, preferably, the nucleic acid sequence encoding the transactivation domain and the nucleic acid sequence encoding the dI-SceI DNA binding domain are each linked, in a mutually exclusive manner, to a nucleic acid sequence encoding a TIR1 or AID protein, or one of the other chemical binding domains described above, to produce the first and second chimeric proteins.

[0489] In one embodiment, the nucleic acid sequence encoding the transactivation domain is selected from the group consisting of a TIR1 protein, an αCaffVHH protein, a PYR Mandi , PYLcs Mandi These linked nucleic acid sequences are linked to nucleic acid sequences encoding the TIR1 protein, the αCaff VHH protein, the PYR protein, or the GID1 protein. Mandi , PYLcs Mandi The gene encoding the GID1 protein encodes a first chimeric protein comprising a transactivation domain fused to the GID1 protein.

[0490] In one embodiment, the nucleic acid sequence encoding the dI-SceI DNA-binding domain is linked to a nucleic acid sequence encoding an AID protein, an αCaff VHH protein, an ABI1 protein, or a GAI protein or modified GAI protein, and these linked nucleic acid sequences encode a second chimeric protein comprising the dI-SceI DNA-binding domain fused to the AID protein, an αCaff VHH protein, an ABI1 protein, or a GAI protein or modified GAI protein.

[0491] Preferably, in such an embodiment, the first chimeric protein comprises a transactivation domain and a TIR1 protein, and the second chimeric protein comprises a dl-SceI DNA binding domain and an AID protein.

[0492] In an alternative embodiment, the first chimeric protein comprises a transactivation domain and an αCaff VHH protein, PYR Mandi Proteins, PYLcs Mandi The second chimeric protein comprises a dl-SceI DNA binding domain and an αCaffVHH protein, an ABI1 protein, or a GAI protein or a modified GAI protein.

[0493] Alternatively, in another embodiment, a nucleic acid sequence encoding a transactivation domain is linked to a nucleic acid sequence encoding an AID protein, and these linked nucleic acid sequences encode a first chimeric protein comprising the transactivation domain fused to the AID protein.

[0494] Alternatively, in another embodiment, a nucleic acid sequence encoding a dI-SceI DNA-binding domain is linked to a nucleic acid sequence encoding a TIR1 protein, and these linked nucleic acid sequences encode a second chimeric protein comprising the dI-SceI DNA-binding domain fused to the TIR1 protein.

[0495] Preferably, in such an embodiment, the first chimeric protein comprises a transactivation domain and an AID protein, and the second chimeric protein comprises a dI-SceI DNA binding domain and a TIR1 protein.

[0496] Thus, similarly, a nucleic acid sequence encoding a transactivation domain may be linked to a nucleic acid sequence encoding an αCaff VHH protein, an ABI1 protein, or a GAI protein or modified GAI protein. Thus, similarly, a nucleic acid sequence encoding a dI-SceI DNA binding domain may be linked to a nucleic acid sequence encoding an αCaff VHH protein, an ABI1 protein, or a GAI protein or modified GAI protein. Mandi , PYLcs Mandi It may be linked to a protein or a nucleic acid sequence encoding the GID1 protein.

[0497] As explained elsewhere herein, it will be understood that the DNA-binding domains used in the first and third constructs, and consequently the binding sites in the second and fourth constructs, are interchangeable. In some embodiments, the first construct, preferably the second chimeric protein, may comprise a GAL4 DNA-binding domain, and the second construct may comprise one or more GAL4 upstream activating sequences.

[0498] In the context of the present invention, the first and second chimeric proteins dimerize in the presence of auxin, or in the presence of caffeine, mandipropamide, or gibberellin. Preferably, the presence of auxin or other compound, if used, allows the transactivation domain to associate with the dI-SceI DNA-binding domain or the GAL4 DNA-binding domain.

[0499] Preferably, dimerization of the first and second chimeric proteins of the invention stimulates transcription. Preferably, transcription of the second construct. Preferably, transcription of the second construct from the effector domain binding site, preferably from the dI-SceI binding site, or, if used, from the GAL4 UAS. Preferably, dimerization of the first and second chimeric proteins of the invention thereby stimulates transcription of the first protein of interest, preferably transcription of the first protein of interest from the second construct.

[0500] It will be understood that a nucleic acid construct according to the present invention may comprise DNA or RNA, and that suitable nucleic acid constructs may consist essentially of DNA, may consist essentially of RNA, or may comprise a combination of DNA and RNA.

[0501] Second construct The second construct according to the present invention comprises a nucleic acid sequence encoding one or more effector domain binding sites and a nucleic acid sequence encoding a protein of interest, and therefore preferably is a second nucleic acid construct.

[0502] Suitably, the nucleic acid sequence encoding the one or more effector domain binding sites is operably linked to the nucleic acid sequence encoding the protein of interest, the term "operably linked" being defined herein above.

[0503] Preferably, the effector domain binding site(s) are suitable for binding to a selected effector domain, and preferably, given that the preferred effector domain is dI-SceI, the effector domain binding site is an ISceI DNA binding site.

[0504] Preferably, each ISceI DNA binding site comprises the sequence: TAGGGATAACAGGGTAAT (SEQ ID NO: 1).

[0505] Preferably, each dI-SceI DNA binding site is an 18 bp sequence consisting of the sequence: TAGGGATAACAGGGTAAT (SEQ ID NO: 1).

[0506] Preferably, the second (or fourth) construct comprises one or more effector domain binding sites, preferably more than one ISceI DNA binding site, preferably a plurality of ISceI DNA binding sites.

[0507] Suitably, the more than one effector domain binding site, preferably ISceI DNA binding site, is in tandem.

[0508] Preferably, the second (or fourth) construct comprises 1 to 15 effector domain-binding sites, preferably arranged in tandem. Preferably, the second construct comprises 1 ISceI DNA-binding site, 2 ISceI DNA-binding sites, 3 ISceI DNA-binding sites, 4 ISceI DNA-binding sites, 5 ISceI DNA-binding sites, 6 ISceI DNA-binding sites, 7 ISceI DNA-binding sites, 8 ISceI DNA-binding sites, 9 ISceI DNA-binding sites, 10 ISceI DNA-binding sites, 11 ISceI DNA-binding sites, 12 ISceI DNA-binding sites, 13 ISceI DNA-binding sites, 14 ISceI DNA-binding sites, or 15 ISceI DNA-binding sites, preferably arranged in tandem.

[0509] In a preferred embodiment, the second (or fourth) construct contains 10 tandem ISceI DNA binding sites.

[0510] Preferably, the second construct may further comprise a promoter. Preferably, the second construct may comprise an effector domain-binding site, preferably a promoter operably linked downstream of the effector domain-binding site. Preferably, the promoter is a minimal promoter. Preferably, the minimal promoter may be selected from, for example, a minimal TATA box promoter, a minimal adenovirus late promoter, a minimal herpes simplex virus (HSV) thymidine kinase promoter, or a minimal c-fos promoter (consisting of nucleotides -53 to +42).

[0511] Therefore, suitably, the effector domain binding site may comprise the sequence set forth in SEQ ID NO: 7. Therefore, suitably, the effector domain binding site may consist of the sequence set forth in SEQ ID NO: 7. Therefore, suitably, the second construct may comprise the sequence set forth in SEQ ID NO: 7.

[0512] The second construct according to the invention may optionally further comprise a recombination site.

[0513] Suitably, the recombination site is located upstream of one or more effector domain binding sites.

[0514] In a preferred embodiment, the recombination site is selected from any serine recombinase site, hi one embodiment, the recombination site is an attB site.

[0515] Preferably, the second construct comprises a nucleic acid sequence encoding a protein of interest. Suitable proteins of interest are as defined elsewhere in this description. Preferably, the nucleic acid sequence encoding the protein of interest is downstream of one or more effector domain binding sites. Preferably, it is downstream of one or more ISceI DNA binding sites.

[0516] In the context of the present invention, a dI-SceI DNA binding domain binds to each of one or more dI-SceI DNA binding sites. Preferably, in some embodiments, each dI-SceI DNA binding domain fused to an auxin binding domain binds to each of one or more dI-SceI DNA binding sites. Preferably, therefore, either the first or second chimeric protein comprising a dI-SceI DNA binding domain fused to an auxin binding domain binds to each of one or more dI-SceI DNA binding sites.

[0517] Suitably, in a preferred embodiment, in the presence of auxin, association of the auxin-binding domains of the first and second chimeric proteins brings the transactivation domain into proximity with the dI-SceI DNA-binding domain bound to the I-SceI binding site in the second construct, thereby stimulating transcription of a downstream nucleic acid encoding a protein of interest.

[0518] It will be appreciated that the effector binding domains of the second and fourth constructs are interchangeable, and therefore, suitably, the second construct may instead comprise one or more GAL4 upstream activating sequences that interact with the GAL4 DNA binding domain as described herein below with respect to the fourth construct.

[0519] It will be understood that a nucleic acid construct according to the present invention may comprise DNA or RNA, and that suitable nucleic acid constructs may consist essentially of DNA, may consist essentially of RNA, or may comprise a combination of DNA and RNA.

[0520] Third construct The third construct according to the present invention comprises a promoter operably linked to nucleic acid sequences encoding the third chimeric protein and the fourth chimeric protein, and therefore preferably is a third nucleic acid construct.

[0521] Preferably, the promoter is a mammalian cell promoter. In a preferred embodiment of the present invention, the mammalian cell promoter is selected from MND, CAG EF1-α, CMV, MSCV, SV40, mouse PGK, human PGK, or UBC. In one embodiment, the promoter is EF1-α.

[0522] Preferably, the promoter of the third construct of the present invention is operably linked to a nucleic acid sequence encoding a third chimeric protein and operably linked to a nucleic acid sequence encoding a fourth chimeric protein.

[0523] The term "operably linked" is as defined above in relation to the first construct.

[0524] Preferably, therefore, the third construct is a bicistronic construct, in that it comprises a first and a second nucleic acid sequence, each of which can be considered a cistron.

[0525] In some embodiments, the third construct according to the present invention may further comprise a cleavable linker.

[0526] Preferably, a cleavable linker links the nucleic acid sequence encoding the third chimeric protein to the nucleic acid sequence encoding the fourth chimeric protein. Preferably, the cleavable linker is positioned between the nucleic acid sequence encoding the third chimeric protein and the nucleic acid sequence encoding the fourth chimeric protein.

[0527] Preferably, the cleavable linker is a nucleic acid sequence encoding a self-cleaving peptide.

[0528] Preferably, a self-cleaving peptide links the third chimeric protein and the fourth chimeric protein. Preferably, when the third construct is transcribed, the linker is automatically cleaved such that the third chimeric protein and the fourth chimeric protein are separated.

[0529] Preferably, the cleavable linker may be a 2A self-cleaving peptide. Preferably, the 2A self-cleaving peptide may be a teschovirus-1 2A (P2A) self-cleaving peptide, a foot-and-mouth disease (F2A) self-cleaving peptide, an equine rhinitis (E2A) self-cleaving peptide, or a thosea asigna (T2A) self-cleaving peptide. Preferably, the self-cleaving peptide may be derived from any particular virus in the groups listed above. In one embodiment, the cleavable linker is a porcine teschovirus P2A self-cleaving peptide.

[0530] In an alternative embodiment, the third nucleic acid construct according to the invention may further comprise an IRES, suitably the IRES may be as described above in relation to the first construct.

[0531] Preferably, the third chimeric protein and the fourth chimeric protein encoded by the nucleic acid sequence of the third construct of the present invention each comprise an abscisic acid-binding domain and an effector domain, and thus the third construct comprises nucleic acid sequences encoding two abscisic acid-binding domains and two effector domains.

[0532] Preferably, the abscisic acid-binding domain of either the third or fourth chimeric protein encoded by the nucleic acid sequence of the third construct of the present invention may be the ABI1 protein, or a fragment or derivative thereof. Thus, the third construct comprises a nucleic acid sequence encoding the ABI1 protein, or a fragment or derivative thereof.

[0533] Preferably, the abscisic acid-binding domain of the third or fourth chimeric protein encoded by the nucleic acid sequence of the third construct of the present invention may be a PYL1 protein, or a fragment or derivative thereof, and thus the third construct comprises a nucleic acid sequence encoding a PYL1 protein, or a fragment or derivative thereof.

[0534] Preferably, the effector domain of either the third or fourth chimeric protein encoded by the nucleic acid sequence of the third construct of the present invention may be a transactivation domain, and thus the third construct comprises a nucleic acid sequence encoding a transactivation domain.

[0535] Preferably, the effector domain of either the third or fourth chimeric protein encoded by the nucleic acid sequence of the third construct of the present invention may be a GAL4 DNA-binding domain, and thus the third construct comprises a nucleic acid sequence encoding a GAL4 DNA-binding domain.

[0536] In the context of the present invention, the abscisic acid binding domain and the effector domains of the third and fourth chimeric proteins are different.

[0537] Therefore, preferably, the nucleic acid sequence encoding the transactivation domain and the nucleic acid sequence encoding the GAL4 DNA binding domain are each linked, in a mutually exclusive manner, to one of the nucleic acid sequences encoding the ABI1 or PYL1 protein to produce the third and fourth chimeric proteins.

[0538] In one embodiment, a nucleic acid sequence encoding a transactivation domain is linked to a nucleic acid sequence encoding a PYL1 protein, and these linked nucleic acid sequences encode a third chimeric protein comprising the transactivation domain fused to the PYL1 protein.

[0539] In one embodiment, the nucleic acid sequence encoding the GAL4 DNA-binding domain is linked to a nucleic acid sequence encoding an ABI1 protein, and these linked nucleic acid sequences encode a fourth chimeric protein comprising the GAL4 DNA-binding domain fused to the ABI1 protein.

[0540] Suitably, in such an embodiment, the third chimeric protein comprises a transactivation domain and a PYL1 protein, and the fourth chimeric protein comprises a GAL4 DNA binding domain and an ABI1 protein.

[0541] Alternatively, in another embodiment, a nucleic acid sequence encoding a transactivation domain is linked to a nucleic acid sequence encoding an ABI1 protein, and these linked nucleic acid sequences encode a third chimeric protein comprising the transactivation domain fused to the ABI1 protein.

[0542] Alternatively, in another embodiment, a nucleic acid sequence encoding a GAL4 DNA-binding domain is linked to a nucleic acid sequence encoding a PYL1 protein, and these linked nucleic acid sequences encode a fourth chimeric protein comprising the GAL4 DNA-binding domain fused to the PYL1 protein.

[0543] Suitably, in such an embodiment, the third chimeric protein comprises a transactivation domain and an ABI1 protein, and the fourth chimeric protein comprises a GAL4 DNA binding domain and a PYL1 protein.

[0544] It will be understood that the DNA-binding domains used in the first and third constructs, and consequently the binding sites in the second and fourth constructs, are interchangeable, as described elsewhere herein. In some embodiments, the third construct, and preferably the fourth chimeric protein, may comprise a dI-SceI DNA-binding domain, and the fourth construct may comprise one or more dI-SceI binding sites.

[0545] In the context of the present invention, the third and fourth chimeric proteins dimerize in the presence of abscisic acid, which preferably allows the transactivation domain to associate with the GAL4 DNA binding domain.

[0546] Preferably, dimerization of the third and fourth chimeric proteins of the invention stimulates transcription. Preferably, transcription of the fourth construct. Preferably, transcription of the fourth construct from the effector domain binding site, preferably from the GAL4 upstream activation sequence. Preferably, dimerization of the third and fourth fusion proteins of the invention thereby stimulates transcription of the protein of interest, preferably transcription of the protein of interest from the fourth construct.

[0547] It will be understood that a nucleic acid construct according to the present invention may comprise DNA or RNA, and that suitable nucleic acid constructs may consist essentially of DNA, may consist essentially of RNA, or may comprise a combination of DNA and RNA.

[0548] Fourth construct The fourth construct according to the present invention comprises a nucleic acid sequence encoding one or more effector domain binding sites and a nucleic acid sequence encoding a protein of interest, and therefore preferably is a fourth nucleic acid construct.

[0549] Suitably, the nucleic acid sequence encoding the one or more effector domain binding sites is operably linked to the nucleic acid sequence encoding the protein of interest, the term "operably linked" being defined herein above.

[0550] Preferably, the one or more effector domain binding sites are suitable for binding of a selected effector domain. Preferably, given that a preferred effector domain is a GAL4 DNA binding domain, the effector domain binding site is a GAL4 DNA binding site, otherwise known as a GAL4 upstream activation sequence.

[0551] Preferably, each GAL4 upstream activating sequence comprises the sequence: CGGAGTACTGTCCTCCG (SEQ ID NO: 28).

[0552] Preferably, each is a GAL4 upstream activation sequence, which is a 17 bp sequence consisting of the sequence CGGAGTACTGTCCTCCG (SEQ ID NO: 28).

[0553] Suitably, the fourth (or second) construct may comprise one or more effector domain binding sites, suitably more than one GAL4 upstream activating sequence, suitably a plurality of GAL4 upstream activating sequences.

[0554] Suitably, more than one effector domain binding site, preferably a GAL4 upstream activation sequence, is in tandem.

[0555] Preferably, the fourth (or second) construct comprises 1 to 15 effector domain binding sites, preferably arranged in tandem. Preferably, the second or fourth construct comprises 1 GAL4 upstream activation sequence, 2 GAL4 upstream activation sequences, 3 GAL4 upstream activation sequences, 4 GAL4 upstream activation sequences, 5 GAL4 upstream activation sequences, 6 GAL4 upstream activation sequences, 7 GAL4 upstream activation sequences, 8 GAL4 upstream activation sequences, 9 GAL4 upstream activation sequences, 10 GAL4 upstream activation sequences, 11 GAL4 upstream activation sequences, 12 GAL4 upstream activation sequences, 13 GAL4 upstream activation sequences, 14 GAL4 upstream activation sequences, or 15 GAL4 upstream activation sequences, preferably arranged in tandem.

[0556] In a preferred embodiment, the fourth (or second) construct contains nine GAL4 upstream activating sequences in tandem.

[0557] Preferably, the fourth construct may further comprise a promoter. Preferably, the fourth construct may comprise an effector domain-binding site, preferably a promoter operably linked downstream of the effector domain-binding site. Preferably, the promoter is a minimal promoter. Preferably, the minimal promoter may be selected from, for example, a minimal TATA box promoter, a minimal adenovirus late promoter, a minimal herpes simplex virus (HSV) thymidine kinase promoter, or a minimal c-fos promoter (consisting of nucleotides -53 to +42).

[0558] Therefore, suitably, the effector domain binding site may comprise the sequence set forth in SEQ ID NO: 19. Therefore, suitably, the effector domain binding site may consist of the sequence set forth in SEQ ID NO: 19. Therefore, suitably, the second construct may comprise the sequence set forth in SEQ ID NO: 19.

[0559] The fourth construct according to the present invention may optionally further comprise a recombination site.

[0560] Suitably, the recombination site is located upstream of one or more effector domain binding sites.

[0561] In a preferred embodiment, the recombination site is selected from any serine recombinase site, hi one embodiment, the recombination site is an attB site.

[0562] Preferably, the fourth construct comprises a nucleic acid sequence encoding a protein of interest. Suitable proteins of interest are as defined elsewhere in this description. Preferably, the nucleic acid sequence encoding the protein of interest is downstream of one or more effector domain binding sites. Preferably, it is downstream of one or more GAL4 upstream activation sequences.

[0563] In the context of the present invention, the GAL4 DNA-binding domain binds to each of one or more GAL4 upstream activating sequences. Preferably, in one embodiment, each GAL4 DNA-binding domain fused to an abscisic acid-binding domain binds to each of one or more GAL4 upstream activating sequences. Therefore, preferably, in a preferred embodiment, either the third or fourth chimeric protein comprising a GAL4 DNA-binding domain fused to an abscisic acid-binding domain binds to each of one or more GAL4 upstream activating sequences.

[0564] Suitably, in a preferred embodiment, in the presence of abscisic acid, association of the abscisic acid binding domains of the third and fourth chimeric proteins brings the transactivation domain into close proximity with the GAL4 DNA binding domain bound to the GAL4 upstream activation sequence in the fourth construct, thereby stimulating transcription of the downstream nucleic acid encoding the protein of interest.

[0565] It will be appreciated that the effector binding domains of the second and fourth constructs are interchangeable, and therefore preferably the fourth construct may comprise one or more dI-SceI binding sites which interact with the dI-SceI DNA binding domain as described above for the second construct.

[0566] It will be understood that a nucleic acid construct according to the present invention may comprise DNA or RNA, and that suitable nucleic acid constructs may consist essentially of DNA, may consist essentially of RNA, or may comprise a combination of DNA and RNA.

[0567] cell The first, second, third and fourth constructs according to the present invention can be introduced into any cell. Therefore, suitably, the present invention relates to a cell comprising the first, second, third and / or fourth construct, preferably all of the first, second, third and fourth constructs.

[0568] Therefore, suitably, the present invention relates to a cell comprising a first chemical-inducible access system and / or a second chemical-inducible access system, preferably a cell comprising a first plant hormone or hormone analogue inducible system, preferably the auxin-inducible system, caffeine-inducible system, mandipropamide-inducible system, or gibberellin-inducible system of the present invention, and / or a second plant hormone or hormone analogue inducible system, preferably the abscisic acid-inducible system of the present invention. Suitably, according to the eighth aspect of the present invention.

[0569] Preferably, the cell can be considered a host cell.

[0570] Preferably, the cell comprising the nucleic acid construct according to the present invention may be an insect, animal, plant, fungal, bacterial, or archaeal cell. Preferably, the cell is an animal cell. Preferably, the cell is a mammalian cell. Preferably, the cell may be a human cell or a non-human cell. Preferably, the cell may be a monkey, dog, cat, mouse, rat, pig, or other animal cell.

[0571] Suitably, the cell may be an immortalized cell or a primary cell. Suitably, the cell may be an immortalized mammalian cell. Suitably, the cell may be an immortalized human or monkey cell. Suitably, the cell comprising the nucleic acid construct according to the present invention may be any mammalian cell line, but preferably may be an HEK293 or CHO-K1 cell.

[0572] Suitably, cells containing a nucleic acid construct according to the present invention may mimic healthy cells or tissues in vivo.

[0573] Advantageously, cells containing a nucleic acid construct according to the invention may mimic diseased cells or diseased tissues in vivo.

[0574] vector The constructs of the present invention may be provided to or introduced into a cell in the form of a vector. Suitably, the first, second, third and / or fourth constructs of the present invention may be provided to or introduced into a vector.

[0575] Generally, the term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends; nucleic acid molecules containing no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and various other polynucleotides known in the art.

[0576] Therefore, suitably, a further aspect of the present invention is a first and / or second construct or vector comprising a first plant hormone inducible system, preferably an auxin inducible system, or a caffeine inducible system, or a mandipropamide inducible system, or a gibberellin inducible system as described herein.Thus, suitably, further provided herein is a third and / or fourth construct or vector comprising a second plant hormone inducible system, preferably an abscisic acid inducible system as described herein.

[0577] Suitably, the one or more vectors may comprise a first, second, third and / or fourth construct.

[0578] In one embodiment, a vector is provided that includes the first and third constructs, which may also be referred to as an inducible vector.

[0579] In one embodiment, a vector is provided that includes the second and fourth constructs, which may also be referred to as a delivery vector.

[0580] Some vectors can direct the expression of genes to which they are operably linked. Such vectors are "expression vectors" and can be selected based on the presence of regulatory elements and the host cells in which expression occurs. This means that the nucleic acid to be expressed is operably linked to regulatory elements, which result in the expression of the nucleotide sequence, whether in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell.

[0581] The term "operably linked" is explained elsewhere in the specification.

[0582] Preferably, the or each vector comprising one or more of the constructs of the invention further comprises one or more regulatory sequences, preferably operably linked to the nucleic acid sequence contained within the or each construct.

[0583] Suitable regulatory sequences control the expression of the nucleic acid sequence in the construct and include, for example, promoters, enhancers, terminators, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and polyU sequences), UTRs, ITRs, introns, etc. For further information, one of ordinary skill in the art is referred to, for example, Goeddel, (1990), Gene Expression Technology in Methods in Enzymology, Vol. 185, Academic Press. Regulatory elements include those that confer direct constitutive expression in many types of host cells and those that direct expression of a nucleotide sequence only in certain cells (i.e., tissue-specific regulatory sequences).

[0584] Tissue-specific promoters direct expression primarily in a desired tissue of interest, such as blood, a specific organ (e.g., liver, pancreas), or a particular cell type. Regulatory elements can also direct expression in a temporally dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific. Promoters useful for this invention include, but are not limited to, constitutive promoters, inducible promoters, developmentally-regulated promoters, tissue-specific / tissue-preferred promoters, etc., as described herein.

[0585] Regulatory elements used herein may be endogenous or heterologous. In some embodiments, endogenous regulatory elements from a subject cell may be inserted into a non-naturally occurring genetic context (e.g., a different location in the genome than that in which they are found in nature), thereby producing a recombinant or modified nucleic acid. In some embodiments, promoters useful for the constructs described herein may be any combination of heterologous and / or endogenous promoters.

[0586] In some embodiments, an inducible promoter can be used. Examples of inducible promoters include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, copper inducible system promoters, salicylic acid inducible system promoters (e.g., PR1a system), glucocorticoid inducible promoters, and ecdysone inducible system promoters.

[0587] Preferably, the promoter operably linked to the first construct, i.e., the promoter operably linked to the nucleic acid sequences encoding the first chimeric protein and the second chimeric protein, is MND, CAG EF1-α, CMV, MSCV, SV40, mouse PGK, human PGK, or UBC. In one embodiment, the promoter is MND. As described above.

[0588] Preferably, the promoter operably linked to the third construct, i.e., the promoter operably linked to the nucleic acid sequences encoding the third and fourth chimeric proteins, is MND, CAG EF1-α, CMV, MSCV, SV40, mouse PGK, human PGK, or UBC. In one embodiment, the promoter is EF1-α.

[0589] Preferably, the promoter operably linked to the second construct is a minimal promoter, preferably selected from a minimal TATA box promoter, a minimal adenovirus late promoter, a minimal herpes simplex virus (HSV) thymidine kinase promoter, or a minimal c-fos promoter (-53 to +42), as described above.

[0590] Preferably, the promoter operably linked to the fourth construct is a minimal promoter, which may be selected from a minimal TATA box promoter, a minimal adenovirus late promoter, a minimal herpes simplex virus (HSV) thymidine kinase promoter, or a minimal c-fos promoter (-53 to +42), as described above.

[0591] Similar to promoters, regulatory elements can include enhancer elements such as the WPRE; the CMV enhancer; the R-U5' segment in the HTLV-I LTR; the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.

[0592] Preferably, the vector may also optionally contain a transcriptional and / or translational termination region (i.e., a termination region) functional in the selected host cell. A variety of transcription terminators are available and are responsible for the termination of transcription across the heterologous nucleotide sequence of interest and accurate mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, native to the operably linked nucleic acid sequence, native to the host cell, or derived from another source (i.e., foreign or heterologous to the promoter, nucleic acid sequence, host, or any combination thereof).

[0593] Preferably, the vector may also contain a nucleotide sequence for a selectable marker that can be used to select transformed host cells. As used herein, a "selectable marker" refers to a nucleotide sequence whose expression confers a distinct phenotype on host cells expressing the marker, thereby allowing such transformed cells to be distinguished from cells that do not possess the marker. Such a nucleotide sequence may encode either a selectable marker or a screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, etc.), or whether the marker is simply a trait that can be identified through observation or testing, such as by screening (e.g., fluorescence). Of course, many examples of suitable selectable markers are known in the art and can be used in the constructs described herein.

[0594] In some embodiments, selectable markers useful in the present invention include polynucleotides encoding polypeptides that confer resistance to antibiotics. Non-limiting examples of antibiotics useful in the present invention include, for example, blasticidin, puromycin, hycromycin, and / or erythromycin. Thus, in some embodiments, a polynucleotide encoding a gene for antibiotic resistance can be introduced into a cell, thereby conferring antibiotic resistance to the cell.

[0595] Non-limiting examples of general classes of vectors include, but are not limited to, viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, or Agrobacterium binary vectors, in double-stranded or single-stranded, linear or circular form, which may or may not be self-infectable or mobilizable. As defined herein, vectors can transform host cells either by integration into the cellular genome or by being present extrachromosomally (e.g., autonomously replicating plasmids with an origin of replication). Additionally, shuttle vectors are included, which refer to DNA vehicles capable of replicating naturally or intentionally in two different host organisms, which may be selected from actinomycetes and related species, bacteria, and eukaryotes (e.g., higher plant, mammalian, yeast, or fungal cells). A plasmid may also be a vector according to this description; a vector is a circular, double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques.

[0596] Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0597] Preferably, in some embodiments of the present invention, the vector is a viral vector. In some embodiments, the first, second, third, and fourth constructs of the present invention may be contained on one or more lentiviral vectors. Preferably, they are contained in one or more lentiviral particles.

[0598] Preferably, according to the sixth aspect of the present invention, a viral particle comprising the first construct and optionally comprising a third construct can be used, and a viral particle comprising the second construct and optionally comprising a fourth construct can be used. Therefore, preferably, the inducer vector and the delivery vector can be contained in the viral particle. Preferably, the viral particle is a lentiviral particle.

[0599] In other cases, one or more of the constructs of the invention may suitably be contained on different types of vectors, such as plasmids, and therefore the method of the invention may suitably use a mix of different vectors to introduce the constructs into the cell.

[0600] Preferably, according to the seventh aspect of the present invention, the first and third constructs of the present invention may be comprised on a lentiviral vector. Therefore, preferably, the delivery vector may be a lentiviral vector. Preferably, the lentiviral vector may be a lentiviral particle. However, preferably, the second and fourth constructs of the present invention may be comprised on a plasmid. Therefore, preferably, the delivery vector may be a plasmid.

[0601] Advantageously, the invention, and in particular the methods of the invention, can utilize several vectors to modify cells so that they contain and express the systems of the invention. Advantageously, the methods of the invention can utilize multiple vectors.

[0602] Advantageously, in methods of the present invention that utilize integration sites within the genome and recombination techniques, additional accessory vectors may be required to facilitate such integration.

[0603] Suitably, in such methods, a vector may be required to generate an integration site or "landing pad" in the genome of the cell into which the construct of the invention can be inserted. Suitably, such a vector may be a lentiviral vector. Suitably, such a vector comprises a first recombination site, which may be paired with a second recombination site present in a vector comprising the construct to be inserted into the genome, such as the delivery vectors described above.

[0604] Suitably, such recombination may be catalysed by a recombinase or integrase enzyme, such as a serine integrase. Suitably, the recombination sites may therefore be attP and attB.

[0605] Advantageously, in such methods, the vector may further need to express a recombinase or integrase enzyme to catalyze the recombination step, such as BXB1 integrase.

[0606] Methods for Producing Cells Comprising a First Plant Hormone-Inducible Access System and / or a Second Plant Hormone-Inducible Access System As discussed elsewhere in the specification, in the context of the present invention, auxin may act as a plant hormone inducer for the chemically induced access system of the present invention. Preferably, auxin induces heterodimerization of TIR1 and AID proteins. Similarly, caffeine may act as a plant hormone inducer for the chemically induced access system of the present invention. Preferably, caffeine induces homodimerization of two αCaffVHH proteins. Similarly, mandipropamide may act as a plant hormone analog inducer for the chemically induced access system of the present invention. Preferably, mandipropamide induces PYR Mandi or PYLcs Mandi and ABI1 protein. Similarly, gibberellins can act as plant hormone inducers for the chemically induced access system of the present invention. Preferably, gibberellins induce heterodimerization of the GID1 protein and the GAI protein or fragments thereof.

[0607] In an aspect of the present invention, there is provided a method for producing a cell comprising a first plant hormone or plant hormone analogue inducible access system of the present invention, preferably an auxin-inducible access system, a caffeine-inducible access system, a mandipropamid-inducible access system, or a gibberellin-inducible access system.

[0608] In an embodiment of the invention, the first and / or second construct of the invention is introduced into the cell via one or more vectors.

[0609] Furthermore, abscisic acid, as described herein, can also act as a plant hormone inducer for the chemically induced access system of the present invention. Preferably, abscisic acid induces heterodimerization of the ABI1 and PYL1 proteins.

[0610] In an embodiment of the present invention, there is provided a method of producing a cell comprising the second plant hormone-inducible access system of the present invention, preferably an abscisic acid-inducible system.

[0611] In one embodiment, the third and / or fourth constructs of the invention are introduced into the cell via one or more vectors.

[0612] Further provided are methods for producing a cell comprising both a first and a second plant hormone inducible system, preferably both an auxin-inducible access system of the present invention and an abscisic acid-inducible access system of the present invention, as well as both a caffeine-inducible access system, a mandipropamid-inducible access system, or a gibberellin-inducible access system of the present invention and an abscisic acid-inducible access system of the present invention.

[0613] In one embodiment, the first, second, third and / or fourth constructs of the invention are introduced into the cell via one or more vectors.

[0614] "Introducing," "introduce," "introduced" (and grammatical variations thereof), in the context of the constructs and cells of the present invention, means presenting a construct of interest to a cell (e.g., a host cell) in such a manner that the construct gains access to the interior of the cell, and includes terms such as "transformation," "transfection," and / or "transduction." As used herein, the terms "transformation," "transfection," and "transduction" refer to the introduction of a construct into a cell. Such introduction into a cell may be stable or transient. Thus, in some embodiments, the host cell is stably transformed with the construct. In other embodiments, the host cell is transiently transformed with the construct.

[0615] In some embodiments, the first, second, third and / or fourth constructs of the present invention may be stably or transiently introduced into a cell.

[0616] In some embodiments, introducing the first, second, third and / or fourth construct into the cell is by transient transfection.

[0617] Suitably, the nucleic acid sequences of the first, second, third and / or fourth constructs introduced by transient transfection can be present in the cell for only a limited time.

[0618] Preferably, the nucleic acid sequence of the construct introduced by transient transfection can be present in the cell for up to 6 hours, up to 12 hours, up to 18 hours, up to 24 hours, up to 30 hours, up to 36 hours, up to 42 hours, up to 48 hours or longer.

[0619] Preferably, the nucleic acid sequence of the construct introduced by transient transfection can remain in the cell for up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days or more.

[0620] Preferably, in such embodiments, the constructs of the invention are introduced into the cell in a vector that remains extrachromosomal. Preferably, the nucleic acid contained in the constructs of the invention is not integrated into the genome of the host cell. Preferably, the constructs of the invention are expressed directly from the vector.

[0621] The term "genome," as used herein, refers to both chromosomes and non-chromosomal elements (i.e., extrachromosomal (e.g., mitochondria, plasmids, and / or extrachromosomal circular DNA (eccDNA))) of a target cell. As used herein, "extrachromosomal" refers to nucleic acid from mitochondria, plasmids, and / or extrachromosomal circular DNA (eccDNA)).

[0622] In another more preferred embodiment, the introduction of the first, second, third and / or fourth construct may be stably introduced or stably transformed.

[0623] The terms "stably introduced" or "stably transformed," as used herein, mean that a nucleic acid sequence is stably incorporated into the genome of a cell, and thus the cell is stably transformed with the construct. Once a construct is stably transformed, and therefore integrated into a cell, the integrated nucleic acid of the construct can be inherited by its progeny, more particularly by its progeny for multiple successive generations.

[0624] Preferably, the first, second, third and / or fourth construct of the present invention may be introduced into the cell by stable transformation. Preferably, the first, second, third and / or fourth construct of the present invention may be integrated into the genome of the cell.

[0625] Advantageously, introduction of the constructs of the present invention into cells can be achieved by any delivery method known in the art, such as standard transfection, electroporation, viral-mediated delivery, transposons, gene editing, etc.

[0626] Suitably, any one or more of the first, second, third and / or fourth constructs of the invention are integrated into the genome of the cell by using any known technique, suitably by using lentiviral integration or by recombination.

[0627] In one embodiment, the constructs of the invention are introduced into the genome by lentiviral integration.

[0628] Therefore, preferably, a method for producing a cell comprising a first and / or second plant hormone-inducible access system, such as an auxin-inducible access system and / or an abscisic acid-inducible access system, comprises the steps of: a) introducing a viral vector comprising the first construct and / or the third construct into the cell; b) introducing a viral vector comprising the second construct and / or the fourth construct into the cell; and b) integrating the first and second constructs, and / or the third and fourth constructs into the genome of the cell.

[0629] In one embodiment, the method of producing a cell comprises both a first plant hormone-inducible access system, such as the auxin-inducible access system of the third aspect, and a second plant hormone-inducible access system, such as an abscisic acid-inducible access system; (a) introducing a viral vector containing the first and third constructs into a cell; (b) introducing a viral vector containing the second and fourth constructs into a cell; (c) integrating the first, second, third, and fourth constructs into the genome of the cell. Includes:

[0630] In one embodiment, the first construct and the third construct may be contained on the same construct, which may be known as an inducer construct. Thus, step (a) may comprise introducing a viral vector comprising the inducer construct into a cell. In one embodiment, the second construct and the fourth construct may be contained on the same construct, which may be known as a delivery construct. Thus, step (b) may comprise introducing a viral vector comprising the delivery construct into a cell.

[0631] In one embodiment, one single viral vector may comprise the first, third, second and fourth constructs. Preferably, the constructs may be comprised in the same viral vector. Preferably, the or each viral vector is a lentivirus. Preferably, a lentiviral particle.

[0632] Preferably, the viral particles are produced by techniques known in the art for producing lentiviral particles. Preferably, the lentiviral particles are produced in producer cells. Preferably, the producer cells are transfected with one or more vectors containing the construct and one or more vectors containing essential viral proteins and cultured under suitable conditions to form viral particles containing the construct. Preferably, the one or more vectors containing essential viral proteins may include an envelope vector and a packaging vector. Preferably, the envelope vector may encode VSV-G. Preferably, the packaging vector may encode Gag, Pol, Rev, and Tat. Thus, preferably, the method may include an initial step of producing viral particles containing a first construct and viral particles containing a second construct, and / or steps of producing viral particles containing a third construct and a fourth construct.

[0633] Preferably, a cell is then transduced with the or each viral particle. Preferably, the cell is transduced with the viral particle under conditions that promote uptake of the viral particle, for example in the presence of a polycation. Preferably, upon transduction, the viral particle releases the construct into the cell, which is then transcribed and integrated into the genome. Preferably, the construct may then be expressed from the genome.

[0634] In another embodiment, the constructs of the present invention are introduced into the genome recombinantly.

[0635] Therefore, suitably, a method for producing a cell comprising a first and / or second plant hormone-inducible access system, such as an auxin-inducible access system and / or an abscisic acid-inducible access system, comprises: (a) introducing the first construct and / or the third construct into a cell; (b) creating an integration site in the genome of the cell, the integration site comprising a first recombination site; (c) introducing into the cell an integration construct comprising the second construct and / or the fourth construct and a nucleic acid sequence encoding an integrase enzyme, wherein the second construct and / or the fourth construct further comprises a second recombination site; (d) integrating the second construct and / or the fourth construct into the genome by recombination between the first and second recombination sites using an integrase enzyme. and Steps (a) and (b) may be performed in any order.

[0636] In one embodiment, the method comprises producing a cell comprising both the first and second plant hormone-inducible access systems, such as both the auxin-inducible access system and the abscisic acid-inducible access system of the third aspect, comprising the steps of: (a) introducing the first and third constructs into a cell; (b) creating an integration site in the genome of the cell, the integration site comprising a first recombination site; (c) introducing into the cell an integration construct comprising the second and fourth constructs and a nucleic acid sequence encoding an integrase enzyme, wherein the second and fourth constructs further comprise a second recombination site; (d) integrating the second construct and the fourth construct into the genome by recombination between the first and second recombination sites using an integrase enzyme. Including, The method may be carried out in any order in steps (a) and (b).

[0637] Suitably, the first and / or third construct may be introduced into the cell in step (a) by any known technique, such as, for example, standard transfection, electroporation, viral-mediated delivery, transposons, gene editing, etc. Suitably, in a preferred embodiment, the first and third constructs are introduced into the cell by viral-mediated delivery, suitably lentiviral delivery, suitably in a lentiviral particle as described above. In one embodiment, the first and third constructs may be contained on the same construct, which may be known as an inducible construct. Thus, step (a) may comprise introducing an inducible construct comprising the first and third constructs into the cell.

[0638] Preferably, the remainder of the method of this embodiment is based on recombinase-mediated integration.

[0639] Suitably, the process of integrating the second and / or fourth construct into the genome of the cell may comprise one or more recombination steps.

[0640] By way of example only, integrating the second and / or fourth construct into the genome may involve three steps. Recombination-based techniques for nucleic acid integration are known, and those skilled in the art will appreciate that any suitable method may be used. The number of steps is for illustrative purposes, and the processes described herein may be combined into one or more "steps."

[0641] Preferably, the first step (b) of creating an integration site (called a "landing pad") in the genome of the cell comprises inserting a first recombination site into the genome of the cell. Preferably, the first recombination site is an integrase site, preferably a serine recombinase site. In one embodiment, the first recombination site is an attP site.

[0642] In an embodiment, the nucleotide sequence of the selectable marker is also integrated into the genome of the cell at the integration site. Thus, preferably, the integration site comprises the first recombination site and the selectable marker.

[0643] Suitably, the selectable marker may be any of the markers described elsewhere in the specification.

[0644] Therefore, preferably, after step (b), the method may comprise screening for cells which comprise the landing pad in their genome. Preferably, such screening comprises exposing the cells to an effective amount of a selection agent and selecting for cells which express the selectable marker. In some embodiments, the selection agent may be an antibiotic and the selectable marker may be an antibiotic resistance gene.

[0645] Preferably, the nucleic acid sequences encoding the recombination sites and / or the nucleic acid sequences encoding the selectable markers are delivered to the cells by any suitable means.

[0646] Preferably by lentiviral integration. Lentiviral integration is described above. Therefore, preferably viral particles, preferably lentiviral particles, are introduced into the cells containing a construct, preferably a nucleic acid construct encoding nucleotide sequences for recombination sites and / or selectable markers.

[0647] In another embodiment, nucleic acid sequences encoding the nucleic acid sequences of recombination sites and / or selectable markers can be delivered to cells by a CRISPR-Cas system. Preferably, the CRISPR-Cas system comprises a Cas nuclease, such as Cas9 or Cas13, operable to cleave genomic DNA, in combination with a guide RNA operable to bind to a target region of genomic DNA. Preferably, the Cas protein and guide RNA are introduced into cells together with a construct, preferably a nucleic acid construct encoding the nucleotide sequence of the recombination site and / or selectable marker. Preferably, the guide RNA directs the Cas protein to cleave genomic DNA at the target region, and the nucleic acid construct encoding the nucleotide sequence of the recombination site and / or selectable marker is introduced into the cleavage site by HDR.

[0648] The second step (c) of introducing the second construct and / or fourth construct and the integrating construct into the cell comprises co-transfecting the second construct, the fourth construct and the integrating construct into the cell, wherein the integrating construct encodes an integrase enzyme.

[0649] Preferably the integration construct comprises a plasmid encoding an integrase enzyme, preferably a serine integrase. Hence, preferably the integrase enzyme is a serine recombinase, preferably a phage-derived serine recombinase.

[0650] Preferably, the phage-derived serine recombinase can be selected from any of C31, Bxb1, φBT1, φC1, MR11, TP901-1, R4, A118, φRV, TG1, φ370.1, Wβ, BL3, SPBc, and K38. In one embodiment, the phage-derived serine recombinase is BXB1.

[0651] Preferably, the second construct and / or the fourth construct may also be contained on a vector. Preferably, the delivery vector can be used to integrate the second and / or fourth construct into the genome of the cell. Preferably, the delivery vector may be a plasmid. Preferably, the delivery vector further comprises, in addition to the second construct and / or the fourth construct, a second recombination site. Preferably, the second recombination site is an integrase site, preferably a serine recombinase site. In one embodiment, the second recombination site is an attB site.

[0652] In one embodiment, the second construct and the fourth construct may be contained on the same construct, which may be known as a delivery construct. Thus, step (c) may comprise introducing into the cell a delivery construct comprising the second and fourth constructs, the delivery construct further comprising a second recombination site. Suitably, in some embodiments, the same construct may also comprise an integrase enzyme.

[0653] Suitable methods of co-transfection include, for example, calcium phosphate-mediated, electroporation, liposome-mediated, exosome-mediated, gene gun, microinjection, and Agrobacterium-mediated transfection. Suitable methods for carrying out such transfection are known to those skilled in the art.

[0654] The third step (d), of integrating the second and / or fourth construct into the genome at the integration site, comprises expressing an integrase enzyme to catalyze recombination b...

Claims

1. A method for screening candidate binding molecules in terms of their biological effects, (a) A step of preparing cells comprising a first inducible system activatable to express a first target protein, and a second inducible system activatable to express a second target protein (optionally); (b) Exposing cells to an effective concentration of a first inducer to induce the expression of a first target protein at a desired level, and optionally exposing cells to an effective concentration of a second inducer to induce the expression of a second target protein at a desired level; (c) A step of bringing cells into contact with candidate binding molecules; (d) A step to determine whether the candidate binding molecule exerts a biological effect on cells expressing the first target protein and, optionally, the second target protein. A method that includes this.

2. A method for determining the minimum level of expression of at least one target protein in a cell in which a candidate binding molecule exerts a biological effect, (a) A step of preparing cells comprising a first inducible system activatable to express a first target protein, and a second inducible system activatable to express a second target protein (optionally); (b) Exposing cells comprising a first inducible system and, optionally, a second inducible system, to several different concentrations of the first inducer to induce several different levels of expression of the first target protein; optionally, exposing cells to several different concentrations of the second inducer to induce several different levels of expression of the second target protein; (c) A step of bringing cells into contact with candidate binding molecules; (d) A step of determining whether the candidate binding molecule exerts a biological effect on cells expressing the first target protein and, optionally, the second target protein, at each level of expression of the first target protein and optionally, the second target protein; (e) A step to determine the minimum expression levels of the first target protein and optionally the second target protein such that the candidate binding molecule exerts a biological effect on cells expressing the first target protein and optionally the second target protein. A method that includes this.

3. The method according to claim 1 or 2, wherein the biological action comprises binding to a first target protein and optionally to a second target protein.

4. The method according to claim 1 or 2, wherein the candidate conjugate molecule is selected from fusion proteins, antibodies or their conjugate fragments, antibody-drug conjugates, aptamers, ankyrin, designed ankyrin repeat proteins (DARPin), peptides, bicyclic peptides, vaccines, cytokines, chemokines, hormones, oncolytic viruses, and bacteria, and the conjugate molecule may be an immunotherapy agent.

5. A method for screening candidate therapeutic agents in terms of their biological effects, (a) a step of preparing cells comprising a first inducible system operable to express a first target protein, and a second inducible system operable to express a second target protein of choice, as well as immune cells; (b) Exposing cells containing a first inducible system and, optionally, a second inducible system, to an effective concentration of the first inducer to induce a desired level of expression of the first target protein; optionally, exposing the cells to an effective concentration of the second inducer to induce a desired level of expression of the second target protein; (c) A step of bringing immune cells into contact with a candidate therapeutic agent; (d) A step to determine whether the contacted immune cells exert a biological effect on cells expressing the first target protein and, optionally, the second target protein. A method that includes this.

6. A method for determining the minimum level of expression of at least one target protein in a cell for immune cells to exert a biological effect in the presence of a candidate therapeutic agent, (a) a step of preparing cells comprising a first inducible system operable to express a first target protein, and a second inducible system operable to express a second target protein of choice, as well as immune cells; (b) Exposing cells comprising a first inducible system and optionally a second inducible system to several different concentrations of the first inducer to induce several different levels of expression of the first target protein, and optionally exposing cells to several different concentrations of the second inducer to induce several different levels of expression of the second target protein; (c) A step of bringing immune cells into contact with a candidate therapeutic agent; (d) A step of determining whether the contacted immune cells exert a biological effect on cells expressing the first target protein and, optionally, the second target protein, at each level of expression of the first target protein and optionally, the second target protein; (e) A step of determining the minimum expression levels of the first target protein and optionally the second target protein such that the contacted immune cells exert a biological effect on cells expressing the first target protein and optionally the second target protein. A method that includes this.

7. The method according to any one of claims 5 or 6, wherein the biological action includes targeting cells expressing a first target protein and, optionally, a second target protein.

8. The method according to claim 5 or 6, wherein the candidate therapeutic agent is a biologic and can be selected from fusion proteins, antibodies or their conjugated fragments, antibody-drug conjugates, aptamers, ankyrin, designed ankyrin repeat proteins (DARPin), peptides, bicyclic peptides, vaccines, cytokines, chemokines, hormones, oncolytic viruses, and bacteria.

9. The method according to claim 5 or 6, wherein the immune cells are selected from T cells, NK cells, B cells, lymphocytes, dendritic cells, and mesenchymal cells, or immortalized cells thereof.

10. A method for screening candidate manipulated immune cells for their biological effects, (a) A step of preparing cells comprising a first inducible system activatable to express a first target protein, and a second inducible system activatable to express a second target protein (optionally); (b) Exposing cells comprising a first inducible system and optionally a second inducible system to an effective concentration of a first inducer to induce a desired level of expression of a first target protein; optionally, exposing cells to an effective concentration of a second inducer to induce a desired level of expression of a second target protein; (c) The step of bringing cells into contact with candidate manipulated immune cells; (d) A step to determine whether the candidate manipulated immune cells exert a biological effect on cells expressing the first target protein and, optionally, the second target protein. A method that includes this.

11. A method for determining the minimum level of expression of at least one target protein in a cell for a candidate manipulated immune cell to exert a biological effect, (a) A step of preparing cells comprising a first inducible system activatable to express a first target protein, and a second inducible system activatable to express a second target protein (optionally); (b) Exposing cells comprising a first inducible system and, optionally, a second inducible system, to several different concentrations of the first inducer to induce several different levels of expression of the first target protein; optionally, exposing cells to several different concentrations of the second inducer to induce several different levels of expression of the second target protein; (c) The step of bringing cells into contact with candidate manipulated immune cells; (d) A step of determining whether the candidate engineered immune cells exert a biological effect on cells expressing the first target protein and, optionally, the second target protein, at each level of expression of the first target protein and optionally, the second target protein; (e) A step of determining the minimum expression levels of the first target protein and optionally the second target protein such that candidate engineered immune cells exert a biological effect on cells expressing the first target protein and optionally the second target protein. A method that includes this.

12. The method according to claim 10 or 11, wherein exerting a biological effect involves targeting cells that express a first target protein and, optionally, a second target protein.

13. The method according to claim 10 or 11, wherein candidate manipulated immune cells are selected from cells expressing CAR or T cell receptor (TCR).

14. The method according to claim 1, 5, or 10, wherein the effective concentration of the first or second derivative is in the range of 0.001 μM to 2000 μM.

15. The method according to claim 1, 5, or 10, comprising the steps of exposing cells to a plurality of different concentrations of a first inducer to induce a plurality of different levels of expression of a first target protein, and optionally exposing cells to a plurality of different concentrations of a second inducer to induce a desired level of expression of a second target protein.

16. The method according to claim 2, 6, or 11, wherein the concentrations of the first derivative and optionally the second derivative are in the range of 0.001 μM to 2000 μM.

17. The method according to claim 2, 6, or 11, wherein cells are exposed to at least two different concentrations of a first inducer and optionally a second inducer, and can be exposed to at least low concentrations in the range of 0.001 to 50 μM and high concentrations in the range of 50 to 2000 μM.

18. The method according to claim 1, 2, 5, 6, 10, or 11, wherein the biological action is selected from binding, targeting, entrapment, trogocytosis, endocytosis, phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), cell disintegration, T cell-mediated cell disintegration, antibody-dependent cell-mediated phagocytosis (ADCP), perforation, cytotoxicity, cytokine / chemokine activity or release, proliferation, cell activation, upmodulation or downmodulation of surface receptors.

19. The method according to claim 1, 2, 5, 6, 10, or 11, wherein the first and second inductive systems are different.

20. The method according to claim 1, 2, 5, 6, 10, or 11, wherein the cell comprises both a first and a second inducible system.

21. The method according to claim 1, 2, 5, 6, 10, or 11, wherein the first inductive system does not interact with the second inductive system.

22. The method according to claim 1, 2, 5, 6, 10, or 11, wherein the first and second inducible systems may be selected from a hypoxia-inducible system, a forskolin-inducible system, a temperature-inducible system, a pH-inducible system, a molar osmotic concentration-inducible system, a carbon source-inducible system, an alcohol-inducible system, an amino acid-inducible system, a steroid-inducible system, a tetracycline-inducible system, a Kumat-inducible system, a 4-hydroxytamoxifen (OHT)-inducible system, a gas-inducible system, a riboswitch system, a ribozyme system, an aptazyme system, a metallothionein-inducible system, a rapamycin-inducible system, a rheoswitch, a CRISPR system, and a chemically induced approach system.

23. The method according to claim 22, wherein the first and second inductive systems are chemically induced access systems (CIP systems).

24. The method according to claim 23, wherein the first and second inducible systems are plant hormone or plant hormone analog inducible access systems.

25. The method according to claim 24, wherein the plant hormone or plant hormone analog-inducible approach system is selected from auxin, abscisic acid, gibberellin, ethene, cytokinin, salicylic acid, jasmonate, brassinosteroid, peptide, and caffeine-inducible approach systems.

26. The method according to claim 24, wherein at least one of the inductive systems is an abscisic acid inductive approach system.

27. The method according to claim 24, wherein the first inducible system is selected from a caffeine-inducible approach system, a mandipropamide-inducible approach system, and a gibberellin-inducible approach system, and the second inducible system is an abscisic acid-inducible approach system.

28. The method according to claim 2, 6, or 11, wherein the candidate exerts a biological effect on cells expressing the first target protein and optionally the second target protein, and the minimum expression level of the first target protein and optionally the second target protein is such that a higher biological effect than the background biological effect is achieved.

29. The method according to claim 28, wherein the minimum expression level of the first target protein and the second target protein, which exerts a biological effect on cells expressing the first target protein and optionally the second target protein, is such that a biological effect is achieved that is at least 3, 4, 5, 6, 7, 8, 9, or 10 standard deviations above the background biological effect.

30. The method according to claim 28, wherein the background biological effect is the candidate biological effect on control cells.

31. The method according to claim 28, wherein the candidate exerts a biological effect on cells expressing the first target protein and optionally the second target protein, and the minimum expression level of the first target protein and optionally the second target protein is the activation threshold for the first target protein and optionally the second target protein.

32. The method according to claim 28, wherein the minimum level of expression of the first target protein and / or the second target protein, or activation threshold, at which the candidate exerts a biological effect on cells expressing the first target protein and / or the second target protein, is calculated using receiver operational characteristic (ROC) curve analysis.

33. (a) a cell comprising a first chemical-inducible approach system and / or (b) a second chemical-inducible approach system, The first chemical substance-guided approach system (a) is, (i) the first chimeric protein, and Second chimeric protein A first construct comprising a promoter operably ligated to a nucleic acid sequence encoding, The first and second chimeric proteins each comprise a first inducer-binding domain and an effector domain; Each first inducer-binding domain is operable to bind to the first inducer; the effector domain comprises a transactivation domain and a first DNA-binding domain; The first construct has different effector domains in the first and second chimeric proteins; and (ii) A second construct comprising nucleic acid sequences encoding one or more first DNA-binding domain binding sites operably ligated to a nucleic acid sequence encoding the first target protein. Includes; The second chemical-inducible approach system (b) is, (i) A third chimeric protein, and The fourth chimeric protein A third construct comprising a promoter operably ligated to a nucleic acid sequence encoding, The third and fourth chimeric proteins each contain a second inducer-binding domain and an effector domain; Each second inducer-binding domain is operable to bind to a second inducer; the effector domain includes a transactivation domain or a second DNA-binding domain; The third construct has different effector domains in the third and fourth chimeric proteins; and (ii) A fourth construct comprising nucleic acid sequences encoding one or more second DNA-binding domain binding sites operably ligated to a nucleic acid sequence encoding a second target protein. Includes; A cell in which the first chemical-inducible approach system does not interact with the second chemical-inducible approach system, and one of the first or second DNA-binding domains is a dI-SceI DNA-binding domain.

34. The cell according to claim 33, wherein the first and / or second chemical-induced approach system is a plant hormone or plant hormone analog-induced approach system.

35. The cell according to claim 34, wherein the plant hormone or plant hormone analog-inducible approach system is selected from auxin, abscisic acid, gibberellin, ethene, cytokinin, salicylic acid, jasmonate, brassinosteroid, peptide, and caffeine-inducible approach systems.

36. The cell according to claim 33, wherein at least one of the chemical-induced approach systems is an abscisic acid-induced approach system.

37. The cell according to claim 33, wherein at least one of the chemical-induced approach systems is selected from an auxin-induced approach system, a caffeine-induced approach system, a mandipropamide-induced approach system, and a gibberellin-induced approach system.

38. The cell according to claim 33, wherein the first chemical-induced approach system is selected from a caffeine-induced approach system, a mandipropamide-induced approach system, and a gibberellin-induced approach system, and the second chemical-induced approach system is an abscisic acid-induced approach system.

39. The auxin-guided approach system The first chimeric protein, and Second chimeric protein A first construct comprising a promoter operably ligated to a nucleic acid sequence encoding Includes, The first and second chimeric proteins each contain an auxin-binding domain and an effector domain; The cell according to claim 37, wherein the auxin-binding domain is selected from transport inhibitor response 1 protein (TIR1) or auxin / indole-3-acetate protein (AID); the effector domain is selected from a transactivating domain or a catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI); and the auxin-binding domain and effector domain of the first and second chimeric proteins are different.

40. Caffeine-induced approach systems The first chimeric protein, and Second chimeric protein A first construct comprising a promoter operably ligated to a nucleic acid sequence encoding Includes, The first and second chimeric proteins each contain a caffeine-binding domain and an effector domain; The cell according to claim 37, wherein the caffeine-binding domain is an anti-caffeine heavy chain antibody fragment (αCaffVHH); the effector domain is selected from a transactivating domain or a DNA-binding domain selected from a Gal4 DNA-binding domain and a catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI); and the effector domains of the first and second chimeric proteins are different.

41. The mandipropamide-induced approach system The first chimeric protein, and Second chimeric protein A first construct comprising a promoter operably ligated to a nucleic acid sequence encoding Includes, The first and second chimeric proteins each contain a mandipropamide (Mandi) binding domain and an effector domain; The mandipropamide-binding domain is a modified pyrabactin receptor (PYR Mandi ), modified pyrabactin-like receptors (PYLcs Mandi ), and abscisic acid-insensitive protein 1 (ABI) are selected; the effector domain is selected from the transactivation domain or from the DNA-binding domain, which is selected from the Gal4 DNA-binding domain and the catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI); The cell according to claim 37, wherein the mandipropamide-binding domain and effector domain of the first and second chimeric proteins are different.

42. Gibberellin-guided approach systems, The first chimeric protein, and Second chimeric protein A first construct comprising a promoter operably ligated to a nucleic acid sequence encoding Includes, The first and second chimeric proteins each contain a gibberellin-binding domain and an effector domain; The gibberellin-binding domain is selected from gibberellin-insensitive dwarf1 protein (GID1) and gibberellin-insensitive (GAI) proteins or fragments thereof; the effector domain is selected from a transactivation domain or a DNA-binding domain selected from a Gal4 DNA-binding domain and a catalytically inactive I-SceI endonuclease DNA-binding domain (dI-SceI); The cell according to claim 37, wherein the gibberellin-binding domain and effector domain of the first and second chimeric proteins are different.

43. The cell according to claim 33, wherein the second construct comprises one or more dI-SceI binding sites or one or more Gal4 upstream activation sequences operably linked to a nucleic acid sequence encoding a first target protein.

44. The abscisic acid-inducible approach system A third chimeric protein, and The fourth chimeric protein A third construct comprising a promoter operably linked to a nucleic acid sequence encoding Includes, The third and fourth chimeric proteins each contain an abscisic acid-binding domain and an effector domain; The abscisic acid binding domain is selected from ABI1 or the pyrabactin resistance-like protein PYL1; the effector domain is selected from the transactivation domain or from the DNA binding domain selected from the Gal4 DNA binding domain and the catalytically inactive I-SceI endonuclease DNA binding domain (dI-SceI); The cell according to claim 36, wherein the abscisic acid-binding domain and the effector domains of the third and fourth chimeric proteins are different.

45. The cell according to claim 33, wherein the fourth construct comprises one or more dI-SceI binding sites or one or more Gal4 upstream activation sequences operably linked to a nucleic acid sequence encoding a second target protein.

46. The cell according to claim 43, wherein one or more dI-SceI binding sites comprise 1 to 15 I-SceI DNA binding sites, and the I-SceI DNA binding sites may be arranged in tandem.

47. The cell according to claim 43, wherein one or more Gal4 upstream activating sequences comprise 1 to 15 GAL4 upstream activating sequences, and the GAL4 upstream activating sequences may be arranged in tandem.

48. The cell according to any one of claims 39 to 47, wherein the above or each transactivation domain is selected from Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4, and p53, NFAT, NF-κB, VP16, or VP34 in yeast.

49. The cell according to any one of claims 33 to 47, wherein the cell is a mammalian cell.

50. The cell according to claim 1, 2, 5, 6, 10, or 11, or claim 33, wherein the first and / or second target protein is an antigen.

51. The method or cell according to claim 50, wherein the antigen is a tumor-associated antigen (TAA).

52. The method or cells according to claim 50, wherein the antigen is selected from CD19, BCMA, CD123, mesothelin, GD2, CD20, CD33, CD47, HER2, CD22, CD13, PSMA, EGFR vIII, EGFR, CD38, EpCAM, PSCA, CEA, HIV, glypican-3, FLT3, NKG2D, claudin 18.2, DLL3, CS1, MUC16, CD3, PD-L1, 4-1BB, PD-1, LAG3, CTLA-4, MUC1, 5T4, CD40, CD155, OX-40, NY-ESO, ROR1, TROP2, VEGFRI, VEGFRII, CLL, CD30, CD70, CD133, TIM-3, L1CAM, ICOS, DLL4, Fr-alpha, WT1, IL13R-alpha, Lewis-Y, or cMET.

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