Receptor- and drug-dependent regulated on-site production of biologics

Abstract

The present invention generally relates to the production of biologics using genetically engineered cells, including genetically engineered immune cells. The genetically engineered cells, e.g., T cells, contain a receptor, e.g., a chimeric antigen receptor, capable of receiving an activation signal, e.g., binding to an antigen. The cells further contain a chimeric protein containing a docking domain, which can bind to the receptor and inhibit signal transduction normally induced when the receptor receives an activation signal. The chimeric protein further contains a drug regulatory protein stability domain. The cells further contain an inducible promoter operably linked to a nucleic acid encoding a protein of interest (e.g., a biologic), such that the inducible promoter is induced when the receptor receives an activation signal in the absence of the chimeric protein. A drug capable of modulating the drug regulatory protein stability domain modulates the stability and levels of the chimeric protein, thereby modulating the production of the biologic.

Description

[Technical Field]

[0001] The present invention generally relates to the production of biologics using genetically engineered cells, including genetically engineered immune cells. The genetically engineered cells, e.g., T cells, contain a receptor, e.g., a chimeric antigen receptor, capable of receiving an activation signal, e.g., binding to an antigen. The cells further contain a chimeric protein containing a docking domain, which can bind to the receptor and inhibit signaling normally induced when the receptor receives an activation signal. The chimeric protein further contains a drug-regulated protein stability domain. The cells further contain an inducible promoter operably linked to a nucleic acid encoding a protein of interest (e.g., a biologic), such that the inducible promoter is induced when the receptor receives an activation signal in the absence of the chimeric protein. A drug capable of modulating the drug-regulated protein stability domain modulates the stability and levels of the chimeric protein, thereby modulating the production of the biologic. [Background technology]

[0002] The Background of the Invention contains information that may be helpful in understanding the present invention. It is not an admission that any of the information presented herein is prior art or relevant to the claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Adoptive T cell therapy has demonstrated significant efficacy in several human malignancies. Specifically, chimeric antigen receptor (CAR) T cell therapy has demonstrated significant clinical efficacy in hematopoietic malignancies, leading to the approval of numerous drugs. Furthermore, the clinical activity of tumor-infiltrating lymphocytes (TILs) has been well established in melanoma. Thus, there is growing evidence that tumor-reactive immune cell products, such as T cell products, are active and useful in the treatment of cancer, particularly solid tumors such as these, although not yet optimal.

[0004] At the same time, the impact of current immune cell-based therapies, such as T cell receptor (TCR)-T cells, CAR-T cells, and TIL therapy, on other solid malignancies has been modest and suboptimal to date. Based on these data, it has been proposed that next-generation immune cell-based therapies, such as adoptive T cell therapy, should not rely solely on the infusion of tumor-reactive T cells. Rather, such cell products may possess the ability to induce intratumoral inflammation. For example, there is evidence, both preclinical and clinical, that local production of (potent) cytokines, such as IL-12, can promote tumor control.

[0005] However, systemic administration or unlimited production of these cytokines from transplanted adoptive cells is precluded by severe toxicity.

[0006] A second challenge in immune cell-based therapeutics, including CAR and TCR T cells, is the lack of sufficiently tumor-selective targets. For example, on-target and off-tumor toxicity can be a life-threatening complication of CAR T cell and bispecific T cell engager (BiTE) therapy products (Edeline et al. J Hematol Oncol. 2021;14:65, Bonifant et al. Mol Ther Oncolytics. 2016 Apr 20;3:16011). Only a few sufficiently specific tumor-specific antigens (TSAs) can be safely targeted with cell-based therapies without causing adverse effects associated with on-target and off-tumor toxicity.

[0007] To improve the therapeutic index of cell-based therapies and mitigate on-target and off-tumor toxicity, attempts have been made to restrict immune responses to the tumor microenvironment. Examples of these methods include SynNotch-based CAR-T cells (Morsut et al. Cell. 2016 Feb 11;164(4):780-91). This method uses a synthetic Notch receptor to detect the presence of a first antigen, which then triggers the expression of an antigen receptor capable of detecting a second antigen. While this method can improve the stringency of tumor detection, the magnitude and duration of the elicited immune response are not controlled. This lack of control can be problematic, as continuous activation of immune cells can lead to excessive immune responses and associated side effects (Brudno et al. Blood Rev. 2019 Mar;34:45-55) and can also lead to cellular exhaustion (Gumber et al. EBioMedicine. 2022 Mar;77:103-941). There are also concerns about the immunogenicity of non-human sequences in genetically engineered cell therapy products, which may lead to, for example, rejection of transplanted adoptive T cells.

[0008] In light of this, novel products, compositions, methods, and uses that allow for spatial (e.g., in or near the tumor, e.g., tumor microenvironment (TME)) and temporal (i.e., ability to control activity levels in time) control of the production of proteins of interest, such as biologics, such as cytokines, preferably simultaneously, are highly desirable but not yet readily available. At the same time, immune cell therapies, such as T cell therapies, that induce immune cell activation in a way that more restricts the immune response to targets, such as the tumor microenvironment, e.g., inducing T cell activation, are highly desirable but not yet readily available. In particular, there is a clear need in the art for reliable, efficient, and reproducible products, compositions, methods, and uses that can be used in immune cell therapies, particularly in the treatment of tumors, such as cancers and solid tumors. It can therefore be seen that the technical problem underlying the present invention is to provide products, compositions, methods, and uses that meet any of the aforementioned needs. This technical problem is solved by the claims and the embodiments characterized herein below. Summary of the Invention

[0009] There is a need to improve the therapeutic window of cell therapies, particularly immune cell therapies such as T cell- and NK cell-based therapies, in the treatment of tumors, e.g., solid tumors. As embodied and broadly described herein, the present invention relates to the surprising discovery that the inventions disclosed herein provide new methods for efficiently delivering genetically engineered cells, genetic constructs, and biological agents, such as cytokines, and other proteins of interest, such as antibodies, T cell engagers, and therapeutic proteins, to tumor cells, particularly solid tumors, thereby improving the therapeutic window of such therapies, e.g., cancer treatment. The protein of interest may be multiple proteins of interest or may be (part of) a protein complex. The present invention is exemplified herein with respect to various immune cells, including T cells and NK cells, and the production of proteins of interest, e.g., in the form of cytokines or bispecific T cell engagers, useful for the treatment of tumors (cancer), particularly solid tumors. Based on the entire disclosure herein, those skilled in the art will readily understand that the present invention is not limited to such examples. Those skilled in the art will be able to apply the inventions disclosed herein to other situations, such as other cells, other receptors, other proteins of interest produced, and other diseases treated, based on the overall teachings provided herein.

[0010] Thus, the present invention provides a novel and improved approach that allows the production of proteins (or biologics) of interest from genetically engineered cells, particularly immune cells (CAR-T, TCR-T, TIL, NK cells, and other suitable cells known to those skilled in the art). Production is strictly dependent on an activating signal, such as an antigen, which activates the receptor that receives the signal, and modulation using the agent (e.g., a small molecule). In some embodiments, the activating signal is specific to (or limited to) a particular microenvironment of the patient, e.g., the tumor microenvironment. By providing the cells of the present invention to a patient, the receptor present in the cells can receive an activating signal provided in the particular microenvironment (e.g., an antigen present in the tumor, preferably a tumor-specific antigen). However, the receptor in the cells of the present invention cannot, or can only transmit the received signal to a very limited extent, and cannot activate the cells, or can only activate them to a limited extent, once the receptor receives an activating signal (e.g., only when a ligand for the receptor is bound), due to the expression and / or presence of a chimeric protein in the cells that can interact with or bind to the receptor. As a result of the interaction or binding of the chimeric protein with a receptor that has received the activation signal (i.e., been activated), the chimeric protein of the present invention can block or inhibit the transmission of the signal received by the receptor. For example, when (a domain of) the chimeric protein binds to the cytoplasmic portion of a receptor that has been activated by receiving the activation signal, e.g., a transmembrane cell surface receptor (including a receptor complex), the activated receptor is inhibited or blocked from transmitting the signal to the cell, thereby, e.g., preventing the cell from responding to the activation signal as it would naturally.

[0011] The chimeric proteins of the present invention further comprise a drug-regulated protein stability domain that degrades the chimeric protein in the presence of a drug, thereby unblocking signal transduction by the receptor. Unblocking this blockage then allows signal transduction through the cell, allowing the cells of the present invention to produce a protein of interest (or biological protein). By providing the drug to a patient with engineered cells of the present invention, the engineered cells can, for the first time, be tightly regulated in their production of a biological substance (protein of interest) both spatially (in a particular microenvironment where an activating signal may be present) and temporally (at a desired time point). Furthermore, by varying the amount of the drug provided to the patient, the production level of a biologic can be tightly regulated. Thus, the present invention allows for tight regulation of cell, e.g., T-cell, activation in a patient, allowing for on-site production (e.g., tumor microenvironment) of a protein of interest (e.g., a therapeutic agent) and delivery to target cells (e.g., tumor cells).

[0012] The present invention, the general teachings of which are illustrated in the Examples, allows for precise control of both the timing and amount of production of a protein of interest, as well as localized (targeted) delivery, using genetically engineered cells, particularly immune cells. This makes it possible, for the first time, to produce appropriate amounts of a biological agent at the site of disease. The present invention makes it possible to optimize the therapeutic window, prevent excessive production of such biological agents to avoid side effects, and simultaneously prevent exhaustion of immune cells upon activation (due to receiving an activation signal). Thus, it has surprisingly been found that the drug-regulating chimeric proteins described herein can control the production level of a protein of interest / biological protein. Furthermore, it has also surprisingly been found that the present invention improves the synthesis of a protein of interest within cells. Furthermore, it has also surprisingly been found that the residual activity of the (activated) cells of the present invention, e.g., T cells, in the absence of a drug that modulates the protein stability domain of the present invention, particularly an immunomodulatory imide drug such as lenalidomide, is significantly lower than that of prior art systems. In other words, the system disclosed herein makes it possible to control the amount of protein produced at the site of interest.

[0013] In the Examples, the inventors created a transgene design in which production of a protein of interest (biologic payload, biologic) is operationally linked to the level of ITAM signaling in immune cells, and the level of ITAM signaling is regulated by a chimeric protein (also referred to herein as a rheostat switch). The rheostat switches used in the present invention and exemplified in the Examples, particularly RheoBrick®, tightly control immune cell activation using orally available, clinically approved drugs or small molecules such as lenalidomide (see also WO 2021 / 080427). The RheoBrick concept is sometimes referred to as "CRASH-IT," as discussed, for example, in Sahillioglu et al. Cancer Immunol Resma. 2021 Sep;9(9):999-1007 and Sahillioglu et al. Hum Gene Ther. 2021 Oct;32(19-20):1029-1043.

[0014] Structurally, the rheostat switch (e.g., RheoBrick®) contained in the cells of the present invention consists of three functional domains: a docking domain that forms a reversible interaction with an antigen receptor and further contains an inhibitory domain or a portion thereof that suppresses ITAM receptor-dependent signaling pathways such as the TCR / CAR / NKR (NK cell receptor) signaling pathway, particularly the TCR / CAR / NKR signaling pathway in T cells (e.g., tumor-infiltrating lymphocytes (TILs)) and NK cells, and a degron domain (or drug-regulated protein stability domain) that controls the stability of the rheostat switch.

[0015] Additionally, the genetically engineered cells of the present invention comprise a transgene under the control of a promoter that is induced / expressed in the cells of the present invention only, or preferentially, upon activation of the cells (via a receptor capable of receiving an activating signal) in the absence of the chimeric protein (e.g., rheostat switch), e.g., after providing the cell / patient with a sufficient drug. The transgene encodes the protein of interest, such that the protein of interest is (preferentially) produced in the absence of the chimeric protein (e.g., rheostat switch), e.g., when the patient is administered a drug that causes degradation of the chimeric protein (e.g., rheostat switch), thereby unblocking signaling of the receptor activated by the activating signal.

[0016] In some embodiments, provided are nucleic acids and / or vectors that (1) encode a chimeric protein of the invention, (2) comprise an inducible promoter operably linked to a nucleic acid encoding a protein of interest, (3) encode a receptor of the invention, or combinations thereof. The nucleic acids and / or vectors may further comprise additional elements, as detailed herein, that further improve expression of the chimeric protein of the invention and the protein of interest, thereby improving precise control of both the timing and dosage of biologic production ex vivo or in vivo (e.g., in a patient).

[0017] Thus, according to a first aspect, there is provided a genetically engineered cell comprising: (a) receptors capable of receiving activating signals (e.g., TCR, CAR and / or NKR and other ITAM receptors); (b) i. a docking domain capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives the activating signal; ii. a drug-regulated protein stability domain; and a chimeric protein comprising: (c) an inducible promoter operably linked to a nucleic acid encoding a protein of interest, the inducible promoter being induced when the receptor receives an activating signal in the absence of the chimeric protein.

[0018] In some embodiments according to the present invention, the engineered cells may be immune cells, which may be selected from the group consisting of T cells, CAR T cells, NK cells, CAR NK cells, macrophages, tumor infiltrating lymphocytes (TILs), and CAR macrophages.

[0019] In an embodiment of the invention, the genetically engineered cell is present in an animal. In an embodiment of the invention, the genetically engineered cell is present in a non-human animal. In an embodiment of the invention, the genetically engineered cell is present in a human. In an embodiment of the invention, an animal is provided comprising at least one genetically engineered cell according to the invention. In an embodiment of the invention, a non-human animal is provided comprising at least one genetically engineered cell according to the invention. In an embodiment of the invention, a human is provided comprising at least one genetically engineered cell according to the invention. In an embodiment of the invention, the animal, non-human animal or human further comprises a tumor. In an embodiment of the invention, the animal, non-human animal or human is suffering from cancer.

[0020] In some embodiments of the present invention, the receptor is a transmembrane receptor, e.g., a cell surface receptor, e.g., a T cell receptor (TCR), a chimeric antigen receptor (CAR), or an NK cell receptor (NKR), and / or the receptor is for an antigen, e.g., a tumor antigen.

[0021] In some embodiments of the present invention, the activation signal is a ligand that can interact with the receptor to activate the receptor and provide an activation signal that transduces a signal throughout the cell. In some embodiments, the ligand is an extracellular ligand. In some embodiments, the ligand is a protein. In preferred embodiments, the activation signal is an antigen, e.g., a tumor-specific antigen.

[0022] In some embodiments of the present invention, the chimeric protein is a cytoplasmic chimeric protein. In some embodiments, the docking domain contained in the chimeric protein binds to a receptor (only or preferentially) when the receptor is activated by an activation signal. In some embodiments, the docking domain contains an SH2 domain that can bind to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) contained in the receptor (the ITAM is phosphorylated after receiving an activation signal (e.g., after the receptor interacts with a ligand, e.g., an antigen)). In some embodiments, according to the present invention, the SH2 domain is derived from a protein selected from the group consisting of Zap70, Syk, and Lck.

[0023] In some embodiments, the ITAM present in the receptor is present in a TCR, a CAR, or an NKR, more preferably the ITAM is derived from or present in the CD3ζ chain, CD3ε chain, CD3δ chain, CD3γ chain, FceRIγ chain, or DAP12.

[0024] In some embodiments, the docking domain further comprises an immunoreceptor tyrosine-based switch motif (ITSM), or an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), preferably the ITIM and / or ITSM are derived from an inhibitory receptor protein, preferably an inhibitory immunoreceptor protein, preferably a protein selected from the group consisting of PD1, BTLA, SIRPalpha, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, and LY9. The ITSM, ITIM, or both contained in the docking domain inhibit signaling induced when the receptor receives an activating signal. In other words, in these embodiments, when the receptor receives an activating signal (e.g., binds to a ligand, e.g., an antigen), the chimeric protein of the present invention can bind to the receptor, e.g., due to the presence of a docking domain comprising an SH2 domain, and subsequently block signaling due to the presence of an inhibitory domain comprising an ITSM, an ITIM, or both, in accordance with the present invention. Although SH2 domains can inhibit ITAM signaling (as an example of signal transduction by the receptor after it receives an activating signal), in some embodiments, the presence of an SH2 domain alone has been shown to partially inhibit ITAM signaling, particularly when ITAM signaling is strong. Thus, to strictly inhibit ITAM signaling, the presence of an ITIM domain, an ITSM domain, or both an ITIM domain and an ITSM domain is preferred.

[0025] In some embodiments of the present invention, the drug-regulated protein stability domain is a CRBN polypeptide substrate domain capable of binding to a CRBN protein in response to a drug, preferably thereby promoting degradation of the chimeric protein via the ubiquitin pathway. In some embodiments, the drug-regulated protein stability domain comprises a Cys2-His2 zinc finger domain capable of drug-inducible binding to a CRBN polypeptide, and preferably, the Cys2-His2 zinc finger domain is a hybrid zinc finger domain (e.g., a fusion protein / domain comprising portions from at least two different zinc fingers, where the fusion protein does not exist in nature). When used to control the level of the chimeric protein, such Cys2-His2 zinc finger domains have been found to allow for tight control and regulation of cellular activation and thereby regulated production of the protein of interest, particularly when the Cys2-His2 zinc finger domain is combined with other preferred features of the chimeric proteins of the present invention and an inducible promoter is operably linked to a nucleic acid encoding the protein of interest.

[0026] In some embodiments, the Cys2-His2 zinc finger domain is a hybrid zinc finger domain composed of a β-hairpin loop derived from a first Cys2-His2 zinc finger domain and an α-helical region derived from a second Cys2-His2 zinc finger domain, and preferably the hybrid zinc finger domain comprises one, two, or more amino acid substitutions in the β-hairpin loop derived from the first Cys2-His2 zinc finger domain and / or the α-helical region derived from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain comprises two amino acid substitutions in the β-hairpin loop derived from the first Cys2-His2 zinc finger domain and / or the α-helical region derived from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain comprises three amino acid substitutions in the β-hairpin loop from the first Cys2-His2 zinc finger domain and / or the α-helical region from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain comprises four amino acid substitutions in the β-hairpin loop from the first Cys2-His2 zinc finger domain and / or the α-helical region from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain is a hybrid zinc finger domain disclosed herein.

[0027] In some embodiments, the drug regulatory protein stability domain comprises an additional Cys2-His2 zinc finger domain, preferably an additional hybrid zinc finger domain.

[0028] In some embodiments, the agent capable of inducing degradation of a chimeric protein of the present invention is an immunomodulatory imid drug (IMiD), preferably the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iberdomide, CC-885, salts and analogs thereof.

[0029] In some embodiments, the nucleic acid encoding the protein of interest encodes a cytokine, interleukin, interferon (in some embodiments, the interferon is interferon gamma), chemokine, receptor, ligand, antibody or antibody fragment, bispecific antibody, T cell engager, bispecific T cell engager (in some embodiments, the bispecific T cell engager is blinatumomab), checkpoint inhibitor antagonist, agonist, enzyme, regulatory element, transcription factor, or DNA-binding domain of a transcription factor. In some embodiments, the cytokine is an interleukin, preferably selected from the group consisting of IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, and IL-21. In embodiments of the invention, the nucleic acid encoding the protein of interest encodes a chemokine. In embodiments of the invention, the chemokine is CCL5, CCL19, or CCL21. In embodiments of the invention, the nucleic acid encoding the protein of interest encodes a cytokine receptor. In an embodiment of the invention, the cytokine receptor is TGFBR (TGF-β receptor) or TGFBR2.

[0030] In some embodiments, the inducible promoter is selected from the group consisting of an NFAT promoter, a synthetic NFAT promoter, a (synthetic) NF-κB promoter, an AP-1 promoter, a (native) IL-2 promoter, an IFNγ promoter, a TNFα promoter, an IL-6 promoter, a CD69 promoter, and a CD137 promoter. In some embodiments, a gene (e.g., a transgene) encoding the protein of interest is introduced into an endogenous locus responsive to signaling from the activating receptor, e.g., expression of the locus is under the control of an inducible promoter according to the present invention. In one such example, for example, a transgene may be introduced into the IFNγ locus under the control of an inducible IFNγ promoter according to the present invention. Those skilled in the art will appreciate that the (trans)gene in embodiments can be introduced anywhere in the genome of a cell, as long as it is under the control of an inducible promoter according to the present invention. Similarly, the inducible promoter is introduced into the genome and operably linked to a gene (e.g., an endogenous or intentionally introduced gene (i.e., as a transgene)).

[0031] In some embodiments, the inducible promoter operably linked to the nucleic acid encoding the protein of interest is not the native promoter of the nucleic acid encoding the protein of interest. In some embodiments, the inducible promoter and the nucleic acid encoding the protein of interest are both contained within a vector, e.g., an expression vector, or e.g., an expression cassette. In some embodiments, the nucleic acid comprising the inducible promoter and the nucleic acid encoding the protein of interest are chimeric nucleic acids. In some embodiments, the nucleic acid comprising the inducible promoter is provided to the cell. In some embodiments, the inducible promoter is present on a vector introduced into the cell. In embodiments of the invention, the inducible promoter is provided to the cell such that the inducible promoter is integrated into the genome of the cell. In some embodiments, the nucleic acid comprising the protein of interest is provided to the cell. In some embodiments, the nucleic acid encoding the protein of interest is present on a vector introduced into the cell. In some embodiments, the nucleic acid encoding the protein of interest is provided to the cell and the inducible promoter is integrated into the genome of the cell. In embodiments of the invention, the inducible promoter and / or the nucleic acid encoding the protein of interest are recombinant inducible promoters and / or nucleic acids encoding the protein of interest. In embodiments of the invention, said inducible promoter and / or nucleic acid encoding said protein of interest is a non-native (with respect to the cell) inducible promoter and / or nucleic acid encoding said protein of interest. In embodiments of the invention, said inducible promoter and / or nucleic acid encoding said protein of interest is not present in the chromosomal location of the cell where such inducible promoter and / or nucleic acid encoding the protein of interest is naturally present in such cell. In other words, in embodiments of the invention, the same inducible promoter and / or nucleic acid encoding the protein of interest may be naturally present in a cell according to the invention independently, but may be present in addition to a (transgenic) inducible promoter and / or (transgenic) nucleic acid encoding a protein of interest according to the invention.

[0032] In some embodiments, the chimeric nucleic acid comprising the inducible promoter and the nucleic acid encoding the protein of interest is not integrated into the genome of the cell, hi some embodiments, the chimeric nucleic acid comprising the inducible promoter and the nucleic acid encoding the protein of interest is integrated into the genome of the cell.

[0033] In some embodiments, nucleic acids (or vectors) comprising a nucleic acid operably linked to a promoter and encoding a chimeric protein of the invention, and / or a nucleic acid operably linked to an inducible promoter and encoding a protein of interest, alone or in combination, are provided and used to form genetically engineered cells of the invention. In some embodiments, vectors are provided that additionally comprise, or further comprise, a nucleic acid operably linked to a promoter and encoding a receptor of the invention.

[0034] In some embodiments, the nucleic acid encoding the protein of interest is operably linked to a nucleic acid encoding an RNA degradation element (RDE), where the RDE is an AU-rich element (ARE) (and the nucleic acid, when transcribed, results in an mRNA transcript encoding the protein linked to the RDE, where the RDE is an RDE that causes RNA stabilization by interacting with an RNA stabilizing protein). In some embodiments, the ARE is or is derived from the 3' untranslated region (UTR) of IL2 or IFNγ.

[0035] According to another aspect of the present invention, there is provided a method of modulating expression of a protein of interest, the method comprising providing an activation signal in vitro or in vivo to a genetically engineered cell of the present invention in the presence of an effective amount of a drug capable of modulating a drug-regulated protein stability domain.

[0036] According to yet another aspect of the present invention, there is provided a method of modulating expression of a protein of interest in a subject, the method comprising administering to the subject an effective amount of an agent capable of modulating a drug-regulated protein stability domain (and thereby the level or degradation of a chimeric protein of the present invention), and the subject comprising a genetically engineered cell of the present invention.

[0037] According to yet another aspect of the invention there is provided a cell according to the invention for use as a medicament, preferably for use in the treatment of cancer and / or the treatment of a tumour in a subject, preferably wherein said treatment comprises administering cells to said subject and, optionally, administering a drug capable of modulating said drug regulatory protein stability domain.

[0038] In some embodiments, the present invention also provides for varying the dosage of a drug capable of modulating a drug-regulated protein stability domain to modulate the expression of said protein of interest.

[0039] In some embodiments, the present invention also provides a drug for use as a pharmaceutical, preferably for use in the treatment of cancer and / or the treatment of a tumor in a subject, said treatment comprising administering to a subject a cell of the present invention and administering said drug, wherein said drug is capable of modulating said drug regulatory protein stability domain. [Brief explanation of the drawings]

[0040] Embodiments of the present invention are further described below with reference to the accompanying drawings.

[0041] [Figure 1]1A and 1B are schematic diagrams illustrating the control of protein of interest (POI) expression via a chimeric protein of the present invention, e.g., a rheostat switch (e.g., RheoBrick®). In this example, a cargo gene encoding the POI is placed downstream of an ITAM signaling-responsive promoter, such as an NFAT promoter. ITAM signaling is provided by an antigen receptor, such as a CAR, T cell receptor, Fc receptor, or NK cell receptor, in the presence of cognate antigen ligation. For reversible and titratable control of POI expression, immune cells are engineered to express a chimeric protein of the present invention, e.g., a rheostat switch (e.g., RheoBrick® switch). A chimeric protein (e.g., a rheostat switch), e.g., RheoBrick®, may have three functional domains. The first is an SH2-based docking domain that enables binding to phosphorylated ITAM motifs present on activated antigen receptors. The second domain contains inhibitory ITIM / ITSM motifs, which allow for the recruitment of inhibitory SHP1 and / or SHP2 phosphatases, thereby downregulating the ITAM signaling pathway. The third domain is a drug-regulated protein stability domain (or degron) that allows for dynamic control of rheostat switches (e.g., RheoBrick®) and protein stability, and consequently, the extent of ITAM signaling in immune cells. The rheostat switches (e.g., RheoBrick®) mediate fine control of ITAM signaling, allowing for antigen-dependent drug (small molecule) modulation of POI expression levels. As a result, POI expression is under AND logic gate control, enabling precise control of POI production levels in the antigen-positive tumor microenvironment. [Figure 2]Figure 2 shows that rheostat switches (e.g., RheoBrick®) can enable antigen-dependent and drug (e.g., lenalidomide)-regulated expression of a protein of interest. A) Schematic of vector design. The rheostat switch (e.g., RheoBrick® switch) in Figure 2A and the following figures refers to the Zap70(2xSH2)-Siglec11 signaling domain-SynFinger®-based switch design (SEQ ID NO: 204). SynFinger encodes a double hybrid degron containing the Q12R / K13V substitution (SEQ ID NO: 205). B) Primary human T cells were engineered with the vector shown in (A). Data show the total EGFP expression levels of CD4 cells (Figures 2B-C) and CD8 cells (Figures 2D-E) when cocultured with CD19-negative K562 cells or CD19-positive Nalm6 cells in the absence or presence of 500 nM lenalidomide. The percentage of EGFP-positive cells was multiplied by the mean fluorescence intensity (MFI) of EGFP expression. A comparison of the fold-induction levels in Nalm6 cell cocultures in the presence versus absence of lenalidomide (Figures 2B and 2D) is shown in Figures 2C and 2E, respectively. Error bars represent standard deviation (n = 3). [Figure 2-1] Same as above description. [Figure 2-2] Same as above description. [Figure 3]Figure 3 shows a schematic diagram of an AU-rich element (ARE) containing an AND logic gate. The stringency and induction fold of POI expression via a chimeric protein (or rheostat switch) of the present invention, e.g., RheoBrick®, can be further increased by placing an ITAM signaling-responsive ARE containing a 3' UTR downstream of the cargo gene (encoding the POI). This demonstrates that rheostats (e.g., RheoBrick®) can control the expression of POIs operatively associated with various ITAM signaling response elements, such as AREs containing a 3' UTR, in addition to the NFAT promoter. In the absence of ITAM signaling, AREs containing a 3' UTR reduce mRNA stability, whereas in the presence of ITAM signaling, RNA-binding proteins (RBPs), such as NF90 and HuR, are exported from the nucleus and bind to AREs containing a 3' UTR, increasing mRNA stability and export from the nucleus, resulting in the production of the POI. [Figure 4] Figure 4 shows that AU-rich elements (AREs) within the 3'UTR of cargo expression cassettes improve induction fold and stringency of expression control. A) Schematic of vector design. B) Primary human T cells were engineered with the vectors shown in (A). Data show secreted IL-12 (p70) upon co-culture with or without CD19-positive Nalm6 cells in the absence or presence of 500 nM lenalidomide. Bar graphs show lenalidomide-induced fold induction of IL-12. Error bars represent standard deviation (n=3). [Figure 4-1] Same as above description. [Figure 5]Figure 5 shows that combinations of ARE elements containing 3'UTRs improve induction folds and stringency of expression control. A) Schematic of vector design. B) Primary human T cells were engineered with the vectors shown in (A). Data show secreted IL12(p70) upon co-culture with or without CD19-positive Nalm6 cells in the absence or presence of 500 nM lenalidomide. Bar graphs show lenalidomide-induced fold induction of IL12 cargo production. Error bars represent standard deviation (n=3). [Figure 5-1] Same as above description. [Figure 6]Figure 6. Effect of vector design on fold induction and stringency of cargo production control. A) Schematic of vector design. B) Primary human T cells engineered with the vectors shown in (A). Data show the percentage of EGFP-positive CD4+ cells when co-cultured with CD19-negative K562 cells or CD19-positive Nalm6 cells in the absence or presence of 500 nM lenalidomide. Error bars represent standard deviation (n=3). Abbreviations: CD19 CAR, second-generation anti-CD19 chimeric antigen receptor containing a CD28 costimulatory signaling domain and a CD3ζ domain; RheoBrick®, a rheostat switch containing a Zap70(2×SH2)-Siglec11 signaling domain and a SynFinger®-based switch design, where SynFinger encodes a double hybrid degron containing a Q12R / K13V substitution; EGFP, enhanced green fluorescent protein; pNFAT, a synthetic NFAT promoter containing an NFAT-binding sequence and four copies of a minimal promoter (Jutz et al. J Immunol Methods. 2016 Mar;430:10-20); pMSCV, a murine stem cell virus U3 promoter derived from the 3-prime long terminal repeat of the MSGV1 vector (Hughes et al. Hum Gene Ther. 2005 Apr;16(4):457-72); pCMV, a cytomegalovirus minimal promoter; IL2 3'UTR, interleukin-2 3 prime untranslated region; IFNg 3'UTR, interferon gamma 3' untranslated region; SV40 pA, simian virus 40 polyadenylation signal; CTE, Mason-Pfizer virus constitutive transport element (CTE) (Bray et al. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1256-1260); WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; BGH pA, bovine growth hormone polyadenylation signal. [Figure 6-1] Same as above description. [Figure 6-2] Same as above description. [Figure 7]Figure 7 shows that the level of secreted IL-12 can be fine-tuned by adjusting the small molecule dose. A) Schematic of vector design. B) Primary human T cells were engineered with the vectors shown in (A). Data show secreted IL12(p70) upon co-culture with or without CD19-positive Nalm6 cells at the indicated concentrations of lenalidomide. Error bars represent standard deviation (n=3). [Figure 7-1] Same as above description. [Figure 8]Figure 8 shows that the invention disclosed herein enables the secretion of antigen-dependent, small molecule regulatory T cell engagers (also known as BiTEs) to redirect unmodified naive T cells against cells expressing a (tumor) antigen of interest. A) Diagram of the logic gate circuit. B) Schematic of the vector design. C) Schematic of the co-culture conditions. Primary human T cells were modified with the vector shown in (B) containing a second-generation CD19 CAR, RheoBrick®, and an NFAT-PSMA BiTE-IL-2 3'UTR expression cassette. The modified T cells were co-cultured with or without CD19-positive Nalm6 cells in the presence or absence of 500 nM lenalidomide (first co-culture). Only in the presence of both Nalm6 target cells and lenalidomide does RheoBrick® (according to the present invention) enable the secretion of PSMA BiTE, as shown on the left. Conditioned medium from the first coculture was collected, filtered, and used for the second coculture, as shown on the right. The presence of PSMA-BiTE in the conditioned medium induced IFNg and TNFa cytokine release and degranulation from unmodified naive T cells only in the presence of PSMA-positive HCT116 tumors, but not in the presence of PSMA-negative HCT116 WT tumors. An overview of the first coculture conditions, predicted BiTE production from modified T cells, and second coculture conditions, as well as predicted degranulation / cytokine production from unmodified naive T cells in the presence of antigen-dependent and small molecule-regulated TCE activity, are shown in (D) and (E), respectively. (F-I) Data show IFNg (F) and TNFa (H-I) production by CD8+ (F-H) and CD4+ (I) T cells, as well as cell surface LAMP1 expression (G), in the second coculture described in (E). Error bars represent standard deviation (n=3). [Figure 8-1] Same as above description. [Figure 8-2] Same as above description. [Figure 8-3] Same as above description. [Figure 8-4] Same as above description. [Figure 9]Figure 9 shows that the sensitivity of zinc-finger degrons to IMiDs can be improved by altering the second zinc finger sequence. (A) Schematic diagram of a zinc-finger degron panel containing the ZFP91 ZF4 β-hairpin and IKZF1 ZF2 α-helix as the hybrid first zinc finger. In this panel, the degron contains a second zinc finger sequence selected from IKZF1 ZF3 (single-hybrid dual-zinc-finger degron), IKZF1 ZF3 β-hairpin-ZFP91 ZF5 α-helix (double-hybrid degron), or no second zinc finger (single-hybrid single-zinc-finger degron). (B-E) Primary human T cells engineered with an HLA class I-restricted CDK4 TCR+ Zap70-PD1-degron CRASH-IT switch (degron sequences selected from (A)) were pretreated with the indicated concentrations of thalidomide. Data show the expression of intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 in CDK4 TCR+, EGFP-high, CD8+ T cells upon coculture with NKIRTIL006 melanoma cells in the continued presence of the indicated concentrations of thalidomide. Error bars represent standard deviation (n=2). Data are representative of two independent experiments. [Figure 9-1] Same as above description. [Figure 10] FIG. 10 is a schematic diagram of a non-limiting embodiment of the present invention. [Figure 10-1] Same as above description. [Figure 11]Figure 11 shows the design of the synthetic zinc finger (SynFinger) library. The SynFinger library contains Zap70-PD1-degron CRASH-IT switch variants that contain one, two, or no amino acid substitutions in the parent zinc finger degron. Figure 11 shows the sequences of the double hybrid degrons used as parent zinc finger sequences in the SynFinger library screen. The amino acids indicated by arrows are substituted with any other genetically encoded amino acid except cysteine. The two cysteines and conserved glycine in the ZFP91 ZF4 β hairpin and the two histidines in the IKZF1 ZF2 α helix were kept constant. Substitution mutations are numbered from the first amino acid (marked with an asterisk) in the ZFP91 ZF4 β hairpin. [Figure 12] Figure 12 shows the enrichment of specific amino acid substitutions in the top 100 degrons. SynFinger degrons were ranked according to their enrichment index (EI) values ​​after removing SynFingers with low sequence reads (see Examples). The graph shows the number of times each amino acid substitution was detected in combination with other amino acid substitutions in the top 100 SynFinger degrons. For example, the Q12R mutation was observed in 11 double amino acid substitution combinations within the top 100 SynFingers, along with any of K13T, L17M, K13V, E4L, L17Y, L22H, C10A, I20R, N15S, E4W, and E4Q. [Figure 13]Figure 13 shows that SynFinger fusion proteins enable delicate control of immune cell function. A subset of SynFingers identified in the SynFinger library screen was individually validated. (A-H) Primary human T cells were engineered with the HLA class I-restricted CDK4 TCR+ Zap70-Siglec11 degron CRASH-IT switch. The degron sequence included either the parent zinc finger degron (the double hybrid degron shown in Figures 9 and 11) or a parent zinc finger degron containing the following mutations: G14N / K21A, G14M / N15R, L17I / K21L, E4R / Q12L, Q12R / K13T, or Q12R / K13V. Primary human T cells engineered with the HLA class I-restricted CDK4 TCR+ vector control served as controls. Cells were pretreated with 5 nM lenalidomide or left untreated. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+, EGFP-intermediate, CD8+ T cells cocultured with NKIRTIL006 melanoma cells in the continued absence (E–H) or presence (A–D) of 5 nM lenalidomide. Dashed horizontal lines indicate cytokine production and degranulation levels in cells engineered with the CRASH-IT switch containing the parental zinc finger degron. Error bars represent standard deviation (n=2). SynFinger and vector control groups were compared to the parental zinc finger group using a one-tailed t-test. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, ns=not significant. Data are representative of two independent experiments. [Figure 13-1] Same as above description. [Figure 14]Figure 14 shows that SynFingers containing a CRASH-IT switch enable restoration of cytokine production and degranulation at lower IMiD concentrations compared to zinc finger degrons not of the present invention. (A-D) Primary human T cells engineered with the HLA class I-restricted CDK4 TCR+Zap70-Siglec11-degron CRASH-IT switch contained a prior art zinc finger degron (single hybrid / dual zinc finger degron shown in Figure 9), a parent zinc finger degron (double hybrid degron shown in Figures 9 and 11), or a parent zinc finger degron containing the following mutations (G14N / K21A, L17I / K21L, or Q12R / K13V) and were pretreated with the indicated concentrations of lenalidomide or left untreated. Data show the expression of intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 in CDK4 TCR+, EGFP intermediate, and CD8+ T cells upon coculture with NKIRTIL006 melanoma cells in the presence or absence of the indicated concentrations of lenalidomide (A-D). Error bars represent standard deviation (n=3). Data are representative of two independent experiments. (E) Table shows the EC50 lenalidomide (nM) values ​​for the cells in (A-D) and the EC50 fold change between the prior art zinc finger degron and SynFinger containing the Q12R / K13V substitution. [Figure 14-1] Same as above description. [Figure 15]Figure 15 shows that combining double substitution sets can further enhance the lenalidomide sensitivity of SynFinger degrons. (A-D) Primary human T cells engineered with the HLA class I-restricted CDK4 TCR+Zap70-Siglec11-degron CRASH-IT switch. In these cells, the degron sequence contained either the parent zinc finger degron (the double hybrid degron shown in Figure 9 and Figure 3) or a parent zinc finger degron with the following mutations (G14N / K21A, Q12R / K13V, or Q12R / K13V / G14N / K21A) and were pretreated with the indicated concentrations of lenalidomide or left untreated. Data show the expression of intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 in CDK4 TCR+, EGFP-intermediate, CD8+ T cells cocultured with NKIRTIL006 melanoma cells in the presence or absence of the indicated concentrations of lenalidomide (A-D). Error bars represent standard deviation (n=3). Data are representative of two independent experiments. [Figure 16] Figure 16 shows the versatility of the RheoBrick-mediated antigen-dependent and small molecule-regulated cargo production platform. A) Schematic of vector design. B) Primary human T cells were engineered with the vectors shown in (A). Data show secreted IL12(p70) upon co-culture with antigen-positive Nalm6, PC3-PSMA, NKIRTIL006, A549, A375, or Mel624 cells in the absence or presence of 500 nM lenalidomide. Fold induction of lenalidomide-induced IL12 cargo production is shown in the bar graph. Error bars represent standard deviation (n=3). [Figure 16-1] Same as above description. [Figure 17]Figure 17 shows that RheoBrick can be used to control the cytotoxicity of CAR-T cell-derived blinatumomab. A) Schematic of vector design. B) Schematic of co-culture conditions. Primary human T cells were engineered with the CC81+CC16 vector containing a second-generation PSMA CAR, RheoBrick, and blinatumomab encoding an ITAM signaling-responsive expression cassette. For normalization of cytotoxicity measurements, T cells were engineered with the CC76+CC16 vector containing a second-generation PSMA CAR, RheoBrick, and an irrelevant cargo (luciferase) encoding an ITAM signaling-responsive expression cassette. Engineered T cells were co-cultured with antigen-positive PC3-PSMA cells or antigen-negative PC3 cells with or without 500 nM lenalidomide for 24 hours (first co-culture). Only in the presence of both PC3-PSMA target cells and lenalidomide does the RheoBrick (according to the present invention) allow secretion of blinatumomab, as shown on the left. Conditioned medium from the first coculture was collected, filtered, and used for the second coculture, as shown on the right. Specific killing of CD19-positive Nalm6 cells was measured as described in "Methods." C) Specific killing of Nalm6 cells after 24 hours of coculture with naive T cells in the presence of serially diluted conditioned medium from the first coculture. D) Positive control for specific killing of aged Nalm6 cells cocultured with naive T cells in the presence of the indicated concentrations of purified blinatumomab. Error bars represent standard deviation (n=3). Data are representative of two independent experiments. [Figure 17-1] Same as above description. [Figure 17-2] Same as above description. [Figure 18]Figure 18 shows that RheoBrick modulated cargo production in vivo. A) Schematic of vector design. B) Schematic of animal experiments. NSG mice were subcutaneously injected with 5 million PC3-PSMA cells. Two weeks later, mice were randomized based on tumor size (approximately 40-50 mm) and injected with human T cells engineered with RheoBrick and the CC83+CC84 vector encoding a PSMA CAR (Groups X1 and X2), or with the CC130+CC131 vector encoding a PSMA CAR without RheoBrick (Groups Y1 and Y2), or left untreated. All cells were equipped with an ITAM signaling-responsive luciferase cargo expression cassette. Groups X1 and Y1 received a daily vehicle control, while groups X2 and Y2 received 1 mg / kg lenalidomide daily (n=6 for groups X1, X2, Y1, and Y2; n=12 for the untreated group). C) Luciferase production was measured using an IVIS imaging system at the indicated time points. D) Luciferase cargo production density was calculated by normalizing IVIS imaging results to tumor size. E) Tumor volume measured with a caliper. F) Survival analysis. G) Graphical summary of the animal study. [Figure 18-1] Same as above description. [Figure 18-2] Same as above description. [Figure 18-3] Same as above description. [Figure 18-4] Same as above description. [Figure 18-5] Same as above description. [Figure 18-6] Same as above description. [Figure 19] Figure 19: Tight regulation of endogenous IFNγ production by the RheoBrick switch. Primary human T cells were transduced with the CC83 vector encoding a PSMA CAR and RheoBrick, or the CC130 vector encoding a PSMA CAR and huEGFRt. The data show that co-culture of the transduced T cells with antigen-positive PC3-PSMA cells or antigen-negative PC3 cells for 24 hours in the absence or presence of 500 nM lenalidomide resulted in endogenous IFNγ production. [Figure 20] Figure 20 shows RheoBrick-mediated antigen- and small molecule-dependent IL-12 cargo production from ovarian cancer tumor-infiltrating lymphocytes (TILs). A) Schematic of vector design. B) TILs derived from an ovarian cancer patient were engineered with RheoBrick and vector CC82, which contains an ITAM signaling-responsive expression cassette encoding scIL-12. Data show secreted IL-12 (p70) upon coculture with ovarian tumor cells or antigen-negative PC3 cells derived from the same patient in the absence or presence of 500 nM lenalidomide. Bar graphs show lenalidomide-induced fold-induction of IL-12 cargo production. Error bars represent standard deviation (n=3). [Figure 20-1] Same as above description.

[0042] explanation definition Portions of this disclosure contain material that is subject to copyright protection, such as diagrams, device photographs, or other aspects of this submission for which copyright protection is or may be available in any jurisdiction. The copyright owner has no objection to anyone copying the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights.

[0043] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0044] Various terms relating to the methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise specified. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0045] In the present invention, the following terms are defined as follows.

[0046] As used herein, the singular terms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "cell" includes a combination of two or more cells, and the like.

[0047] As used herein, "about" and "approximately," when referring to measurable values ​​of amounts, durations, and the like, are meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as appropriate for practicing the disclosed invention.

[0048] As used herein, the term "and / or" refers to a situation in which one or more of the listed cases may occur alone or in combination with at least one of the listed cases, up to all of the listed cases.

[0049] As used herein, the term "at least" a particular value means greater than or equal to that particular value. For example, "at least 2" is understood to be the same as "2 or greater," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. As used herein, the term "up to" a particular value means less than or equal to that particular value. For example, "up to 5" is understood to be the same as "less than or equal to 5," i.e., 5, 4, 3, -10, -11, etc.

[0050] As used herein, the term "comprising" is to be interpreted as inclusive and open-ended, rather than exclusive. Specifically, this term and its variations mean that the specified features, steps, or components are included. These conditions should not be interpreted to exclude the presence of other features, steps, or components. Furthermore, the more restrictive meaning of "consisting of" is also encompassed.

[0051] As used herein, "prior art" or "methods known to those skilled in the art" refers to situations in which it would be clear to one of ordinary skill in the art how to carry out the prior art used in the methods of the present invention. The practice of prior art techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medicine, pharmacology, immunology, and related fields is well known to those skilled in the art and is described in various handbooks and reference works.

[0052] The word "exemplary" as used herein means "serving as an example, instance, or illustration," and should not be interpreted as excluding other configurations disclosed herein.

[0053] The term "cancer" as used herein refers to a physiological condition in mammals typically characterized by unregulated cell growth. The terms "cancer," "neoplasm," and "tumor" are often used interchangeably to describe cells that have undergone pathological malignant changes in the host organism. Primary cancer cells can be distinguished from non-cancerous cells by techniques known to those skilled in the art. As used herein, cancer cells include not only primary cancer cells, but also cancer cells (including metastatic cancer cells) derived from such primary cancer cells and cell lines derived from cancer cells. Examples include solid tumors, non-solid tumors, or hematological tumors. Preferably, the tumor is a solid tumor. Examples of cancer include, but are not limited to, leukemia, lymphoma, sarcoma, and carcinoma (e.g., colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, melanoma, lymphoma, non-Hodgkin's lymphoma, colon cancer, (malignant) melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung cancer, and lung adenocarcinoma). As is well known, tumors can metastasize from their primary site to one or more other body tissues or sites. References to treating a patient for a "neoplasm," "tumor," or "cancer" include treatment of the primary cancer and, where appropriate, treatment of metastases.

[0054] As used herein, the term "chimeric gene" or "chimeric nucleic acid" refers to a gene or nucleic acid that is not normally found in nature, particularly a gene or nucleic acid in which one or more portions of the nucleotide sequence are not naturally associated with each other. For example, a promoter or 3'UTR is not naturally associated with part or all of the transcribed region or another regulatory region, or different portions of a transcribed region are not naturally associated. The term "chimeric gene" is understood to include an expression construct in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences. In some embodiments, a chimeric gene of a chimeric nucleic acid can be used to generate a chimeric protein.

[0055] As used herein, the terms "chimeric polypeptide," "chimeric protein," or "fusion protein" refer to a polypeptide not normally found in nature, particularly a polypeptide in which one or more portions of the amino acid sequence are not associated with each other in nature. For example, a chimeric protein can include an N-terminal portion consisting of a first amino acid sequence and a C-terminal portion consisting of a second amino acid sequence that are not associated with each other in nature and / or that are not associated with each other in this order in nature. A chimeric protein can result, for example, from transcription and translation of a chimeric gene or nucleic acid.

[0056] As used herein, "immunomodulatory drug," "immunomodulatory imidrug," or "IMiD" refers to compounds known in the art. IMiDs include thalidomide, pomalidomide, lenalidomide, iverdomide (CC-220), avadomide (CC-122), and CC-885, or pharmaceutically acceptable salts thereof. These compounds are sometimes referred to as cereblon modulators (CRBN modulators). Thalidomide, lenalidomide, and pomalidomide have each been approved for the treatment of various diseases, while other IMiDs or cereblon modulators are currently under review. The compounds may be in the form of a free acid or free base, or a pharmaceutically acceptable salt.

[0057] As used herein, the term "isolated" when referring to a polynucleotide (nucleic acid) or polypeptide (protein) refers to a protein or nucleic acid that is in a non-native environment, e.g., that has been separated from its naturally occurring environment. For example, an isolated protein or polypeptide according to the present invention relates to a protein that is no longer in its natural environment, e.g., in vitro or within a recombinant host cell. These terms also refer to such proteins or nucleic acids that, apart from being isolated from a naturally occurring source, are artificially or synthetically produced. It will be clear to one of skill in the art whether reference to a protein, polypeptide, nucleic acid, or polynucleotide in the context of the present invention includes reference to an "isolated" protein, polypeptide, nucleic acid, or polynucleotide.

[0058] As used herein, the term "immunoreceptor tyrosine-based activation motif (ITAM)" refers to a conserved sequence of four amino acids repeated twice in the cytoplasmic tail (i.e., endodomain) of certain cell surface proteins of the immune system. A half ITAM contains a tyrosine residue (Y) separated from a leucine residue (L) or an isoleucine residue (I) by any two other amino acids. The consensus sequence for a half ITAM is YxxL / l. Typically, two half ITAMs are separated by 6-8 amino acids to form a complete ITAM. The consensus sequence for an ITAM is YxxL / lx(6-8)YxxL / l. ITAMs play an important role in immune cell signaling, particularly in the cytoplasmic tails of cell signaling molecules of the T cell receptor complex (CD3ε, CD3δ, CD3γ, and / or CD3ζ chains). In NK cells, ITAMs are present in the NK cell receptor complex, which includes the CD3ζ chain, the gamma (γ) chain of the immunoglobulin receptor FcεRI, and DAP12 (Lanier et al, Nat Immunol. 2008 May; 9(5): 495-502). ITAMs are also present in chimeric antigen receptor (CAR) complexes, including the CD3ζ chain (Abate-Daga et al., Mol Ther Oncolytics. 2016;3:16014), CD3ε chain (Nolan et al., Clin Cancer Res. 1999 Dec;5(12):3928-41), the gamma (γ) chain of the immunoglobulin receptor FcεRI (Ren-Heidenreich et al., Cancer Immunol Immunother. 2002 Oct;51(8):417-23), and DAP12 (Topfer et al., J Immunol. 2015 Apr 1;194(7):3201-12).

[0059] As used herein, the term "immunoreceptor tyrosine-based inhibitory motif (ITIM)" generally refers to a conserved sequence of amino acids found in the cytoplasmic tails of many inhibitory receptors of the immune system. An ITIM motif contains a serine (S), isoleucine (I), valine (V), or leucine (L) residue, separated from a tyrosine (Y) residue by any other amino acid residue (x), and further separated from the isoleucine (I), valine (V), or leucine (L) residue by any other two amino acids. The consensus signature is S / l / V / LxYxxl / V / L. In vivo, when inhibitory receptors bearing ITIMs interact with ligands, the ITIM motifs are phosphorylated by Src kinase enzymes, which can recruit SH2-containing protein tyrosine phosphatases (PTPs) such as SHP-1 and SHP-2 (Coxon et al., Blood. 2017 Jun 29;129(26):3407-3418) and lipid phosphatases such as SHIP-1. PTPs oppose the positive regulatory effects of protein tyrosine kinases (PTKs) such as Lck and Zap70, thereby negatively regulating T cell signaling (Lorenz et al., Immunol Rev. 2009 Mar;228(1):342-359). PTPs can reverse the activating effects of ITAM phosphorylation by dephosphorylating ITAMs in TCRs, CARs, and other immune receptors. Lipid phosphatases regulate cell signaling by altering the concentration of lipid phosphates relative to their dephosphorylated products.

[0060] As used herein, the term "immunoreceptor tyrosine-based switch motif (ITSM)" refers to a conserved sequence of amino acids found in the cytoplasmic tail (or cytoplasmic domain, intracellular domain, or endodomain; in other words, the portion of the protein present in the cytoplasm (rather than the membrane and / or extracellular space)) of many inhibitory receptors of the immune system. The ITSM motif contains a threonine (T) residue, separated from a tyrosine (Y) residue by any other amino acid residue, and separated from a valine (V) or isoleucine (I) residue by any other two amino acids. The consensus signature is TxYxxV / I. Similar to inhibitory receptors with ITIMs, inhibitory receptors with ITSMs interact with ligands, and the ITIM motif is phosphorylated by Src kinase enzymes, thereby recruiting SH2-containing phosphates such as SHP-1 and SHP-2 (Lorenz et al., Immunol Rev. 2009 Mar;228(1):342-359). Some studies have reported that both ITIM and ITSM motifs contribute to the inhibitory signaling of PD1 (Boussiotis et al., Cancer J. 2014 Jul-Aug;20(4):265-271, Peled et al., Proc Natl Acad Sci U S A. 2018 Jan 16;115(3):E468-E477). Other studies have shown that ITSM motifs are primarily responsible for the inhibitory effect of PD1, while ITIM motifs have only a limited effect (Chemnitz et al., J Immunol. 2004 Jul 15;173(2):945-54, Yokosuka et al., J Exp Med. 2012 Jun 4;209(6):1201-17).

[0061] As used herein, the term "linker," as used with respect to a portion of a protein, refers to an amino acid sequence that connects two portions of a protein, for example, in a fusion protein. Generally, such molecules have no specific biological activity other than to connect or maintain a minimum distance or other spatial relationship between the proteins. However, in certain embodiments, the linker may be selected to influence some property of the linker and / or the protein, such as the folding, net charge, or hydrophobicity of the linker.

[0062] As used herein, the term "non-naturally occurring" refers to a polypeptide, polynucleotide, or a domain contained in such a polypeptide or polynucleotide that is not known to occur in nature, e.g., a domain that is not known to occur in a (human) cell, meaning that one or more portions of the polypeptide, polynucleotide, or domain are not associated with each other in nature. An example is a fusion protein or chimeric protein of the invention.

[0063] As used herein, the term "pharmaceutical composition" refers to a composition formulated into a pharmaceutically or physiologically acceptable composition for administration to a cell or a subject. The compositions of the present invention can be administered to a subject in combination with other agents, so long as the additional agents do not adversely affect the ability of the composition to perform its intended therapeutic function. The pharmaceutical compositions often contain one or more pharmaceutically acceptable carriers (or excipients) in addition to the pharmaceutically active agent. The pharmaceutical compositions may be specifically formulated for administration in solid or liquid form, including those suitable for the following uses: (1) oral administration, e.g., as a drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, or paste; (2) parenteral administration, e.g., as a sterile solution or suspension, e.g., by subcutaneous, intramuscular, or intravenous injection; (3) topical application, e.g., as a cream, ointment, or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; or (5) as an aerosol, e.g., as an aqueous aerosol, liposomal formulation, or solid particles containing the compound. The drugs, therapeutic agents, medicaments, and pharmaceutical compositions of the present invention can be formulated for administration by several routes, including, but not limited to, parenteral, intravenous, intraarterial, intramuscular, intratumoral, and oral. The drugs, therapeutic agents, medicaments, and compositions can be formulated in liquid or solid form. Liquid formulations may be formulated for administration by injection into selected areas of the human or animal body.

[0064] As used herein, the terms "protein" and "polypeptide" refer to molecules consisting of chains of amino acids, regardless of a particular mechanism of action, size, three-dimensional structure, or origin. Thus, a "fragment" or "portion" or "part" of a polypeptide may still be referred to as a "polypeptide." An "isolated protein" or "isolated polypeptide" is used to refer to a protein or polypeptide that is no longer present in its natural environment, such as in vitro or in a recombinant host cell.

[0065] As used herein, the term "SH2 domain" refers to an SRC homology 2 domain. SH2 domains are structurally conserved protein domains found in Src oncoproteins and many other intracellular signaling proteins. SH2 domains enable proteins containing them to dock with phosphorylated tyrosine residues on other proteins. Thus, SH2 domains are modular protein domains that function as adaptors, binding to phosphorylated peptides within their respective protein-binding partners to mediate protein-protein interactions.

[0066] As used herein, "subject" or "patient" refers to an organism to which, for example, administration is intended to be treated. The subject, according to the present invention, can be any subject, including, but not limited to, a human, male, female, infant, child, adolescent, adult, young adult, middle-aged adult, or elderly, and / or other primates or mammals. Preferably, the subject is a human patient. The subject may be diagnosed with cancer or suspected of having cancer.

[0067] As used herein, "treatment," "treating," "palliating," "alleviating," and "ameliorating," in the context of a subject being treated, all refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit. A therapeutic effect refers to the eradication or amelioration of the underlying disease being treated. A therapeutic effect is also achieved by the eradication or amelioration of one or more physiological symptoms associated with the underlying disease, such that an improvement in the patient's condition is observed, even if the patient continues to suffer from the underlying disease. DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention is defined in this specification, and particularly in the appended claims. Subject matter not included in the claims does not form part of the present invention.

[0069] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use, or composition described herein. Embodiments described in connection with a method, use, and / or composition according to the present invention can also be used with respect to other methods, uses, or compositions described herein. Thus, embodiments with respect to one method, use, or composition can also be applied to other methods, uses, and compositions according to the present invention.

[0070] References herein to methods of treatment also refer to the compounds, pharmaceutical compositions and medicaments of the invention for use in such methods for treating the human (or animal) body by therapy.

[0071] As embodied and broadly described herein, the present invention relates to the surprising discovery that for the first time, it is possible to provide genetically engineered cells that can tightly regulate the production of a biological substance (a protein of interest) both spatially (in a specific microenvironment) and temporally (at a desired time point).

[0072] Thus, the present invention allows for tight regulation of cell, e.g., T cell, activation in a patient, allowing for on-site (e.g., tumor microenvironment) production of a protein of interest (e.g., a therapeutic agent) and delivery to target cells (e.g., tumor cells). This, for the first time, makes it possible to produce desired amounts of a biologic at the site of disease. Thus, the present invention allows for the optimization of the therapeutic window of cells, particularly immune cells, and for controlling the production of such biologics to avoid or prevent side effects, while simultaneously preventing immune cell exhaustion upon activation (due to receiving an activation signal). Furthermore, in a preferred embodiment, the chimeric protein of the present invention is composed of human protein domains. Furthermore, in a preferred embodiment, the protein of interest is composed of human protein domains. According to a first aspect of the invention, there is provided a cell, such as a genetically engineered cell, comprising: (a) a receptor capable of receiving an activating signal; (b) i. a docking domain capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives the activating signal; ii. a drug-regulated protein stability domain; and a chimeric protein comprising: (c) an inducible promoter operably linked to a nucleic acid encoding a protein, the inducible promoter being induced when the receptor receives an activating signal in the absence of the chimeric protein; A cell comprising:

[0073] The cell may be any cell, e.g., an engineered cell, such as a genetically engineered cell. In other words, the cell of the present invention is a cell that has been modified to contain the inducible promoter operably linked to a nucleic acid encoding the receptor, the chimeric protein, and a protein of interest, and / or a nucleic acid comprising an inducible promoter operably linked to a nucleic acid encoding the receptor of the present invention, a nucleic acid encoding the chimeric protein of the present invention, and / or a nucleic acid encoding a protein of interest.

[0074] In some embodiments, the original cell has been modified by providing the cell with a nucleic acid encoding a chimeric protein of the present invention, or such a chimeric protein, preferably operably linked to a promoter. In some embodiments, the original cell has been (further) modified by providing the cell with a nucleic acid comprising said inducible promoter and further comprising a gene encoding said protein of interest, said inducible promoter being operably linked to a gene encoding said protein of interest. In some embodiments, the original cell has been (further) modified by providing the cell with said receptor, or a nucleic acid encoding a receptor, preferably operably linked to a promoter.

[0075] The receptor may be any suitable receptor capable of receiving an activating signal, and preferably, upon receiving an activating signal, the receptor activates an intracellular pathway, resulting in the transmission of the activating signal through the cell. The receptor may be a cytoplasmic receptor or a transmembrane receptor, and preferably is a transmembrane receptor, and preferably a transmembrane receptor has at least one extracellular domain, at least one transmembrane domain, and at least one cytoplasmic domain. The receptor is preferably a cell surface receptor, preferably a cell surface transmembrane receptor, and is capable of interacting with its cognate ligand (e.g., an antigen) expressed on the cell surface of other cells or present in the extracellular environment (e.g., secreted by other cells, e.g., a specific extracellular matrix protein, cytokine, hormone, or antibody). In some embodiments, the receptor is capable of interacting with or binding to a ligand (which provides an activating signal) provided to a patient during the course of treating the patient's disease.

[0076] The activation signal of the present invention may be any signal that can interact with or bind to the receptor, thereby causing activation of the receptor. In the context of the present invention, activation of the receptor means, for example, the induction of cell-mediated signal transduction through the interaction of the activation signal with the receptor. Non-limiting examples of such activation signals include any cognate binding partner that can activate the receptor within the context of the present invention, such as antigens, tumor antigens, proteins, antibodies (including agonist or antagonist antibodies), other cells, nucleic acids, drugs, and small molecules. Preferably, the activation signal is a ligand that binds to or interacts with the receptor, i.e., the cognate binding partner of the receptor. Preferably, the activation signal is an antigen, preferably a tumor antigen. Preferably, the activation signal is an extracellular activation signal, such as an antigen that is present outside the cell of the present invention, for example, an antigen present on the cell surface of a tumor cell, or an antigen secreted or present in the extracellular environment.

[0077] The chimeric proteins of the present invention are preferably cytoplasmic chimeric proteins. Said chimeric proteins are expressed in the cells of the present invention and can be provided to the cells of the present invention, for example, by providing the cells with a nucleic acid encoding the chimeric protein of the present invention, wherein the nucleic acid encoding the chimeric protein of the present invention is operably linked to any suitable promoter. Preferably, said chimeric proteins are composed of or consist of human protein domains.

[0078] As described in detail below, the chimeric protein includes a docking domain that can bind to the receptor and inhibit signal transduction induced when the receptor receives the activating signal. The binding between the docking domain and the receptor is preferably reversible, meaning that the docking domain contained in the chimeric protein and the receptor can dissociate. The docking domain contained in the chimeric protein can bind to or interact with any part of the receptor, but in the case of a transmembrane cell surface receptor, such as a TCR, CAR, or NKR (NK cell receptor), it preferably interacts with or binds to the cytoplasmic part of the receptor.

[0079] As described in more detail below, the chimeric protein further comprises a drug-regulated protein stability domain (sometimes referred to herein as a "degron"). The drug-regulated protein stability domain can be any suitable drug-regulated protein stability domain, various suitable examples of which are described herein and / or known to those of skill in the art. The drug-regulated protein stability domain included in the chimeric protein of the present invention allows for modulation of the stability of the chimeric protein in response to a drug. The drug is an agent capable of interacting with or binding to the drug-regulated protein stability domain, thereby modulating the stability of the chimeric protein of the present invention, for example, by modulating the degradation of the chimeric protein of the present invention. Modulating the stability of the chimeric protein of the present invention can modulate the degree of binding of the chimeric protein of the present invention to the receptor, thereby modulating signal transduction through a cell of a receptor activated by an activation signal, for example, signal transduction upon binding of the receptor to a ligand (e.g., an antigen recognized by the receptor). Non-limiting examples of chimeric proteins include amino acid sequences having at least 80% identity, preferably at least 81%, 83%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, to the amino acid sequences set forth in SEQ ID NOs: 178-179, 180, 181-191, or 192-201. As will be appreciated by those skilled in the art, sequences defined above in which 1, 2, 3, 4, 5, 6, 10, or 15 amino acids have been deleted, substituted, or inserted are also included. The chimeric polypeptides may also contain additional amino acids (extensions) flanking the amino acids defined above, as long as the chimeric polypeptides of the invention remain functional within the context of the present invention. Other preferred chimeric proteins are as described herein.

[0080] As described in more detail below, the cells of the present invention further comprise an inducible promoter operably linked to a nucleic acid encoding a protein of interest, the inducible promoter being induced when the receptor receives an activation signal in the absence of the chimeric protein. As will be appreciated by those of skill in the art, the protein of interest may be any suitable protein selected by one skilled in the art for the purpose of, for example, using the cells of the present invention to treat a disease, condition, or disorder, such as a tumor, in a patient. The inducible promoter may be any promoter that is induced when the receptor is activated by an activation signal, allowing signal transduction through the cell in response to receptor activation. In other words, the inducible promoter is a promoter that is induced when the receptor is activated and subsequent signal transduction through the cell occurs. In embodiments of the present invention, signal transduction by the receptor activated by an activation signal is inhibited, reduced, or prevented in the presence of the chimeric protein of the present invention. In such embodiments, induction of the inducible promoter is also inhibited, reduced, or prevented, and expression of the protein of interest is similarly inhibited, reduced, or prevented.

[0081] Preferably, the inducible promoter and the nucleic acid encoding the protein of interest are provided as a transgene, i.e., intentionally introduced into a cell according to the present invention. Preferably, the nucleic acid encoding the protein of interest is not operably linked to the inducible promoter under natural conditions; e.g., to provide a cell according to the present invention, an inducible promoter operably linked to a nucleic acid encoding a protein of interest is introduced into the cell. Preferably, the operably linked inducible promoter and the nucleic acid encoding the protein of interest are provided as a chimeric nucleic acid. In some embodiments, the operably linked inducible promoter and the nucleic acid encoding the protein of interest are integrated into the genome. In some embodiments, the operably linked inducible promoter and the nucleic acid encoding the protein of interest are provided to the cell in a vector, preferably not integrated into the genome of the cell. However, in other embodiments, the gene encoding the protein of interest may be integrated into the genome, for example, at a locus responsive to activated receptor signaling, i.e., under the control of an inducible promoter according to the present invention. In other embodiments, the inducible promoter and / or the protein of interest are introduced / integrated into the genome. In some embodiments, an endogenous protein can be placed under the control of an inducible promoter in accordance with the present invention by introducing / integrating said inducible promoter into the genome. In some embodiments, said protein of interest is operably linked to an inducible promoter according to the present invention, which promoter is an endogenous promoter / promoter integrated into the genome.

[0082] As will be appreciated by those of skill in the art, suitable inducible promoters for use in accordance with the present invention will depend on the receptor and the signaling pathway activated by activation of the receptor, and those of skill in the art will know how to identify or select suitable inducible promoters for use in the present invention and based on the disclosure herein.

[0083] According to the present invention, the inducible promoter can be any promoter that is induced upon activation of the receptor and subsequent signal transduction through the cell. In some embodiments, the inducible promoter operably linked to the nucleic acid encoding the protein of interest is any promoter that is activated in response to a transcription factor that increases upon specific activation of immune cells by activation of the receptor. In some embodiments, the inducible promoter is a promoter that responds to ITAM-mediated signaling (see, e.g., Love et al. Cold Spring Harb Perspect Biol. 2010 Jun;2(6):a002485. doi:10.1101 / cshperspect.a002485), such as signaling initiated by phosphorylation of a conserved motif contained within the cytoplasmic domain (ITAM) or transmembrane cell surface receptor (e.g., TCR, CAR, NKR receptor).

[0084] In some embodiments, the inducible promoter is a human promoter.

[0085] In a preferred embodiment of the invention, the inducible promoter is selected from the group consisting of NFAT promoter, NF-κB promoter, AP-1 promoter, CD69 promoter, CD137 promoter, IFNγ promoter, TNFα promoter, GM-CSF promoter, IL-2 promoter, IL-4 promoter, IL-6 promoter, IL-8 promoter, IL-13 promoter, and IL-17 promoter. For example, in a preferred embodiment of the present invention, the inducible promoter is selected from the group consisting of a (synthetic) NFAT promoter (Jutz et al. J Immunol Methods. 2016 Mar;430:10-20, Zhang et al. Mol Ther. 2011 Apr;19(4):751-759), a (synthetic) NF-κB promoter (Jutz et al. J Immunol Methods. 2016 Mar;430:10-20), and a (synthetic) AP-1 promoter (Jutz et al. J Immunol Methods. 2016 Mar;430:10-20). As will be understood by those skilled in the art, such NFAT promoters, NF-κB promoters, and AP-1 promoters are also referred to as synthetic promoters because they (may) contain multiple copies of the NFAT-binding motif, NF-κB-binding motif, or AP-1-binding motif.Similarly, in a preferred embodiment of the invention, the inducible promoter is the (native) CD69 promoter (Redondo-Anton et al. Front Genet. 2020 Oct 27;11:552949), the (native) CD137 promoter (Kim et al. FEBS Lett. 2003 Apr 24;541(1-3):163-70), the (native) IFNγ promoter (Gonsky et al. J Immunol. 2000 Feb 1;164(3):1399-407), the (native) TNFα promoter (Goldfeld et al. J Exp Med. 1993 Oct 1;178(4):1365-1379), the (native) GM-CSF promoter (Cockerill et al. Mol Cell Biol. 1995 Apr;15(4): 2071-2079), (natural) IL-2 promoter (Skerka et al. J Biol Chem. 1995 Sep 22;270(38):22500-6), (natural) IL-4 promoter (Davydov et al. J Immunol. 1995 Dec 1;155(11):5273-9, Macian et al. EMBO J. 2000 Sep 1;19(17):4783-95), (natural) IL-6 promoter (Faggioli et al. Biochim Biophys Acta. 2004 May 28;1692(1):17-24), (natural) IL-8 promoter (Okamoto et al. J Biol Chem. 1994 Mar 18;269(11):8582-9), (natural) IL-13 promoter (Dolganov et al. Blood. 1996 Apr 15;87(8):3316-26), and the (native) IL-17 promoter (Liu et al. J Biol Chem. 2004 Dec 10;279(50):52762-71). These promoters are well known to those skilled in the art and include, for example, those described in WO 2020 / 141106.

[0086] Specifically, for example, an NFAT promoter refers to one or more NFAT response elements linked to a minimal promoter of any gene expressed by a T cell, for example. In some embodiments, the minimal promoter of a gene expressed by a T cell is a minimal human IL-2 promoter (see, e.g., Zhang et al. Mol Ther. 2011 Apr; 19(4): 751-759). The NFAT response element includes one or more binding motifs to which NFAT proteins, such as NFAT1, NFAT2, NFAT3, and / or NFAT4, bind. The NFAT promoter can include any number of binding motifs, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or up to 12 binding motifs. In a preferred embodiment, the sequence of the NFAT binding motif is ggaggaaaaactgtttcatacagaaggcgt (SEQ ID NO: 202). In embodiments, the NFAT promoter comprises an NFAT binding motif. In particularly preferred embodiments, the NFAT promoter nucleotide sequence comprises or consists of ggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtcgcgaattcgcggagactctagagggtatataatggaagctcgatttccag (SEQ ID NO: 203), which contains four NFAT binding sites and a minimal promoter (see also Jutz et al. J Immunol Methods. 2016 Mar;430:10-20).

[0087] According to the present invention, the cells of the present invention are provided with a nucleic acid comprising an inducible promoter, for example, a promoter that is induced upon signal transduction via ITAM, for example, when a TCR, CAR, or NKR is activated by an activation signal, for example, upon binding of a receptor to a ligand such as an antigen. The inducible promoter contained in the nucleic acid provided to the cells of the present invention is operably linked to a nucleic acid encoding a protein of interest. In a preferred embodiment of the present invention, the nucleic acid encoding the protein of interest is further operably linked to a nucleic acid encoding an RNA degradation element (RDE), and the RDE is preferably an AU-rich element (ARE). The 3' untranslated region (UTR) used in the present invention is preferably the 3' untranslated region (UTR) of IL-2, IL-3, IL-4, IL-6, IL-13, IL-17, GATA-3, IFNγ, TNFα, CSF2, FasL, or c-fos. Preferably, the ARE is or is derived from the 3' untranslated region (UTR) of IL-2, IL-3, IL-4, IL-6, IL-13, IL-17, GATA-3, IFNγ, TNFα, CSF2, FasL, or c-fos. Preferably, the ARE is or is derived from the 3' untranslated region (UTR) of IL2 or IFNγ. Preferably, the RDE, preferably an ARE, is located in the 3' untranslated region (UTR) of a nucleic acid encoding a protein of interest. Preferably, multiple RDEs, preferably AREs, are used in the nucleic acids of the present invention, for example, one, two, three, four, or more AREs are used in the 3' UTR. In some embodiments, the 3' untranslated region (UTR) comprises a combination of RDEs, preferably AREs (see also Examples), for example, two or more (sometimes referred to as tandem 3' UTRs).

[0088] RNA destabilizing elements (RDEs) are known to those skilled in the art and are nucleic acids that affect or maintain the stability or translational kinetics of RNA molecules. Some RDEs are bound by polypeptides. Preferably, the RDE is an RDE bound by an RDE-binding polypeptide that stabilizes RNA and extends the half-life of the RNA. Such an RDE can be used to control the expression of the protein of interest operably linked to the inducible promoter. In other words, in such a preferred embodiment, the RDE is an RDE that causes RNA stabilization by interacting with an RNA stabilizing protein.

[0089] In the context of the present invention, the RDE can contribute to the regulation of expression of the protein of interest. Examples of RDEs include, for example, AU-rich elements, U-rich elements, GU-rich elements, and certain stem-loop elements. Exemplary RDEs are described in Ovarik et al., Cytokine 89:21-26 (2017), Ray et al., Nature 499:172-177 (2013), Castello et al., Cell 149:1393-1406 (2012), Vlasova et al., Molc. Cell. 29:263-270 (2008), Barreau et al., Nucl. Acids Res. vol 33, doi:10.1093 / nar / gki1012 (2006), Meisner et al., ChemBioChem 5:1432-1447 (2004), Guhaniyogi et al., Gene 265:11-23 (2001), and WO 2018 / 045177.

[0090] Preferably, the RDE is an AU-rich element (ARE). Such adenylate-uridylate-rich elements are found in the 3' untranslated regions (UTRs) of many messenger RNAs (mRNAs), including those encoding protooncogenes, nuclear transcription factors, and cytokines. AREs are one of the most common factors determining RNA stability in mammalian cells. AREs are defined as regions in mRNA where adenine and uridine bases frequently occur. AREs are generally classified into three sequence-distinct classes (see Goss et al. Encyclopedia of Cell Biology Volume 1, 2016, Pages 341-345; doi: / 10.1016 / B978-0-12-394447-4.10040-9; Ripin et al. PNAS 2019:116(8):2935-2944; Benjamin et al. Expert Opin. Biol. Ther. (2007) 7(10):1515-1529). The RDE can be a class I AU-rich element, a class II AU-rich element, or a class III AU-rich element. Class I AREs contain several AUUUA motifs dispersed across the 3'UTR within a largely U-rich background. Class II AREs contain multiple copies of the canonical AUUUA pentamer, sometimes overlapping as AUUUAUUUA nonamer sequences. In contrast, class III AREs are negatively defined as not containing the AUUUA motif, but are generally U-rich. The best-characterized adenylate-uridylate (AU)-rich elements have a core sequence of AUUUA within a U-rich sequence.

[0091] In a preferred embodiment, multiple RDEs, preferably AREs, are used. Repeats of the core AUUUA element are often required for function in a base sequence.

[0092] In a preferred embodiment, the RDE may be a class I AU-rich element, preferably originating from the 3'UTR of a gene encoding, for example, c-myc, c-fos, beta-AR, PTH, interferon-γ, MyoD, p21, cyclin A, cyclin B1, cyclin D1, PAI-2, or NOS HANOS.

[0093] In a preferred embodiment, the RDE may be a class II AU-rich element, preferably arising from the 3'UTR of a gene encoding, for example, GM-CSF, TNF-α, interferon α, COX-2, IL-2, IL-3, bcl-2, interferon β, or VEG-F. In a preferred embodiment, the RDE may be a class III AU-rich element, preferably arising from the 3'UTR of a gene encoding, for example, c-jun, GLUT1, p53, hsp 70, myogenin, NF-M, or GAP-43.

[0094] In a highly preferred embodiment of the present invention, said RIDEs, preferably AREas, preferably AREas, are bound by RNA-binding proteins (RBPs), such as NF90 and HuR. The 3' untranslated region (UTR) of IL-2 is a ubiquitous 3' untranslated region of IL-2 (NM_000586.4), IL-3 (NM_000588.4), IL-4 (NM_000589.4), IL-6 (NM_000600.5), IL-13 (NM_002188.3), IL-17 (NM_002190.3), GATA-3 (NM_001002295.2), IFNg (NM_000619.3), The 3' untranslated regions (UTRs) of TNFα (NM_000594.4), CSF2 (NM_000758.4), FasL (NM_000639.3), c-fos (NM_005252.4), and others known to those skilled in the art contain AU-rich elements (AREs), which respond to ITAM signaling by increasing mRNA stability and nuclear transport (Salerno et al., 2011). et al. Nat Immunol. 2018 Aug; 19(8): 828-837, Casolara et al. J Allergy Clin Immunol. 2008 Apr;121(4):853-9.e4, Dean et al. Mol Cell Biol. 2001 Feb;21(3):721-30, Ouhara et al. Clin Exp Immunol. 2018 Jun;192(3):325-336, Fan et al. EMBO J. 1998 Jun 15;17(12):3448-60, Karginov et al. RNA Biol. 2019 May;16(5):686-695, Drury et al. J Biol Chem. 2010 Oct 8;285(41):31130-8, Chen et al. J Immunol. 2013 Dec 1;191(11):5441-50, Stellato et al. J Immunol. 2011 Jul 1;187(1):441-9). In a preferred embodiment, AU-rich elements (AREs) from these 3' untranslated regions (UTRs) are used in the present invention.

[0095] Specifically, induction of ITAM signaling increases the nuclear export of mRNA-stabilizing factors, such as HuR and NF90, which bind to AREs in the 3'UTR and displace pre-bound mRNA destabilizing factors (Shim et al. Mol Cell. 2002 Dec;10(6):1331-44, Wang et al. J Immunol. 2006 Feb 15;176(4):2105-13, Nicolet et al. Immunol Rev. 2021 Nov;304(1):10-29). Displacement of mRNA destabilizing factors improves mRNA stability and protein expression levels. Importantly, control of payload production via the rheostat (e.g., RheoBrick®) of the present invention surprisingly works for different classes of regulatory gene elements (e.g., promoters and / or 3'UTRs) that share the property of responding to ITAM signaling.

[0096] In particular, the RDE, preferably an ARE, is an RDE, preferably an ARE, to which an RNA-binding protein (RBP) binds, thereby increasing mRNA stability. In preferred embodiments, the RBP is a protein that is exported from the nucleus in response to ITAM signaling, such as NF90 and HuR. In the absence of ITAM signaling, AREs containing the 3'UTR reduce mRNA stability, whereas in the presence of ITAM signaling, RNA-binding proteins (RBPs) such as NF90 and HuR are exported from the nucleus and bind to AREs containing the 3'UTR, increasing mRNA stability and export of mRNA from the nucleus, resulting in the production of POI. In some preferred embodiments, the ARE is or is derived from the 3' untranslated region (UTR) of IL2 or IFNγ.

[0097] Those skilled in the art know how to provide a nucleic acid comprising an inducible promoter according to the invention and further comprising a nucleic acid encoding a protein of interest, said nucleic acid being operably linked to a nucleic acid encoding an RDE, preferably an ARE, which is transcribed (using the inducible promoter) to give a transcript (mRNA) encoding the protein linked to the RDE.

[0098] The present invention is not particularly limited by the type of cell, e.g., the type of cell that has been or will be modified to provide the genetically engineered cells of the present invention. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. Preferably, the cell is a eukaryotic cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an animal cell or a human cell, e.g., an immune cell. In some embodiments, the cell is a human cell, a rodent cell, a rabbit cell, a rat cell, a mouse cell, or a primate cell. In some embodiments, the cell is a stem cell, e.g., a pluripotent stem cell, e.g., an induced pluripotent stem cell. In preferred embodiments, the cell is a human immune cell. In preferred embodiments, the cell is an immune cell, preferably selected from the group consisting of a T cell, a TCR-expressing T cell, a TCR-modified T cell, a CAR T cell, a NK cell, a CAR NK cell, a tumor-infiltrating lymphocyte (TIL), and a macrophage. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CAR T cell. In some embodiments, the cell is an NK cell. In some embodiments, the cells are CAR NK cells. In some embodiments, the cells are macrophages, including CAR macrophages (see, e.g., www.pennmedicine.org / news / news-releases / 2022 / january / first-in-human-trial-with-car-macrophages-shows-the-cell-therapy-safe-feasible-for-solid-tumors and eBioMedicine 2022;76:103873 or doi.org / 10.1016 / j.ebiom.2022.103873). In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the cells are human cells.Preferably, the immune cells are selected from the group consisting of human T cells, human CART cells, human TCR-modified T cells (sometimes referred to as modified T cell receptor T cells), human NK cells, human CARNK cells, human tumor-infiltrating lymphocytes (TILs), and human macrophages.

[0099] Those skilled in the art are well aware of the existence of different cells, including different immune cells, and know how to recognize such cells. For example, T cells or T lymphocytes play a central role in cell-mediated immunity. Lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) or chimeric antigen receptors (CARs) on their surface. CAR T cells are T cells that express CAR complexes. There are various types of T cells, including, but not limited to, T helper cells (TH cells), cytolytic T cells, and regulatory T cells. TH cells express CD4 on their surface and are activated when peptide antigens are presented on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes that secrete different cytokines to promote different types of immune responses. Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize targets by binding to antigens associated with MHC class I, which is present on the surface of all nuclear cells. Regulatory T cells (Tregs) suppress immune responses by secreting molecules such as IL-10, and these cells are characterized by the expression of the transcription factor FOXP3.

[0100] Another example is memory T cells, a subset of antigen-specific T cells that persist long after an infection has subsided. Upon re-exposure to the cognate antigen, these rapidly expand into large numbers of effector T cells, presenting the immune system with a "memory" of past infection. Memory cells are CD4 + or CD8 + Preferably, the T cells are CD4-positive T cells. Preferably, the T cells are CD8-positive T cells.

[0101] Natural killer cells (or NK cells) are a type of cytolytic cell that is part of the innate immune system. NK cells respond to innate signals from virus-infected cells in a peptide-MHC-independent manner. NK cells are defined as large granular lymphocytes and constitute the third cell type differentiated from common lymphoid progenitors that generate B and T lymphocytes. NK cells are known to differentiate and mature in, for example, the bone marrow, lymph nodes, spleen, tonsils, and thymus. CAR NK cells are NK cells that express the CAR complex (see, e.g., Zhang et al., Biomarker Research volume 10, Article number: 12 (2022)).

[0102] Macrophages are a type of white blood cell of the immune system that engulf and digest pathogens such as cancer cells, microorganisms, cellular debris, and foreign bodies that lack proteins on their surfaces that are characteristic of healthy body cells. Another type of immune cell that may be suitably used in accordance with the present invention is tumor-infiltrating lymphocytes, or TILs, preferably human tumor-infiltrating lymphocytes. These and other cells are well known to those skilled in the art.

[0103] Combined with a drug-regulated protein stability domain that allows dose-dependent expression of a chimeric protein in the genetically engineered cells, e.g., T cells, of the present invention, this system provides an efficient and reliable method for precisely regulating T cell activation in response to an activation signal (e.g., an antigen) that activates a receptor (e.g., a TCR), thereby regulating the induction of an inducible promoter and the expression (production) of a protein (protein of interest). Furthermore, cytokine production and secretion can also be regulated in this manner. This method not only allows for regulating T cell activity, leading to regulated production of a POI, but also allows for the maintenance, restoration, safe, and controllable function of T cells both in vitro and in vivo (i.e., in the treatment of other conditions, such as cancer or autoimmune diseases, that rely on the use of T cells, including T cells engineered to express new or additional TCRs and / or CARs). Similarly, in addition to regulating the production of a protein of interest (biological or payload), NK cell function can also be regulated.

[0104] Immune cells, e.g., T cells (including CAR T cells) or NK cells (including CAR NK cells), comprising a receptor operably linked to a protein of interest, a chimeric protein, and an inducible promoter, and expressing or containing the molecules of the invention, can be obtained using the patient's own peripheral blood or from donor peripheral blood. Alternatively, the cells can be obtained using ex vivo expansion and / or differentiation of immune cell precursor stem cells using conventional methods known to those of skill in the art.

[0105] According to the present invention, the receptor may be any suitable receptor. The receptor is capable of receiving an activation signal, which causes the receptor to induce, e.g., via a signal transduction pathway, an inducible promoter operably linked to a nucleic acid encoding a protein of interest. In other words, the receptor must be capable of receiving an activation signal, such as a binding event, and transmitting the signal to, i.e., through, the cell, thereby allowing the inducible promoter to be induced and leading to the expression (production) of the protein of interest. In some embodiments, the receptor is a membrane-bound receptor. In some embodiments, the receptor is a single protein receptor. In some embodiments, the receptor is a receptor complex (e.g., comprising a complex of different proteins). In some embodiments, the receptor is a cell surface receptor. In some embodiments, the receptor is an intracellular receptor. Preferably, the receptor is a surface receptor. This allows, for example, reception of an extracellular signal (herein referring to a signal not originating from the cell containing the receptor). In a preferred embodiment of the invention, the receptor is selected from the group consisting of a T cell receptor (TCR), a chimeric antigen receptor (CAR), and an NK cell receptor (NKR), and / or the receptor is directed against an antigen, such as a tumor antigen.

[0106] According to the present invention, the docking domain can be any suitable docking domain. The docking domain is capable of binding (preferably reversibly binding) to the receptor, e.g., the intracellular or cytoplasmic domain of a receptor, and by binding to the receptor, inhibits, reduces, or prevents signal transduction induced when the receptor receives an activating signal (e.g., prevents signal transmission when a CAR binds to an antigen). The docking domain inhibits, reduces, or prevents signal transmission from the receptor to a cell, thereby inhibiting, reducing, or preventing activation of the cell despite the receptor being activated by an activating signal (e.g., an antigen such as a tumor antigen, or other suitable receptor-activating molecule).

[0107] In a preferred embodiment of the invention, the chimeric proteins of the invention and the chimeric proteins comprised in the genetically engineered cells of the invention are characterized by the presence of a docking domain comprising (as a first moiety) an SH2 domain from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM). In a further preferred embodiment of the invention, the docking domain comprises an SH2 domain capable of binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) comprised in said receptor, preferably after receiving an activation signal, and preferably the SH2 domain is from a protein selected from the group consisting of Zap70, Syk, and Lck.

[0108] Examples of such docking domains (and drug-regulating protein stability domains) containing suitable SH2 domains are provided in detail in WO 2021 / 080427, and such docking domains or SH2 domains disclosed therein are particularly contemplated and preferred in the present invention. Preferably, the SH2 domain of the present invention may be any SH2 domain from a protein capable of binding to (phosphorylated) ITAMs. Those skilled in the art will be well aware of SH2 domains suitable for use in the chimeric proteins of the present invention and / or will be able to readily identify such suitable SH2 domains or proteins containing such SH2 domains capable of binding to phosphorylated (ITAMs).

[0109] As will be understood by those skilled in the art, a suitable SH2 domain can be selected taking into account the ITAM contained in the receptor of the present invention and capable of receiving an activating signal, such as in a TCR / CD3 complex and / or a CAR and / or an NK cell receptor (NKR) complex for which the chimeric protein or polypeptide of the present invention is designed. In other words, the chimeric protein of the present invention comprises an SH2 domain from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), such as that contained in a TCR or CAR complex targeted in the context of the present invention. In other words, in these preferred embodiments, according to the present invention, when the receptor (e.g., TCR) receives an activating signal (e.g., upon antigen binding), the ITAM contained in the receptor is phosphorylated, allowing the SH2 domain contained in the docking domain to bind to the phosphorylated ITAM contained in the receptor.

[0110] Indeed, SH2 domains typically bind to phosphorylated tyrosine residues within the context of longer peptide motifs within target proteins. SH2 domains themselves lack intrinsic catalytic activity but serve to localize bound functional domains within polypeptides near appropriate substrates, activators, or inhibitors (Ngoenkametal, Immunology. 2018 Jan; 153(1): 42-50). For example, some SH2 domains interact with proteins containing phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs), while others interact with proteins containing phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIMs). In the present invention, SH2 domains from proteins that bind to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) are preferred. Surprisingly, it was found that while SH2 domains from proteins that bind to phosphorylated ITAMs are important for regulating T cell or NK cell activity by the chimeric proteins of the present invention, SH2 domains that interact with ITIMs are less suitable for use in the chimeric proteins of the present invention.

[0111] In a preferred embodiment, the SH2 domain is derived from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) present in the TCR complex, the NKR complex, and / or a CAR.

[0112] In some embodiments, the SH2 domain is the SH2 domain annotated as βA-αA-βB-βC-βD-βE-βF-αB-βG for 120 known human SH2 domains by Liu et al. (Mol Cell. 2006;22(6):851-868. doi:10.1016 / j.molcel.2006.06.001), where β refers to a β-strand and α refers to an α-helix (see also Eck et al. Nature. 1993;362(6415):87-91. doi:10.1038 / 362087a0). SH2 domains can be found, for example, in various databases known to those skilled in the art (see, for example, smart.embl.de / smart / do_annotation.pl?DOMAIN=SM00252 or www.ebi.ac.uk / interpro / entry / InterPro / IPR000980 / ). In some embodiments, the SH2 domain embodiments comprise or consist of an amino acid sequence having at least 80% identity, preferably at least 81%, 83%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, to the amino acid sequence according to SEQ ID NOs: 164-168, or alternatively, an amino acid sequence having at least 80% identity, preferably at least 81%, 83%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, to the amino acid sequence according to SEQ ID NOs: 169-171. As will be appreciated by those skilled in the art, sequences as defined above in which 1, 2, 3, 4, 5, 6, or 10 amino acids have been deleted, substituted, or inserted are also included.

[0113] Furthermore, in the cells of the present invention, the SH2 domain is derived from a protein selected from the group consisting of Zap70, Syk, and Lck. In particular, chimeric proteins of the present invention, which comprise a first portion comprising an SH2 domain from Zap70, Syk, or Lck, have been found to be highly suitable for the present invention.

[0114] ZAP70 is a protein typically expressed near the plasma membrane of T cells and natural killer cells. It plays an important role in T cell signal transduction. It has a molecular weight of 70 kDa and is composed of two N-terminal SH2 domains and one C-terminal kinase domain. It is a member of the protein tyrosine kinase family. The UniProtKB accession number for human ZAP70 protein is P43403. This sequence is 619 amino acids long and is shown as SEQ ID NO: 153. Syk is expressed in thymocytes, intraepithelial γδ T cells, naive αβ T cells, and B cells (Latour et al., Mol Cell Biol. 1997 Aug; 17(8): 4434-4441). Syk is highly homologous to ZAP70 and shares the same domain structure of two N-terminal SH2 domains and one C-terminal kinase domain. In B cells, Syk deficiency is reconstituted by Zap70 (Kongetal, Immunity. 1995 May;2(5):485-92). Similarly, ZAP70 deficiency can be reconstituted by Syk in T cells (Williams et al., Mol Cell Biol. 1998 March;18(3):1388-99). The UniProtKB accession number for the human Syk protein is P43405. This sequence is 635 amino acids long and is shown as SEQ ID NO:154.

[0115] Lck (also known as p56-LCK) is expressed in lymphocytes. It plays a key role in the TCR signaling pathway. In T cells, it constitutively binds to the cytoplasmic domains of the CD4 and CD8 coreceptors. Upon TCR activation by peptide-MHC complexes, Lck is recruited to the TCR complex and phosphorylates ITAM residues within the CD3 subunit. The phosphorylated ITAM serves as a docking site for the SH2 domain of Zap70 (Simeoni, Oncotarget. 2017 Nov 28; 8(61): 102761-102762). The domain structure of Lck is SH4-unique domain (UD)-SH3-SH2-kinase domain. The SH2 domain is essential for interaction with phosphorylated ITAM, and the SH4 domain is essential for membrane binding (Ngoenkametal, Immunology. 2018 Jan; 153(1): 42-50). The UniProtKB accession number for the human Lck protein is P06239. The sequence is 509 amino acids in length and is shown as SEQ ID NO: 155. Preferably, the Zap70, Syk, and Lck are human Zap70, Syk, and Lck.

[0116] In a further preferred embodiment of the present invention, the ITAM present in the receptor is present in a TCR, a CAR, or an NKR, more preferably the ITAM is derived from or present in the CD3ζ chain, CD3ε chain, CD3δ chain, CD3γ chain, FceRIγ chain, or DAP12.

[0117] In a preferred embodiment, the engineered cells are T cells expressing a TCR complex and / or a CAR complex, preferably comprising an ITAM-containing CD3 zeta chain domain or other ITAM-containing domains disclosed herein.

[0118] In another preferred embodiment, the engineered cells are NK cells expressing an NKR complex and / or a CAR complex, preferably comprising an ITAM-containing CD3 zeta chain domain or other ITAM-containing domains disclosed herein.

[0119] In some embodiments of the present invention, the ITAM is an ITAM contained in a T cell receptor (TCR) complex and / or a chimeric antigen receptor (CAR) and / or an NKR complex, and is preferably an ITAM contained in the CD3ζ chain, CD3ε chain, CD3δ chain, CD3γ chain, the γ chain of the immunoglobulin receptor FceRI, and DAP12.

[0120] As previously mentioned, the SH2 domain may be derived from a protein that binds to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs), which are present in the intracellular domains of cell signaling molecules such as the CD3 zeta (ζ), epsilon (ε), gamma (γ), and delta (δ) chains of the T cell receptor complex and Fc receptors (Love et al., Cold Spring Harb Perspect Biol. 2010 Jun;2(6):a002485), including FceRI.

[0121] The chimeric proteins are designed to interact with phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within the TCR / CD3 complex and / or CAR and / or NK cell receptor (NKR) and / or macrophage CAR complex, including ITAM-containing signaling molecules such as DAP12, the gamma (γ) chain of the immunoglobulin receptor FceRI, or the CD3ζ chain (Lanier et al, Nat Immunol. 2008 May; 9(5): 495-502).

[0122] The tyrosine residues within the ITAM motif are phosphorylated after interaction of a receptor molecule with its ligand (activation signal), forming a docking domain for other proteins involved in cellular signaling pathways. Interaction of the chimeric protein of the present invention with a TCR and / or CAR inhibits T cell activation (and subsequent cytotoxic effects and / or cytokine secretion). Similarly, interaction of the chimeric protein of the present invention with an NKR and / or CAR in NK cells inhibits NK cell activation (and subsequent cytotoxic effects and / or cytokine secretion).

[0123] In NK cells, specific activating NK cell receptors (NKRs) form complexes with ITAMs containing signaling molecules such as the CD3ζ chain, the gamma (γ) chain of the immunoglobulin receptor FceRI, and DAP12. For example, the NK cell receptors (NKRs) NKp46 and NKp30 bind to the gamma (γ) chain of the immunoglobulin receptor FceRI and the CD3ζ chain, and NKp44 binds to the signaling adaptor DAP12 (Barrow et al., Front Immunol. 2019; 10: 909). Thus, in one embodiment of the present invention, the cells of the present invention are NK cells.

[0124] ITAM-bearing domains are also used in the design of chimeric antigen receptors (CARs). The CD3ζ chain contains three ITAMs, while the CD3ε chain, the gamma (γ) chain of the immunoglobulin receptor FceRI, and the DAP12 signaling domain contain one ITAM, and these have been used in various CAR designs (Ren-Heidenreich et al., Cancer Immunol Immunother. 2002 Oct;51(8):417-23, Nolan et al., Clin Cancer Res. 1999 Dec;5(12):3928-41, Topfer et al., J Immunol. 2015 Apr 1;194(7):3201-12).

[0125] Half-ITAM signatures are easily recognized as a tyrosine separated from a leucine or isoleucine by two other amino acids, resulting in the signature YxxL / l. Two of these signatures are separated by 6–8 amino acids, forming the ITAM consensus sequence YxxL / lx(6–8)YxxL / l.

[0126] In a preferred embodiment, the ITAM-containing domain may be or comprise a CD3 ζ chain domain. In another preferred embodiment, the ITAM-containing domain may be or comprise a CD3 ε chain domain. However, in another preferred embodiment, the ITAM-containing domain may be or comprise the gamma (γ) chain of the immunoglobulin receptor FceRI. However, in another preferred embodiment, the ITAM-containing domain may be or comprise a DAP12 domain.

[0127] In a preferred embodiment, the chimeric protein is designed to interact with phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within the TCR / CD3 complex and / or CAR and / or NK cell receptor (NKR) complex, including ITAM-containing signaling molecules such as DAP12, the gamma (γ) chain of the immunoglobulin receptor FceRI, or the CD3ζ chain (Lanier et al, Nat Immunol. 2008 May; 9(5): 495-502).

[0128] Tyrosine residues within these ITAM motifs are phosphorylated after interaction of the receptor with its ligand (activation signal), forming docking sites for other proteins involved in cellular signaling pathways. Interaction of the chimeric proteins of the present invention with TCR and / or CAR inhibits T cell activation (and subsequent cytotoxic effects and / or cytokine secretion). Similarly, interaction of the chimeric proteins of the present invention with NKR and / or CAR in NK cells inhibits NK cell activation (and subsequent cytotoxic effects and / or cytokine secretion).

[0129] In some embodiments, the ITAM is an ITAM contained in SEQ ID NOs: 172-177 or an ITAM that binds to an amino acid sequence having at least 80% identity, preferably at least 81%, 83%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NOs: 172-177.

[0130] According to a preferred embodiment of the present invention, the docking domain of the chimeric protein further comprises an immunoreceptor tyrosine-based switch motif (ITSM) and / or ITIM, preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), in addition to an SH2 domain capable of binding to a phosphorylated ITAM motif (e.g., present in receptors that have received an activating signal). Such motifs are present, for example, in the inhibitory tail of PD1, and have been suggested to be involved in the immunosuppressive effects of PD1 (Boussiotis et al., Cancer J. 2014 Jul-Aug;20(4):265-271).

[0131] The presence of these ITSM, ITIM, or ITSM and ITIM motifs in the docking domain further improves the blockade of receptor-mediated signaling, in this case ITAM signaling, by the chimeric protein of the present invention. Thus, while the presence of an SH2 domain already blocks ITAM signaling, the presence of these ITSM, ITIM, or ITSM and ITIM motifs in the docking domain further improves this blockade, allowing for tighter regulation of signaling through the cell, and subsequently, because the inducible promoter used in such cases is a promoter that is dependent on or induced by this signaling (here, ITAM signaling), tighter regulation of the production of the biological substance (protein of interest).

[0132] Surprisingly, it has been found that the presence of such ITSMs, preferably such ITSMs and such ITIMs, in the docking domain of the chimeric protein enables the chimeric protein to effectively inhibit signaling by a receptor, e.g., a TCR, an NKR, or a CAR (upon binding of a ligand to the receptor). The inventors have shown that the use of these ITSMs, preferably ITSMs and ITIMs present within the inhibitory tail of an inhibitory immunoreceptor protein, e.g., PD1, can inhibit TCR and / or CAR signaling in T cells and NKR signaling in NK cells, without the need for the extracellular domain of the inhibitory protein to be present or for it to interact with its ligand (e.g., PD-L1 in the case of PD1). As described herein, the system of the present invention, when combined with a drug-regulated protein stability domain that allows dose-dependent expression of the chimeric protein in cells (e.g., T cells), provides an efficient and reliable method for precisely regulating T cell activation, thereby regulating the induction of an inducible promoter of the present invention and the production of a protein of interest.

[0133] According to a preferred embodiment, the docking domain comprises an immunoreceptor tyrosine-based switch motif (ITSM), an immunoreceptor tyrosine-based inhibitory motif (ITIM), or preferably a portion comprising an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0134] The order of the SH2 domain and ITSM, ITIM, or ITSM and ITIM contained in the chimeric protein is not important. For example, in some embodiments, the SH2 domain (hereinafter referred to as the first portion), the drug-related protein stability domain (as described elsewhere herein, hereinafter referred to as the second portion), or the ITSM, ITIM, or ITSM and ITIM (hereinafter referred to as the third portion) is fused (e.g., genetically linked) to the N-terminus or C-terminus of the chimeric protein, or is present internally in the chimeric protein. In other words, in the chimeric protein, the first, second, or third portion may be at the C-terminus, the N-terminus, or may be adjacent to another portion at the C-terminus and / or N-terminus. Examples of suitable ordering of the first portion (P1), second portion (P2), and third portion (P3) generally include xP1xP2xP3x, xP1xP3xP2x, xP2xP1xP3x, xP2xP3xP1x, xP3xP1xP2x, or xP3xP2xP1x, where x at any position may independently refer to the absence of an additional amino acid residue or the presence of one or more additional amino acid residues that do not form part of P1, P2, and / or P3. Similar to the example above, with respect to the ordering of the first and second portions, e.g., in embodiments where the third portion is absent, examples of suitable ordering of the first portion (P1) and second portion (P2) generally include xP1xP2x or xP2xP1x, where x at any position may independently refer to the absence of an additional amino acid residue or the presence of one or more additional amino acid residues that do not form part of P1 and / or P2.

[0135] At the same time, one skilled in the art will understand that the first portion can include, in addition to the SH2 domain of a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), one or more of the amino acids that are normally adjacent (on one or both sides) to the SH2 domain of a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM).

[0136] At the same time, one skilled in the art will appreciate that the second portion may comprise additional domains or amino acids on one or both sides in addition to the drug regulatory protein stability domain.

[0137] At the same time, those skilled in the art will understand that the third portion may comprise, in addition to the immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), further domains or amino acids, for example, one or more of the amino acids that typically flank (on one or both sides of) these motifs.

[0138] One skilled in the art will understand that the first, second, and third portions of the chimeric proteins of the present invention can be present in any order within the chimeric protein, so long as the drug-regulated protein stability domain is arranged within the chimeric protein such that upon contact with a drug that interacts with the drug-regulated protein stability domain, the chimeric protein is degraded, and degradation of the chimeric protein relieves inhibition of signaling by, for example, an activating receptor (e.g., a TCR, NKR, or CAR), thereby leading to signal transduction, activation of the T cell, induction of the inducible promoter, and subsequent production of the protein of interest.

[0139] Thus, the present disclosure can provide for precise regulation of, for example, T cell and NK cell activation by the chimeric proteins of the present invention due to the presence of a drug-regulated protein stability domain that is utilized to regulate (e.g., decrease or increase) expression of the chimeric proteins of the present invention in a time-dependent and / or dose-dependent manner.

[0140] Preferably, the ITIM and / or ITSM are derived from an inhibitory receptor protein, preferably an inhibitory immunoreceptor protein, preferably a protein selected from the group consisting of PD1, BTLA, SIRPα, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, and LY9. Preferably, the inhibitory receptor protein, the inhibitory immunoreceptor protein, or the protein selected from the group consisting of PD1, BTLA, SIRPα, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, and LY9 is of human origin.

[0141] PD1 (also known as PD-1) is encoded by the PDCD1 gene. PD1 is a type I membrane protein. Interaction with its ligands PD-L1 / PD-L2 leads to downregulation of cytotoxic T cell effector function. The UniProtKB accession number for human PD1 is Q15116. The sequence is 288 amino acids long. The cytoplasmic domain of PD1 contains ITIM and ITSM motifs. Phosphorylated ITSMs in the cytoplasmic domain of PD1 recruit SHP-2 phosphatase, which dephosphorylates key signaling molecules in the TCR signaling pathway, such as ZAP70, PKCθ, and CD3ζ (CD247), resulting in downregulation of TCR signaling (Bardhan et al., Front Immunol. 2016;7:550) and CD28-mediated costimulation (Hui et al., Science. 2017 Mar 31;355(6332):1428-1433). For example, a suitable portion of an immunoreceptor tyrosine-based switch motif (ITSM), preferably comprising an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), that can be used in the chimeric proteins / proteins of the present invention is characterized by SEQ ID NO: 156 (representing the cytoplasmic domain of PD1).

[0142] B and T lymphocyte attenuator (BTLA) is primarily expressed in T cells, B cells, and mature lymphocytes (Yue et al., Front Immunol. 2019;10:617). It is an immunoregulatory receptor that plays an important role in immune tolerance. Like PD1, BTLA is a type I transmembrane glycoprotein. Engagement of the BTLA receptor induces SHP-1 / SHP-2 recruitment and downregulation of IL-2 secretion in T cells (Watanabe et al., Nat Immunol. 2003 Jul;4(7):670-9). The UniProtKB accession number for human BTLA protein is Q7Z6A9. The sequence is 289 amino acids long. The cytoplasmic domain of BTLA contains ITIM and TSM motifs.

[0143] For example, a suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), that can be used in the chimeric proteins of the present invention is characterized by SEQ ID NO: 157 (representing the cytoplasmic domain of BTLA).

[0144] SIRPA (also known as SIRPalpha, SIRPα, BIT, MFR, MYD1, PTPNS1, SHPS1, and SIRP) is expressed in myeloid cells. Upon binding to its ligand CD47, it negatively regulates phagocytosis, mast cell activation, and dendritic cell activation (Timms et al., Curr Biol. 1999 Aug 26;9(16):927-30, Latour et al., J Immunol. 2001 Sep 1;167(5):2547-54, Matlung et al., Immunol Rev. 2017 Mar;276(1):145-164. doi: 10.1111 / imr.12527). In macrophages, SIRPA primarily associates with SHP-1 (Veillette et al, J Biol Chem. 1998 Aug 28;273(35):22719-28). SIRPA is a type I membrane protein. The UniProtKB accession number for human SIRPA protein is P78324. The sequence is 504 amino acids long. The cytoplasmic domain of SIRPA contains two ITIM and one ITSM motif.

[0145] For example, a suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), that can be used in the chimeric protein of the present invention is characterized by SEQ ID NO: 158 (representing the cytoplasmic domain of SIRPa).

[0146] PECAM1 (also known as PECAM-1 or CD31) is expressed on T cells, B cells, platelets, monocytes, macrophages, and neutrophils (Newton-Nash et al., J Immunol. 1999 Jul 15;163(2):682-8). PECAM1 inhibits T and B cell signaling through the recruitment of SHIP1, SHP-1, and SHP-2 (Marelli-Berg et al., J Cell Sci. 2013 Jun 1;126(Pt 11):2343-52). In macrophages, ligand binding to PECAM1 leads to the recruitment of SHP-1 and SHP2, downregulation of TNF-α, IL-6, and IFN-β production, and TLR4 signaling (Rui et al., J Immunol. 2007 Dec 1;179(11):7344-51). PECAM1 negatively regulates platelet signaling pathways (Jones et al, FEBS Lett. 2009 Nov 19;583(22):3618-24). PECAM1 is a type I membrane protein. The UniProtKB accession number for the human PECAM1 protein is P16284. This sequence is 738 amino acids long. The cytoplasmic domain of PECAM1 contains ITIM and ITSM motifs. For example, a suitable portion containing an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), that can be used in the chimeric proteins of the present invention is characterized by SEQ ID NO: 159 (representing the cytoplasmic domain of PECAM1).

[0147] Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a group of immunoregulatory receptors primarily expressed on hematopoietic cells (Bornhufft et al., Dev Comp Immunol. 2018 Sep;86:219-231). SIGLEC5 (also known as CD33L2 or OBBP2) is expressed on monocytes, neutrophils, and B cells, SIGLEC9 is expressed on neutrophils, monocytes, dendritic cells, and NK cells, and SIGLEC11 is expressed on macrophages (Macauley et al., Nat Rev Immunol. 2014 Oct;14(10):653-666). Most Siglecs contain inhibitory ITIM / ITSM motifs that recruit SHP1 and SHP2 and function as negative regulators of the immune system (Crocker et al., Nat Rev Immunol. 2007 Apr;7(4):255-66, Avril et al., J Biol Chem. 2005 May 20;280(20):19843-51, Haas et al., Cancer Immunol Res. 2019 May;7(5):707-718, Angata et al., J Biol Chem. 2002 Jul 5;277(27):24466-74). SIGLEC5, SIGLEC9, and SIGLEC11 are type I membrane proteins. They contain ITIM and ITSM motifs. The UniProtKB accession number for the human SIGLEC5 protein is O15389. The sequence is 551 amino acids long. The UniProtKB accession number for the human SIGLEC9 protein is Q9Y336. This sequence is 463 amino acids in length. The UniProtKB accession number for the human SIGLEC11 protein is Q96RL6. This sequence is 698 amino acids in length. For example, suitable portions comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), that can be used in the chimeric proteins of the present invention are characterized by SEQ ID NO: 161, SEQ ID NO: 162, or SEQ ID NO: 160 (representing the cytoplasmic domains of SIGLEC5, 9, and 11, respectively).

[0148] The T lymphocyte surface antigen Ly-9 (also known as LY9, SLAMF3, or CD229) is expressed on thymocytes and mature T and B lymphocytes (de la Fuente et al., Blood. 2001 Jun 1;97(11):3513-20). It interacts with SHIP-1 and SHP-2 (Punet-Ortiz et al., Front Immunol. 2018 Nov 16;9:2661) and has been reported to function as a negative regulator of immune responses, contributing to peripheral cell tolerance (de Salort et al., Front Immunol. 2013;4:225). LY9 is a type I membrane protein. The UniProtKB accession number for the human LY9 protein is Q9HBG7. The sequence is 655 amino acids long. The cytoplasmic domain of LY9 contains two ITSM motifs. For example, a suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), that can be used in the chimeric protein of the present invention is characterized by SEQ ID NO: 163 (representing the cytoplasmic tail of LY9).

[0149] Thus, the ITSM and / or ITIM contained in the third portion of the chimeric polypeptide can be an ITSM and / or ITIM contained in SEQ ID NOs: 156-163, or an ITSM and / or ITIM having a sequence with at least 80% identity, preferably at least 81%, 83%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NOs: 156-163. As will be understood by those skilled in the art, the above-defined sequences, in which 1, 2, 3, 4, 5, 6, or 10 amino acids have been deleted, substituted, or inserted, are also included. The third portion may also include additional amino acids (extensions) adjacent to the above-defined amino acids, as long as the third portion of the chimeric polypeptide maintains its functionality within the context of the present invention.

[0150] In some embodiments, the ITSM and ITIM are obtained or derived from the same inhibitory protein, while in other embodiments, the ITSM and ITIM are each derived from different inhibitory proteins.

[0151] Furthermore, in the cell of the present invention, the chimeric protein comprises an ITSM and an ITIM.

[0152] Surprisingly, it has been found that the presence of both an ITSM and an ITIM in the third moiety contained in the chimeric protein according to the invention is particularly advantageous (see examples).

[0153] In some embodiments of the invention, the drug-regulated protein stability domain is a CRBN polypeptide substrate domain that is capable of binding to a CRBN protein in response to a drug, preferably thereby promoting degradation of the chimeric protein via the ubiquitin pathway.

[0154] In this embodiment, the chimeric protein of the present invention comprises a drug-regulated protein stability domain that can interact with and bind to a CRBN protein in the presence of a drug. For example, various IMiDs, including those described herein, have been shown to bind to CRBN proteins, thereby promoting the interaction between the CRBN protein and its target (see also Buhimschi et al. Biochemistry 2019, 58, 861-864), ubiquitination of the target protein, and subsequent degradation.

[0155] CRBN (cereblon) is a 442-amino acid protein that forms an E3 ubiquitin ligase complex with damaged DNA-binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1; Angers et al. Nature 443: 590-593). This complex ubiquitinates numerous other proteins. Thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdomide), and CC-885 have each been shown to bind to CRBN (see, e.g., Lopez-Girona et al. Leukemia 26: 2326-2335).

[0156] Those skilled in the art will recognize CRBN polypeptide substrate domains that are capable of binding to a CRBN protein in response to an agent, thereby promoting degradation of the chimeric protein via the ubiquitin pathway, and are suitable for use in the present invention.

[0157] In a preferred embodiment, the drug-regulated protein stability domain comprises a Cys2-His2 zinc finger domain capable of drug-inducible binding to a CRBN polypeptide, and preferably, the Cys2-His2 zinc finger domain is a hybrid zinc finger domain. Such a drug-regulated protein stability domain comprises a Cys2-His2 zinc finger domain and is also referred to as a "zinc finger degron."

[0158] The Cys2-His2-like fold (C2H2) is a well-characterized class of zinc finger domains highly common in mammalian transcription factors. These domains adopt a simple ββα fold, forming two short β-strands connected by a turn (zinc knuckle, β-turn) followed by a short helix, and share the amino acid sequence motif X2-Cys-X2,4-Cys-X12-His-X3,4,5-His (Pabo et al. Annual Review of Biochemistry (2001). 70: 313-40).

[0159] In some embodiments, the chimeric protein comprises a CRBN polypeptide substrate domain comprising one or more zinc fingers, i.e., zinc finger degrons.

[0160] Although not particularly limited to a particular CRBN polypeptide substrate domain, particularly a C2H2 zinc finger domain, in some embodiments, the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, ZFN654, ZNF787, ZNF653, ZFP91, ZNF276, ZNF827, and fragments thereof capable of small molecule-induced binding to a CRBN polypeptide, preferably the fragment is selected from the group consisting of IKZF1 ZF2-3 (SEQ ID NO: 136), IKZF3 ZF2-3 (SEQ ID NO: 137), ZFP91 ZF4-5 (SEQ ID NO: 138), ZNF276 ZF4-5 (SEQ ID NO: 139), ZNF653 ZF4-5 (SEQ ID NO: 140), and ZNF692 ZF4-5 (SEQ ID NO: 141).

[0161] In another preferred embodiment, the CRBN polypeptide substrate domain comprises a hybrid fusion polypeptide comprising at least a first fragment of a first C2H2 zinc finger protein and a second fragment from a second C2H2 zinc finger protein, wherein the combination of the first and second fragments within the hybrid fusion polypeptide is capable of drug-induced binding to a CRBN polypeptide. For example, in some embodiments, a β-turn (formed by two short β-strands) from a first C2H2 zinc finger protein can be fused to an α-helix of a second C2H2 zinc finger protein. Cells of the present invention also comprise chimeric proteins of the present invention comprising such small molecule regulatory protein stability domains.

[0162] While the present invention is not particularly limited to a particular hybrid fusion polypeptide that may be formed or contained within a CRBN polypeptide substrate domain, in a preferred embodiment, the hybrid fusion polypeptide comprises a first fragment selected from a β-turn of ZFP91 ZF4 (LQCEICGFTCR, SEQ ID NO: 142), ZFN653 ZF4 (LQCEICGYQCR, SEQ ID NO: 143), ZNF276 ZF4 (LQCEVCGFQCR, SEQ ID NO: 144), or ZNF827 ZF1 (FQCPICGLVIK, SEQ ID NO: 145), and a second fragment selected from an α-helix of IKZF1 ZF2 (QKGNLLRHIKLH, SEQ ID NO: 146), in any possible combination. Preferably, the hybrid fusion polypeptide comprises a β-turn of ZFP91 ZF4 and an α-helix of IKZF1 ZF2, and preferably, the hybrid fusion polypeptide comprises one selected from SEQ ID NOs: 147-151. The cells of the present invention also include chimeric proteins of the present invention that include such small molecule regulatory protein stability domains.

[0163] According to another embodiment, the CRBN polypeptide substrate-binding domain used in the methods of the present invention comprises or further comprises IKZF1 ZF3 (FKCHLCNYACRRRDALTGHLRTH, SEQ ID NO: 152), and preferably, the CRBN polypeptide substrate-binding domain comprises the β-turn of ZFP91 ZF4, the α-helix of IKZF1 ZF2, and IKZF1 ZF3. Preferably, IKZF1 ZF3 is at the C-terminus of the second portion of the chimeric protein of the present invention. Additionally, the cells of the present invention comprise the chimeric protein of the present invention comprising such a small molecule regulatory protein stability domain.

[0164] As will be appreciated by those skilled in the art, in some embodiments, the chimeric proteins of the present invention include one or more CRBN polypeptide substrate domains that are capable of binding to CRBN in response to a drug, thereby promoting degradation of the chimeric proteins of the present invention via the ubiquitin pathway. The zinc finger degron polypeptide domains (CRBN polypeptide substrate domains that are capable of binding to CRBN in response to a drug, thereby promoting degradation of the chimeric proteins of the present invention via the ubiquitin pathway) can be included as a single degron polypeptide domain or as multiple degron polypeptide domains, optionally linked in tandem or in an array, using polypeptide linkers known in the art.

[0165] As will be appreciated by those of skill in the art, within a CRBN polypeptide substrate-binding domain used in the methods of the invention, different portions (e.g., β-turns and α-helices) may be directly adjacent to each other or may be linked using polypeptide linkers (comprising small stretches of amino acids), such as those known in the art. Those of skill in the art will be familiar with zinc finger degrons suitable for use in the present invention.

[0166] Suitable agents (e.g., small molecules) for modulating the degradation of chimeric proteins of the invention comprising one or more of such C2H2 zinc finger proteins, fragments, or domains include so-called immunomodulatory imide drugs (IMiDs), including, but not limited to, thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdomide), and CC-885 (see, e.g., Matyskiela et al. J. Med. Chem. 2018, 61, 2, 535-542; 2017; doi.org / 10.1021 / acs.jmedchem.6b01921 and Gao et al. Biomarker Research (2020) 8:2; doi.org / 10.1186 / s40364-020-0182-y). Those of skill in the art will know how to select suitable agents, such as appropriate IMiDs, for use in the methods of the present invention.

[0167] Thus, in the cell of any one of the preceding claims, the agent by which the CRBN polypeptide substrate domain binds to the CRBN protein and promotes degradation of the chimeric protein via the ubiquitin pathway is an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdomide), and CC-885.

[0168] Immunomodulatory imid drug (IMiD)-inducible zinc finger degron systems (i.e., comprising exemplary drug-regulated stability domains of the present invention, such as Cys2-His2 zinc finger domains, e.g., the hybrid Cys2-His2 zinc finger domains (or hybrid zinc finger domains) described above) are known per se in the prior art and are described, for example, in Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572. As mentioned above, in the context of the present invention, the degron system uses a fusion of at least the docking domain of the chimeric protein of the present invention with a short zinc finger degron (which may also be referred to as a (hybrid) zinc finger domain, Cys2-His2 zinc finger domain, and / or zinc finger polypeptide) that promotes recruitment of the chimeric protein of the present invention to the IMiD / CRBN E3 ligase complex, for example, in the presence of a small molecule known as an IMiD. Generally, IMiDs bind to cereblon (CRBN), a substrate receptor for the CRL4CRBN E3 ubiquitin ligase. CRBN can recruit (fusion) proteins containing zinc-finger degrons (as drug-regulated protein stability domains) through interactions with zinc-finger degrons. This interaction is thought to be mediated by IMiDs such as thalidomide and its derivatives. Because this zinc-finger degron system is based on human protein sequences, it limits the risk of immune-mediated rejection. Furthermore, protein stability can be regulated by clinically approved small molecules such as thalidomide, pomalidomide, and lenalidomide (as examples of IMiDs), thereby facilitating the clinical development of these systems.

[0169] In preclinical studies, this protein stability control system has demonstrated its value in clinically relevant applications, such as modulating CAR-T cell activity when such zinc finger degrons are fused to CARs (Jan et al. Sci Transl Med. 2021 Jan 6;13(575):eabb6295). Furthermore, the inventors have reported the use of an IMiD / zinc finger system in the chemically regulated SH2-delivered inhibitory tail (CRASH-IT) switch platform. This switch platform, in a preferred embodiment, is also used in the present invention (referred to as a rheostat switch, e.g., RheoBrick®, which can control the cellular activity levels of various cell therapy platforms, such as CAR-T cells, TCR-T cells, and NK cells) (Sahillioglu et al., WO 2021 / 080427).

[0170] The fact that zinc-finger degrons are composed of human sequences and can be regulated using approved molecules facilitates clinical application of this system. However, clinical use of these IMiD molecules in the treatment of patients with hematological cancers is associated with significant side effects at therapeutically effective concentrations. For example, a study evaluating high-dose (25 mg daily) and low-dose (5 mg daily) lenalidomide maintenance therapy in patients with multiple myeloma showed that lenalidomide dose correlated with both toxicity and efficacy, with dose reductions due to toxicity, particularly neutropenia, occurring more frequently in the high-dose lenalidomide cohort (Fenk et al. Clin Cancer Res. 2020 Nov 15;26(22):5879-5886).

[0171] Therefore, preferably, within the context of the present invention, a drug-related protein stability domain is used that responds to lower drug doses, preferably a drug-related protein stability domain comprising a Cys2-His2 zinc finger domain capable of drug-induced binding to a CRBN polypeptide.

[0172] Thus, in preferred embodiments, the Cys2-His2 zinc finger domain is a hybrid zinc finger domain (or hybrid zinc finger degron) composed of a β-hairpin loop derived from a first Cys2-His2 zinc finger domain and an α-helical region derived from a second Cys2-His2 zinc finger domain, and preferably, the hybrid zinc finger domain comprises one, two, or more amino acid substitutions in the β-hairpin loop derived from the first Cys2-His2 zinc finger domain and / or the α-helical region derived from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain comprises two amino acid substitutions in the β-hairpin loop derived from the first Cys2-His2 zinc finger domain and / or the α-helical region derived from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain comprises three amino acid substitutions in the β-hairpin loop from the first Cys2-His2 zinc finger domain and / or the α-helical region from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain comprises four amino acid substitutions in the β-hairpin loop from the first Cys2-His2 zinc finger domain and / or the α-helical region from the second Cys2-His2 zinc finger domain. In some embodiments, the hybrid zinc finger domain is a hybrid zinc finger domain disclosed herein.

[0173] Such zinc finger degrons (as drug-regulatory stable domains) allow for the use of low concentrations of small molecules or drugs to modulate the drug-regulatory stable domains (particularly IMiDs) to control the proteolysis, expression and / or stability (and thus, e.g., cellular activity (total activity within a cell)) of the chimeric protein.

[0174] Thus, such zinc finger degrons (degron tags) of the present invention may provide better control of the proteolysis, expression, and / or stability of chimeric proteins at similar or reduced concentrations of small molecules, particularly IMiDs, and may also allow for more precise regulation of the degradation, expression, levels, and / or stability of chimeric proteins.

[0175] The drug-regulated stability domain preferably comprises a Cys2-His2 zinc finger domain, and the Cys2-His2 zinc finger domain is preferably a hybrid zinc finger domain comprising a first portion and a second portion. The first portion and the second portion each independently comprise an amino acid sequence. The first portion and the second portion of the hybrid zinc finger domain may or may not be directly adjacent to each other. In some embodiments, the first portion and the second portion are connected via a linker peptide, e.g., of 1, 2, 3, 4, 5, or more amino acids. Thus, in some embodiments, the first portion and the second portion may be connected to each other via an additional linker peptide, e.g., of 1, 2, 3, 4, 5, 6, 7, or more amino acids. However, in some embodiments, no additional linker peptide is present between the first portion of the hybrid zinc finger domain and the second portion of the hybrid zinc finger domain. In such embodiments, the first portion and the second portion are directly adjacent to each other. Preferably, said first portion and said second portion are immediately adjacent to each other.

[0176] The first portion of the hybrid zinc finger domain may be N-terminal or C-terminal to the second portion of the hybrid zinc finger domain, hi a preferred embodiment, the first portion of the hybrid zinc finger domain is N-terminal to the second portion of the hybrid zinc finger domain.

[0177] In a preferred embodiment, the first portion consists of 8 to 30 amino acids, in increasing order of priority 11 to 30, 10 to 20, 11 to 20, 10 to 14, 11 to 14, 10 to 11, and most preferably 11 amino acids.

[0178] In a preferred embodiment, the second portion consists of 8 to 30 amino acids, in increasing order of priority 12 to 30, 11 to 20, 12 to 20, 11 to 14, 12 to 14, and most preferably 12 amino acids.

[0179] In some embodiments, the non-naturally occurring hybrid zinc finger domain preferably consists of about 16-60 amino acids, preferably about 20-36 amino acids, about 20-30 amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In certain embodiments, the hybrid zinc finger domain comprises a Cys2-His2 (C2H2) domain, and the hybrid zinc finger domain comprises at least two subdomains (referred to herein as portions), each subdomain being derived from a different wild-type zinc finger. For example, the first portion of the hybrid zinc finger domain is derived from a first wild-type zinc finger, and the second portion of the hybrid zinc finger domain is derived from a second, different wild-type zinc finger domain. Based on the disclosure herein, one of skill in the art can readily select the first portion of the hybrid zinc finger domain from a first wild-type zinc finger and / or the second portion of the hybrid zinc finger domain from a second wild-type zinc finger.

[0180] For example, such moieties can be suitably selected from wild-type zinc finger domains (particularly wild-type Cys2-His2(C2H2) zinc finger domains) available from various scientific publications and publicly known gene and protein databases (e.g., Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572), which are known or predicted to destabilize proteins (e.g., wild-type proteins comprising such wild-type zinc finger domains) in the presence of an IMiD such as thalidomide, thereby controlling the degradation of such proteins.

[0181] In certain embodiments, the first portion of the hybrid zinc finger domain has the sequence X1X2C3X4X5C6X7X8X9X 10 X 11 wherein each X (X1, X2, ..., etc.) independently represents any amino acid, and more specifically, each X represents any naturally occurring or proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). Another way to represent the amino acid sequence contained in the first portion of the hybrid zinc finger domain is (X)2C(X)2C(X)5. However, because amino acid substitutions at specific positions, such as in the first portion of the hybrid zinc finger domain, are part of the present invention, the following notation is used to represent the individual amino acids and their positions: X1X2C3X4X5C6X7X8X9X. 10 X 11 It is preferred to use

[0182] Those skilled in the art will understand that, for example, X1 may represent a different amino acid than, for example, X2, or may represent the same amino acid. Those skilled in the art will understand that C3 indicates the presence of a cysteine ​​at the third position of this amino acid sequence in the first portion of the hybrid zinc finger domain. Similarly, C6 indicates the presence of a cysteine ​​at the sixth position. In a preferred embodiment, the first portion of the hybrid zinc finger domain has the amino acid sequence shown above (X1X2C3X4X5C6X7X8X9X 10 X 11 ), which amino acid sequence is that of a first Cys2-His2 zinc finger domain (e.g., a wild-type Cys2-His2 zinc finger domain). In some embodiments, the depicted amino acid sequence of the first portion of the hybrid zinc finger domain can include an additional short stretch of amino acids at its N-terminus, e.g., 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 amino acids, e.g., the first or second Cys2-His2 zinc finger domain. For example, FIG. 11 shows an example of a zinc finger degron (i.e., a drug-regulated protein stability domain) of the invention (with the positions of amino acid substitutions indicated), in which the N-terminus of the hybrid zinc finger domain is provided with a short stretch of amino acids, GERP (in this case, derived from IKZF1; in other embodiments, the extension may be derived from another wild-type zinc finger domain, e.g., GEKP). Experiments have confirmed that such short stretches can be included but do not substantially affect the results obtained with the degron (ie, drug-regulated protein stability domain).

[0183] As described in detail elsewhere herein, in a preferred embodiment, the hybrid zinc finger domain included in the drug regulatory protein stability domain of the present invention is a hybrid zinc finger comprising a Cys2-His2 (C2H2) zinc finger domain, wherein the hybrid zinc finger domain comprises at least two subdomains, i.e., a first portion and a second portion, and the amino acid sequence of the first portion is derived from a wild-type zinc finger, more specifically, a wild-type Cys2-His2 (C2H2) zinc finger.

[0184] In certain embodiments, the second portion of the hybrid zinc finger domain has the sequence X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 Each X(X 12 , X 13 , ..., etc.) independently represent any amino acid, and more specifically, each X represents any natural or proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). Another way to represent the amino acid sequence contained in the second portion of the hybrid zinc finger domain is (X)7H(X)3H. However, because amino acid substitutions at specific positions, such as in the second portion of the hybrid zinc finger domain, are part of the present invention, the individual amino acids and their positions can be represented by X. 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X21 X 22 H 23, It is also contemplated that in some embodiments, the second portion of the hybrid zinc finger domain is (X)6H(X)3H.

[0185] Those skilled in the art will recognize, for example, X 12 But for example, X 13 It will be understood that these terms may refer to different amino acids than, or may refer to the same amino acid as, H. 19 is understood to indicate the presence of a histidine at position 19 of this amino acid sequence contained in the second portion of the hybrid zinc finger domain. 23 indicates the presence of a histidine-cysteine ​​residue at position 23 of this sequence. In a preferred embodiment, the second portion of the hybrid zinc finger domain has the amino acid sequence shown above (X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 ), the amino acid sequence of the second Cys2-His2 zinc finger domain.

[0186] In some embodiments, the designated amino acid sequence of the second portion of the hybrid zinc finger domain may include an additional short stretch of amino acids at its C-terminus, e.g., 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 amino acids, e.g., the second Cys2-His2 zinc finger domain. In preferred embodiments, the first portion of the hybrid zinc finger domain is N-terminal to the second portion of the hybrid zinc finger domain. In such embodiments, the second portion is C-terminal to the first portion.

[0187] In other words, the hybrid zinc finger domain included in the zinc finger degron (i.e., drug-regulating protein stability domain) of the present invention comprises, in a preferred embodiment, a Cys2-His2 (C2H2) domain, and the hybrid zinc finger domain comprises at least two subdomains, i.e., a first portion and a second portion, and the amino acid sequence of the first portion is similar to that of a wild-type zinc finger, more specifically, a wild-type Cys2-His2 (C2H2) zinc finger (i.e., a first Cys2-His2 zinc finger domain). The amino acid sequence of the second portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2-His2 (C2H2) zinc finger (i.e., a second Cys2-His2 zinc finger domain), and preferably the second Cys2-His2 zinc finger domain is different from the first Cys2-His2 zinc finger domain (e.g., the first Cys2-His2 zinc finger domain is a wild-type zinc finger that is different from the second Cys2-His2 zinc finger domain). Preferably, the amino acid sequence of the first portion is X1X2C3X4X5C6X7X8X9X 10 X 11 Contains or X1X2C3X4X5C6X7X8X9X 10 X 11 and / or the amino acid sequence of the second portion is X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 Contains or X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23. is.

[0188] As indicated above, the first portion and the second portion together form the hybrid zinc finger domain, where the first portion is preferably a first Cys2-His2 zinc finger domain, e.g., a wild-type, naturally occurring Cys2-His2 zinc finger domain, and the second portion is preferably a second, different Cys2-His2 zinc finger domain, e.g., a different wild-type, naturally occurring Cys2-His2 zinc finger domain. In such embodiments, the hybrid zinc finger domain formed by the first portion and the second portion may be referred to as a hybrid Cys2-His2 (C2H2) zinc finger domain, e.g., a wild-type zinc finger protein and / or a non-naturally occurring Cys2-His2 (C2H2) zinc finger domain.

[0189] Typically, a C2H2 zinc finger domain, including, for example, a hybrid zinc finger domain of the present invention, comprises a β-hairpin subdomain and an α-helix subdomain. Typically, a C2H2 zinc finger domain consists of about 20 to 36 amino acids, about 20 to 30 amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. A C2H2 zinc finger domain typically comprises an N-terminal β-hairpin containing two conserved cysteine ​​residues and an α-helix containing two conserved histidine residues at its C-terminus (see, e.g., Fedotova et al., Acta Naturae, 2017 Apr-Jun; 9(2): 47-58). These motifs are also present in the zinc finger domain of the present invention.

[0190] In a preferred embodiment, the zinc finger degron (i.e., drug-regulated protein stability domain) of the present invention is further characterized by the presence of two amino acid substitutions in the hybrid zinc finger domain, wherein the amino acid substitutions in the first portion of the hybrid zinc finger domain are relative to the first portion of the first Cys2-His2 zinc finger domain, and the amino acid substitutions in the second portion of the hybrid zinc finger domain are relative to the second portion of the second Cys2-His2 zinc finger domain, and the substitutions are at positions C3, C6, X7, and H. 19 rank, or H 23 In some embodiments, X7 is not present at any of the positions 1 to 7. In some embodiments, X7 is glycine.

[0191] As discussed herein, in embodiments, the hybrid zinc finger domain is comprised of a first portion from a first Cys2-His2 zinc finger domain, particularly a first wild-type Cys2-His2 zinc finger domain, wherein the first portion comprises the amino acid sequence X1X2C3X4X5C6X7X8X9X 10 X 11 (i.e., present in the first wild-type Cys2-His2 zinc finger domain). As discussed herein, in embodiments, the hybrid zinc finger domain comprises a second portion from a second Cys2-His2 zinc finger domain, particularly a second wild-type Cys2-His2 zinc finger domain, wherein the second portion comprises the amino acid sequence X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 (i.e., present in the second wild-type Cys2-His2 zinc finger domain).

[0192] In a preferred embodiment, the Cys2-His2 zinc finger domain of the present invention is further characterized by the presence of two amino acid substitutions in the hybrid zinc finger domain, the amino acid substitutions being in the first portion of the hybrid zinc finger domain relative to the first portion of the first Cys2-His2 zinc finger domain from which the sequence was derived. For example, if X2 is valine in the first (wild-type) Cys2-His2 zinc finger domain from which the sequence was derived and this valine is substituted with lysine in the zinc finger degron (i.e., drug-regulated protein stability domain) of the present invention, then in the zinc finger degron of the present invention, X2 is lysine, and the amino acid substitution to lysine is relative to the valine contained in the wild-type Cys2-His22 zinc finger domain from which it was derived. Similarly, amino acid substitutions in the second portion of the hybrid zinc finger domain are defined herein. For example, X in the second (wild-type) Cys2-His2 zinc finger domain from which the sequence of the second portion was derived. 20 is proline, and this proline is replaced by, for example, leucine in the zinc finger degron of the present invention, then in the zinc finger degron of the present invention, X 20 becomes leucine, and the amino acid substitution to leucine is for the proline contained in the wild-type Cys2-His2 zinc finger domain from which it was derived.

[0193] As disclosed above, the hybrid zinc finger domain contains two amino acid substitutions compared to portions of the wild-type first and second Cys2-His2 zinc finger domains. One skilled in the art can provide, prepare, and recognize such amino acid substitutions, for example, by comparing the amino acid sequence of the first and / or second portions of the hybrid zinc finger domain with the wild-type first and second Cys2-His2 zinc finger domains.

[0194] In some embodiments, two amino acid substitutions (i.e., substitutions of an amino acid in the wild-type sequence with another, different amino acid) are both present in the first portion. In some embodiments, two amino acid substitutions are both present in the second portion. In other embodiments, one amino acid substitution is present in the first portion and one amino acid substitution is present in the second portion. Preferably, the hybrid zinc finger domain contains no more than two amino acid substitutions.

[0195] In a preferred embodiment, the substitutions are at the C3 position, the C6 position, the X7 position, H 19 rank, or H 23 In some embodiments, X7 is not present at any of the positions 1 to 7. In some embodiments, X7 is G7.

[0196] Surprisingly, it has been found that introducing two amino acid substitutions into the first and / or second portions of the hybrid zinc finger domain relative to the first portion of the (wild-type) first Cys2-His2 zinc finger domain and the second portion of the (wild-type) second Cys2-His2 zinc finger domain can provide a Cys2-His2 zinc finger domain with significantly enhanced or increased sensitivity to an IMiD molecule, such as thalidomide or an analog, relative to the wild-type zinc finger domain and relative to a hybrid zinc finger domain composed of wild-type subdomains (as described herein) that does not contain the two amino acid substitutions. Interestingly, it has been found that introducing a single amino acid substitution can also enhance or increase sensitivity to an IMiD molecule, but, as shown in the Examples and described herein, unexpectedly, introducing additional amino acid substitutions can further enhance or increase sensitivity to an IMiD molecule.

[0197] Thus, according to this aspect of the present invention, there is provided a hybrid Cys2-His2 zinc finger domain that contains two amino acid substitutions relative to (a part of) the wild-type zinc finger domain that constitutes the hybrid zinc finger domain, and that has enhanced or increased sensitivity to IMiD molecules.

[0198] In some embodiments, the two amino acid substitutions are located immediately adjacent to each other, and in other embodiments, the positions of the amino acid substitutions are separated from each other by at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 or more amino acids (i.e., unsubstituted amino acids), for example, as shown in the accompanying tables, figures, and examples.

[0199] While in principle any amino acid substitution is permissible, preferably, the amino acid substitution at a specific position within the hybrid zinc finger domain is as described herein (either with respect to the substituted amino acid or the substituting amino acid). Based on the information disclosed herein, those skilled in the art can provide such amino acid substitutions and the Cys2-His2 zinc finger domain of the present invention without undue burden.

[0200] Also provided are hybrid Cys2-His2 zinc finger domains, wherein the first portion is a β-hairpin loop of a first Cys2-His2 zinc finger domain and the second portion is an α-helical region of a second, different Cys2-His2 zinc finger domain. Such hybrid Cys2-His2 zinc finger domains are not naturally occurring in the cells of the invention, and preferably are not naturally occurring.

[0201] Zinc finger domains contain a β-hairpin loop and an α-helical region, and those skilled in the art will know how to provide the β-hairpin loop and / or α-helical region portions of a (wild-type) Cys2-His2 zinc finger domain. Representative examples are well known to those skilled in the art and include, for example, those disclosed and described herein. Examples of zinc fingers containing a β-hairpin loop and an α-helical region include, but are not limited to, (human) IKZF1, IKZF2, IKZF3, SALL4, ZFP91, GZF1, ZNF653, ZNF692, ZNF827, ZBTB39, WIZ, ZNF98, ZNF654, ZNF787, ZNF 276, ZNF582, ZNF517, and E4F1 (see also Uniprot accession numbers: Q13422, Q9UKS7, Q9UKT9, Q9UJQ4, Q96JP5, Q9H116, Q96CK0, Q9BU19, Q17R98, O15060, O95785, A6NK75, Q8IZM8, Q6DD87, Q8N554, Q96NG8, Q6ZMY9, Q66K89).

[0202] As disclosed above, zinc finger degrons and / or hybrid zinc finger polypeptides of the invention can include one or more amino acid residues N-terminal to the β-hairpin portion, one or more amino acid residues between the β-hairpin portion and the α-helix portion, and one or more amino acid residues C-terminal to the α-helix portion, provided that the zinc finger degrons and / or hybrid zinc finger polypeptides of the invention are substrates for the CRBN-IMiD complex and / or have enhanced or increased sensitivity to an IMiD molecule (e.g., compared to wild-type). These additional amino acids may correspond to residues within a native Cys2-His2 zinc finger domain or may be different, so long as the zinc finger degron maintains a zinc finger-like fold and exhibits the properties disclosed herein.

[0203] In embodiments of the present invention, zinc finger degrons and / or hybrid zinc finger polypeptides of the present invention are provided, in which a first substitution is in the second portion and a second substitution is in either the first portion or the second portion. While two amino acid substitutions can both be in the first portion, or both in the second portion, or one in the first portion and one in the second portion, in preferred embodiments, at least one amino acid substitution is in the second portion. Particularly suitable zinc finger degrons of the present invention are provided, in which at least one of the amino acid substitutions is found in the second portion, i.e., the α-helical portion of the hybrid zinc finger domain. The second amino acid substitution can be in the second portion or in the first portion (i.e., the β-hairpin loop).

[0204] Also provided are zinc finger degrons and / or hybrid zinc finger polypeptides of the invention, wherein at least one substitution is selected from the group consisting of X, X, X, 12 , X 13 , X 14 , X 15 , X 17 , X 21 , and X 22. and (iii) at a position selected from the group consisting of: (i) at one of these positions within the hybrid zinc finger domain; and (ii) at one of these positions within the hybrid zinc finger domain; and (iii) at one of these positions within the hybrid zinc finger domain; and (iv) at one of these positions within the hybrid zinc finger domain; and (v) at one of these positions within the hybrid zinc finger domain; and (vi) at one of these positions within the hybrid zinc finger domain; and (vi) at one of these positions within the hybrid zinc finger domain; and (vi) at one of these positions within the hybrid zinc finger domain; and

[0205] In a preferred embodiment, a zinc finger degron and / or hybrid zinc finger polypeptide of the present invention is provided, wherein at least one of the substitutions present in the zinc finger degron and / or hybrid zinc finger polypeptide of the present invention (or present in the hybrid zinc finger domain) is selected from those listed in Table 1. Table 1 lists various positions X1 through X2. 22 Table 1 lists the positions where substitutions may occur in the zinc finger degrons and / or hybrid zinc finger polypeptides of the present invention, and in preferred embodiments, the positions where substitutions may occur in the zinc finger degrons and / or hybrid zinc finger polypeptides of the present invention are shown along with the substituting amino acid (i.e., the amino acid contained at that position in the zinc finger degron and / or hybrid zinc finger polypeptide of the present invention, in one-letter code). Preferably, both amino acid substitutions (both the positional amino acid and the substituting amino acid) are selected from those listed in Table 1. Those skilled in the art will understand that when both amino acid substitutions are selected from those listed in Table 1 (or other tabular listings presented herein), each amino acid substitution is at a different position. At the same time, those skilled in the art will understand that this means that the substituting amino acid represents an amino acid that is different from a natural amino acid (i.e., different from an amino acid present in the wild-type Cys2-His2 zinc finger domain used in the first or second portion of the hybrid zinc finger domain). In that regard, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, second portion (and / or the third and fourth portions discussed herein).

[0206] Similarly, the present invention encompasses the use of the positions and substitutions listed in Table 1 (or any other tabular listing provided herein) in any suitable first or second portion of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a hybrid zinc finger domain of the present invention. In other words, the positions and substituting amino acids listed in Table 1 (or any other tabular listing provided herein) are applicable to any suitable hybrid zinc finger domain, so long as the zinc finger degron and / or hybrid zinc finger polypeptide of the present invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., susceptibility to IMiDs in terms of proteolysis).

[0207] However, in a preferred embodiment, the positions and substitutions are relative to the hybrid zinc finger domain used in the Examples herein, i.e., the first portion of the hybrid zinc finger domain is the β-hairpin region of ZFP91 ZF4 and the second portion of the hybrid zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11, which shows a schematic representation of the hybrid zinc finger domain of the "parent zinc finger degron" used in the Examples).

[0208] [Table 1]

[0209] In a preferred embodiment, a zinc finger degron and / or hybrid zinc finger polypeptide according to the present invention is provided, in which two substitutions are selected from those listed in Table 2. Table 2 lists preferred combinations of two amino acid substitutions at two different positions in the hybrid zinc finger domains described herein (each row shows a preferred combination of two positions in the hybrid zinc finger domain and the corresponding substituting amino acid). For example, 4R and 12L indicate that in this combination, the amino acid at position 4 of the hybrid zinc finger domain is substituted with arginine (R) and the amino acid at position 12 of the hybrid zinc finger domain is substituted with leucine (L). In other words, the original amino acids of the portion derived from a wild-type Cys2-His2 zinc finger domain are substituted with arginine at position 4 and leucine at position 12. In a preferred embodiment of the present invention, the two substitutions are selected from any of the combinations shown in Table 2.

[0210] [Table 2]

[0211] Those skilled in the art will understand that substituting amino acids is meant to refer to amino acids that are different from the natural amino acids (i.e., different from the amino acids present in the wild-type Cys2-His2 zinc finger domain used in the first or second portions of the hybrid zinc finger domain). In that regard, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, second portion (and / or the third and fourth portions discussed herein).

[0212] Similarly, the present invention encompasses the use of the positions and substitutions listed in Table 2 (or any other tabular listing provided herein) in any suitable first or second portion of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a hybrid zinc finger domain of the present invention. In other words, the positions and substituting amino acids listed in Table 2 (or any other tabular listing provided herein) are applicable to any suitable hybrid zinc finger domain, so long as the zinc finger degron and / or hybrid zinc finger polypeptide of the present invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., susceptibility to IMiDs in terms of proteolysis).

[0213] However, in a preferred embodiment, the positions and substitutions are relative to the hybrid zinc finger domain used in the Examples herein, i.e., the first portion of the hybrid zinc finger domain is the β-hairpin region of ZFP91ZF4 and the second portion of the hybrid zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11, which shows a schematic representation of the hybrid zinc finger domain of the "parent zinc finger degron" used in the Examples).

[0214] Furthermore, the frequently observed primary amino acid substitutions Q12R, Q12K, N15R, and L22R in the Examples, when combined with the secondary amino acid substitutions listed in Table 8 (see below), synergistically further enhanced susceptibility to IMiDs (i.e., EI Q12R / K13V=0.006877267 while EI Q12R=0.167217557). Thus, particularly preferred embodiments of the invention use / provide combinations of amino acid substitutions listed in Table 8, such as 12R and 13T, or 22R and 14L.

[0215] In a preferred embodiment, a zinc finger degron and / or hybrid zinc finger polypeptide according to the invention is provided, wherein the substituted amino acid in the first portion and / or the substituted amino acid in the second portion are selected from those listed in Table 3.

[0216] Table 3 lists preferred amino acids to be substituted in the hybrid zinc finger domain of a zinc finger degron and / or hybrid zinc finger polypeptide of the present invention. In other words, in a preferred embodiment of the present invention, the positions and / or positions and substituted amino acids are selected from those listed in Table 3. Thus, in a preferred embodiment of the present invention, two amino acid substitutions are selected from combinations of positions 1, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, and 22, and the substituted amino acids are combinations of two amino acids listed in Table 3.

[0217] [Table 3]

[0218] Preferably, both the substituted amino acids (both the positional amino acid and the substituting amino acid) are selected from those listed in Table 3. As will be appreciated by one of skill in the art, amino acids, e.g., the preferred amino acids shown in Table 3, may be substituted with any suitable amino acid, so long as it provides a zinc finger degron and / or hybrid zinc finger polypeptide of the present invention. However, in preferred embodiments, amino acids, preferably combinations of amino acids listed in Table 3, are replaced with corresponding amino acids listed in Table 1 or corresponding combinations of amino acids shown in Table 2 (e.g., corresponding refers to corresponding positions within the hybrid zinc finger domains disclosed herein).

[0219] Those skilled in the art will understand that when both substituted amino acids are selected from those listed in Table 3 (or other tabular listings provided herein), each amino acid substitution will be at a different position. At the same time, those skilled in the art will understand that a substituted amino acid is intended to refer to an amino acid that is substituted with another amino acid and is different from the natural amino acid (i.e., different from that present in the wild-type Cys2-His2 zinc finger domain used in the first or second portion of the hybrid zinc finger domain). In that regard, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, second portion (and / or the third and fourth portions discussed herein).

[0220] Similarly, the present invention encompasses the use of the positions and substitutions listed in Table 3 (or any other tabular listing provided herein) in any suitable first or second portion of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a hybrid zinc finger domain of the present invention. In other words, the positions and substituted amino acids listed in Table 3 (or any other tabular listing provided herein) are applicable to any suitable hybrid zinc finger domain, so long as the zinc finger degron and / or hybrid zinc finger polypeptide of the present invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., susceptibility to IMiDs in terms of proteolysis).

[0221] However, in a preferred embodiment, the positions and substitutions are relative to the hybrid zinc finger domain used in the Examples herein, i.e., the first portion of the hybrid zinc finger domain is the β-hairpin region of ZFP91 ZF4 and the second portion of the hybrid zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11, which shows a schematic representation of the hybrid zinc finger domain of the "parent zinc finger degron" used in the Examples).

[0222] In a preferred embodiment, a zinc finger degron and / or hybrid zinc finger polypeptide according to the invention is provided, wherein the two amino acids of the first portion and / or the second portion that are substituted are selected from those listed in Table 4.

[0223] Table 4 lists preferred combinations of two amino acids to be substituted at two different positions in the hybrid zinc finger domains described herein according to the present invention (each row represents a preferred combination of two positions in the hybrid zinc finger domain and the corresponding substituting amino acid). For example, E4 and Q12 indicate that in this combination, the substituted amino acid at position 4 of the hybrid zinc finger domain is glutamic acid (E) and the substituted amino acid at position 12 is glutamine (Q). In other words, the original amino acids of the portion derived from the wild-type Cys2-His2 zinc finger domain are glutamic acid at position 4 and glutamine at position 12. In a preferred embodiment of the present invention, the two substituted amino acids are selected from any of the combinations shown in Table 4. As will be understood by those skilled in the art, amino acids, such as the preferred amino acids shown in Table 4, may be substituted with any suitable amino acid, as long as the substitution provides a zinc finger degron and / or hybrid zinc finger polypeptide according to the present invention. However, in preferred embodiments, amino acids, preferably combinations of amino acids listed in Table 4, are replaced with the corresponding amino acids listed in Table 1 or the corresponding combinations of amino acids shown in Table 2 (where corresponding refers, e.g., to corresponding positions within the hybrid zinc finger domains disclosed herein).

[0224] [Table 4]

[0225] Those skilled in the art will understand that a substituting amino acid is meant to refer to an amino acid that is different from the naturally occurring amino acid being substituted (i.e., different from the amino acid present in the wild-type Cys2-His2 zinc finger domain used in the first or second portion of the hybrid zinc finger domain). In that regard, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, second portion (and / or the third and fourth portions discussed herein).

[0226] Similarly, the present invention encompasses the use of the positions and substitutions listed in Table 4 (or any other tabular listing provided herein) in any suitable first or second portion of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a hybrid zinc finger domain of the present invention. In other words, the positions and substituted amino acids listed in Table 4 (or any other tabular listing provided herein) are applicable to any suitable hybrid zinc finger domain, so long as the zinc finger degron and / or hybrid zinc finger polypeptide of the present invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., susceptibility to IMiDs in terms of proteolysis).

[0227] However, in a preferred embodiment, the positions and amino acids substituted are relative to the hybrid zinc finger domain used in the Examples herein, i.e., the first portion of the hybrid zinc finger domain is the β-hairpin region of ZFP91 ZF4 and the second portion of the hybrid zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11, which shows a schematic representation of the hybrid zinc finger domain of the "parent zinc finger degron" used in the Examples).

[0228] In a further embodiment, there is provided a zinc finger degron and / or hybrid zinc finger polypeptide according to the invention, wherein the first Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF2, IKZF3 ZF2, ZFP91 ZF4, ZNF654 ZF1, ZNF787 ZF5, ZNF653 ZF4, ZNF276 ZF4, ZNF692 ZF4, ZNF582 ZF9, ZNF517 ZF10, E4F1 ZF2, and ZNF827 ZF1, preferably ZFP91 ZF4, and / or the second Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF2, IKZF3 ZF2, ZFP91 ZF4, ZNF654 ZF1, ZNF787 ZF5, ZNF653 ZF4, ZNF276 ZF4, ZNF692 ZF4, ZNF582 ZF9, ZNF517 ZF10, E4F1 ZF2, and ZNF827 ZF1, preferably ZFP91 ZF4. ZF4, ZNF692 ZF4, ZNF582 ZF9, ZNF517 ZF10, E4F1 ZF2, and ZNF827 ZF1, preferably IKZF1 ZF2.

[0229] In other words, the amino acid sequence of the Cys2-His2 zinc finger domain for selection of the first portion and / or the second portion (see below) may, in some embodiments, be any of SEQ ID NO:116 (IKZF1 ZF2 - FQCNQCGASFTQKGNLLRHIKLH), SEQ ID NO:117 (IKZF3 ZF2 - FQCNQCGASFTQKGNLLRHIKLH), SEQ ID NO:118 (ZFP91 ZF4 - LQCEICGFTCRQKASLNWHMKKH), SEQ ID NO:119 (ZNF654 ZF1 - FACVICGRKFRNRGLMQKHLKNH), SEQ ID NO:120 (ZNF787 ZF5 - FVCPRCGRGFSQPKSLARHLRLH), SEQ ID NO:121 (ZNF653 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:122 (ZNF276 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:123 (ZNF276 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:124 (ZNF276 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:125 (ZNF276 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:126 (ZNF276 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:127 (ZNF276 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:128 (ZNF276 ZF4 - LQCEICGYQCRQRASLNWHMKKH), SEQ ID NO:129 (ZNF276 ZF4 - LQCEVCGFQCRQRASLKYHMTKH), SEQ ID NO:123 (ZNF692 ZF4 - LQCEICGFTCRQKASLNWHQRKH), SEQ ID NO:124 (ZNF582 ZF9 - YQCKVCGRAFKRVSHLTVHYRIH), SEQ ID NO:125 (ZNF517 ZF10 - YRCRACGRACSRLSTLIQHQKVH), SEQ ID NO:126 (E4F1 ZF2 - HECKLCGASFRTKGSLIRHHRRH), and / or SEQ ID NO:127 (ZNF827 ZF1 - FQCPICGLVIKRKSYWKRHMVIH).

[0230] In a highly preferred embodiment, the β-hairpin loop (or first portion) of the hybrid zinc finger domain is derived from ZFP91 ZF4 and the α-helix (or second portion) of the hybrid zinc finger is derived from IKZF1 ZF2. In an even more highly preferred embodiment, the β-hairpin loop (or third portion) of the further hybrid zinc finger domain is derived from IKZF1 ZF3 and the α-helix (or fourth portion) of the further hybrid zinc finger is derived from ZFP91 ZF5.

[0231] As briefly mentioned above, one embodiment of the present invention also provides a zinc finger degron of the present invention, in which two amino acid substitutions are introduced into the hybrid zinc finger domain. The first portion of the hybrid zinc finger domain has the amino acid sequence of SEQ ID NO: 101 (LQCEICGFTCR - ZFP91 ZF4 (first portion)), and / or the second portion has the amino acid sequence of SEQ ID NO: 102 (QKGNLLRHIKLH - IKZF1 ZF2 (second portion)), and / or the hybrid zinc finger domain has the amino acid sequence of SEQ ID NO: 103 (LQCEICGFTCRQKGNLLRHIKLH - (ZFP91 ZF4 / IKZF1 ZF2 hybrid zinc finger domain)). In a preferred embodiment, the two amino acid substitutions of the present invention can be introduced into these sequences.

[0232] In a further preferred embodiment, a zinc finger degron according to the present invention is provided having two amino acid substitutions introduced therein, the zinc finger degron having the amino acid sequence of SEQ ID NO: 107 (LQCEICGFTCRQKGNLLRHIKLHSGEKPFKCHLCNYACRRKDSVVAHKAKSH). In a preferred embodiment, the two amino acid substitutions according to the present invention can be introduced into these sequences.

[0233] In a preferred embodiment, a zinc finger degron and / or hybrid zinc finger polypeptide according to the present invention is provided, wherein the hybrid zinc finger domain comprising two substitutions is selected from those listed in Table 5. Table 5 lists preferred hybrid zinc finger domains (or hybrid zinc finger polypeptides) according to the present invention. These preferred zinc finger domains exhibited the most beneficial EI index (enrichment index) when calculated according to the Examples section. The results are summarized in Table 6.

[0234] [Table 5]

[0235] [Table 6-1] [Table 6-2]

[0236] Indeed, it has been surprisingly found that two substitutions within the hybrid zinc finger domains described herein provide zinc finger degrons of the present invention with enhanced or increased IMiD sensitivity, particularly compared to those containing only a single substitution within the hybrid zinc finger domain. For example, when the hybrid zinc finger domain used in the Examples contained only one mutation (substitution L1Y, providing the sequence YQCEICGFTCRQKGNLLRHIKLH (SEQ ID NO: 108)), the corresponding EI value was at least 10-15 times higher than the EI value determined for SEQ ID NO: 36 (and at least 5 times higher than SEQ ID NO: 100) (indicating a zinc finger degron and / or hybrid zinc finger polypeptide with reduced IMiD sensitivity). Thus, despite the introduction of a second substitution into the hybrid zinc finger domain, the sensitivity of the resulting hybrid zinc finger domain (with two substitutions) is surprisingly and dramatically improved. Exemplary EI values ​​obtained with various single mutations in the hybrid single domains used in the Examples herein are shown in Table 7.

[0237] [Table 7]

[0238] In preferred embodiments, zinc finger degrons of the present invention comprise a hybrid zinc finger domain comprising any of the sequences listed in Table 5. However, it is contemplated that the hybrid zinc finger domain of a zinc finger degron of the present invention may comprise either a β-hairpin of a hybrid zinc finger domain listed in Table 5 or an α-helix of a hybrid zinc finger domain listed in Table 5, and further comprise different α-helix and β-hairpin portions. For example, in preferred embodiments, the hybrid zinc finger domain will be comprised of a combination of a β-hairpin portion of any of the sequences listed in Table 5 and an α-helix portion of any of the sequences listed in Table 5. In some embodiments, the hybrid zinc finger domain will be comprised of a β-hairpin portion of any of the sequences listed in Table 5 in combination with an additional α-helix portion not necessarily listed in Table 5. In some embodiments, the hybrid zinc finger domain is comprised of a β-hairpin portion of any one of the sequences listed in Table 5 in combination with an additional α-helical portion not necessarily listed in Table 5.

[0239] In some embodiments, the hybrid zinc finger domain of a zinc finger degron and / or zinc finger polypeptide of the present invention is any of those listed in Table 5, and preferably any one of SEQ ID NOs: 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, or 1-10. In other embodiments, the hybrid zinc finger domain of a zinc finger degron and / or zinc finger polypeptide of the present invention is any one of SEQ ID NOs: 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, or 91-100. In a further embodiment, the hybrid zinc finger domain shown in FIG. 11 (ZFP91 ZF4-IKZF1 ZF2) is substituted for any one of the sequences listed in Table 5.

[0240] Another aspect of the invention is a method for providing a zinc finger degron of the invention, a non-natural hybrid zinc finger polypeptide of the invention, a fusion protein of the invention, or a nucleic acid encoding the same, comprising the steps of: (A) providing a hybrid zinc finger domain comprising a first portion and a second portion, (1) The first portion has the amino acid sequence X1X2C3X4X5C6X7X8X9X of a first Cys2-His2 zinc finger domain. 10 X 11 wherein X represents any amino acid, (2) the second portion comprises the amino acid sequence X of a second Cys2-His22 zinc finger domain; 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23wherein X represents any amino acid, and the second Cys2-His2 zinc finger domain is different from the first Cys2-His2 zinc finger domain; (3) the second portion is C-terminal to the first portion; (B) introducing two different amino acid substitutions into the hybrid zinc finger domain, the substitutions being C3, C6, X7, H 19 , or H 23 and (C) preparing a zinc finger degron of the present invention, a non-natural hybrid zinc finger polypeptide of the present invention, a fusion protein of the present invention, or a nucleic acid encoding any of them, using the hybrid zinc finger domain obtained in step (B) or a nucleic acid sequence encoding the hybrid zinc finger domain obtained in step (B).

[0241] Those skilled in the art are well aware of methods that allow for the preparation of zinc finger degrons of the invention, non-natural hybrid zinc finger polypeptides of the invention, fusion proteins of the invention, or nucleic acids encoding them, and such methods are readily available in the prior art and include, for example, those described in the Examples.

[0242] Those skilled in the art will understand that the same considerations, features, and preferences described elsewhere herein apply with respect to the first portion, the second portion, and the first and second Cys2-His2 zinc finger domains.

[0243] Those skilled in the art will also understand that the two different amino acid substitutions introduced in the methods of the present invention can be at any position within a hybrid zinc finger domain comprising a first portion and a second portion, and that the substitution can be for any amino acid (as long as the substituted amino acid is different from the amino acid it replaces). Those skilled in the art will also understand that in preferred embodiments, the positions, substituted amino acids, and / or substituting amino acids are those described in connection with any one of Tables 1-6 and 8 (including combinations of two substitutions and the indicated priorities).

[0244] The method may include testing the hybrid zinc finger domain comprising the two substitutions, and / or the zinc finger degron of the invention, the non-natural hybrid zinc finger polypeptide of the invention, the fusion protein of the invention, or nucleic acids encoding same, for susceptibility to an IMiD obtained using a method of the invention, e.g., as described in the Examples, e.g., by determining an EI value or index as described herein.

[0245] Based on the EI value or index, it may be determined whether the resulting hybrid zinc finger domain containing the two substitutions, and / or zinc finger degron of the invention, non-natural hybrid zinc finger polypeptide of the invention, fusion protein of the invention, or nucleic acid encoding the same, should be discarded. Thus, in a preferred embodiment, the method comprises analyzing the resulting hybrid zinc finger domain containing the two substitutions, and / or zinc finger degron of the invention, non-natural hybrid zinc finger polypeptide of the invention, fusion protein of the invention, or nucleic acid encoding the same, for sensitivity to an IMiD, e.g., as described in the Examples section, to determine its usefulness as a zinc finger degron and the like.

[0246] Accordingly, embodiments are provided for determining the susceptibility of a zinc finger degron of the invention, a non-natural hybrid zinc finger polypeptide of the invention, or a fusion protein of the invention (e.g., obtained using a method of the invention) to immunomodulatory imide drug (IMiD)-induced degradation, preferably wherein the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iverdomide, salts and analogs thereof.

[0247] The foregoing description of specific embodiments makes the basic nature of the present invention fully clear, and those skilled in the art can, by applying knowledge within the skill of the art (including the contents of the references cited herein), without undue experimentation, readily modify and / or adapt such specific embodiments to various applications without departing from the basic concepts of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0248] According to some embodiments, there is also provided a drug regulatory protein stability domain, preferably the drug regulatory protein stability domain comprises a Cys2-His2 zinc finger domain, preferably said Cys2-His2 zinc finger domain is a hybrid zinc finger domain, e.g., a Cys2-His2 zinc finger domain and / or a hybrid zinc finger domain as disclosed herein, further comprising a second Cys2-His2 zinc finger domain, preferably a further hybrid zinc finger domain.

[0249] With regard to the further or second zinc finger domain (Cys2-His2 zinc finger domain), it has surprisingly been found that preferably, the second zinc finger domain included in the zinc finger degron (drug-regulated protein stability domain) of the present invention is also a hybrid zinc finger domain, similar to the above description for the (first) hybrid zinc finger domain (Cys2-His2 zinc finger domain). The second zinc finger domain may be the same as or different from the first hybrid zinc finger domain. Preferably, the second hybrid zinc finger domain is different from the first hybrid zinc finger domain.

[0250] With respect to the second zinc finger domain (second Cys2-His2 zinc finger domain), the second hybrid zinc finger domain, and / or the third or fourth portions described herein, in some embodiments, they themselves (i.e., in the absence of the first zinc finger degron (Cys2-His2 zinc finger domain)) may or may not be sensitive to an IMiD, i.e., may or may not destabilize proteins in the presence of an IMiD, such as thalidomide, thereby controlling the degradation of such proteins. Preferably, they are not sensitive to an IMiD themselves.

[0251] In addition to the Cys2-His2 zinc finger domain, the drug-regulated stability domain can also include a second naturally occurring or non-naturally occurring zinc finger domain, e.g., a hybrid zinc finger domain (Cys2-His2 zinc finger domain). In the zinc finger degrons of the present invention, the second zinc finger domain is located C-terminal to the first (hybrid or non-hybrid) zinc finger domain. In such embodiments, the first (hybrid or non-hybrid) zinc finger domain of the zinc finger degron of the present invention is N-terminal to the second (hybrid or non-hybrid) zinc finger domain.

[0252] In some embodiments, the first zinc finger domain and the second zinc finger domain are directly adjacent to each other, and no peptide linker is present. In some embodiments, the first (hybrid or non-hybrid) zinc finger domain and the second (hybrid or non-hybrid) zinc finger domain are not directly adjacent to each other, but are connected to each other via a short peptide linker. In embodiments in which the first (hybrid or non-hybrid) zinc finger domain and the second (hybrid or non-hybrid) zinc finger domain are connected via a linker peptide, such linker peptide can be composed of, for example, 1, 2, 3, 4, 5, 6, 7, or more amino acids.

[0253] For example, in some embodiments, the linker may be a short stretch of amino acids derived from a wild-type zinc finger domain, e.g., the N-terminal portion or a portion adjacent to the N-terminal portion of the wild-type zinc finger domain. Such an amino acid sequence may be, for example, the SGEKP sequence (see FIG. 11 ) (in this case, derived from the IKZF1 protein, the SGEKP sequence being the endogenous linker between IKZF1 ZF2 and IKZF1 ZF3 within the IKZF1 protein; similarly, other endogenous linkers, e.g., equivalent endogenous linkers from other C2H2 zinc finger proteins, may be used as linkers within the context of the present invention; in some other embodiments, the extension may be derived from another wild-type zinc finger domain, e.g., GERP). Experiments have confirmed that such short stretches may be included without substantially affecting the results obtained with the zinc finger degron, and that those skilled in the art are fully capable of providing additional linkers suitable within the context of the present invention.

[0254] With respect to the second (hybrid or non-hybrid) zinc finger domain, it has been found that the inclusion of such a second (hybrid or non-hybrid) zinc finger domain can further enhance the sensitivity of the zinc finger degron of the present invention in regulating protein degradation, protein levels, and / or cellular activity. Thus, according to this aspect of the present invention, there is provided a zinc finger degron comprising the first and second (hybrid or non-hybrid) zinc finger domains detailed herein. According to a preferred embodiment, the present invention provides a zinc finger degron comprising the first and second zinc finger domains (Cys2-His2 zinc finger domains) detailed herein.

[0255] The second zinc finger domain comprises a third portion and a fourth portion. The third portion and the fourth portion each independently comprise an amino acid sequence. The third portion and the fourth portion of the second zinc finger domain may or may not be directly adjacent to each other. In some embodiments, the third portion and the fourth portion are connected via a linker peptide, e.g., of 1, 2, 3, 4, 5, or more amino acids. Thus, in some embodiments, the third portion and the fourth portion may be connected to each other via an additional linker peptide, e.g., of 1, 2, 3, 4, 5, 6, 7, or more amino acids. However, in some embodiments, no additional linker peptide is present between the third portion of the second zinc finger domain and the fourth portion of the second zinc finger domain. In such embodiments, the third portion and the fourth portion are directly adjacent to each other. Preferably, the third portion and the fourth portion are directly adjacent to each other.

[0256] The third portion of the second zinc finger domain may be N-terminal or C-terminal to the fourth portion of the second zinc finger domain, hi a preferred embodiment, the third portion is N-terminal to the fourth portion.

[0257] In a preferred embodiment, the third portion consists of 8 to 30 amino acids, and in increasing order of priority is 11 to 30, 10 to 20, 11 to 20, 10 to 14, 11 to 14, 10 to 11, and most preferably 11 amino acids.

[0258] In a preferred embodiment, the fourth portion consists of 8 to 30 amino acids, in increasing order of priority 12 to 30, 11 to 20, 12 to 20, 11 to 14, 12 to 14, and most preferably 12 amino acids.

[0259] In some embodiments, the non-naturally occurring second zinc finger domain preferably consists of about 16-60 amino acids, preferably about 20-36 amino acids, about 20-30 amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In certain embodiments, the second zinc finger domain comprises a Cys2-His2 (C2H2) domain, and the hybrid zinc finger domain comprises at least two subdomains (referred to herein as portions), each subdomain being derived from a different wild-type zinc finger. For example, a third portion of the second zinc finger domain is derived from a third wild-type zinc finger, and a fourth portion of the second zinc finger domain is derived from a fourth, different, wild-type zinc finger domain. Based on the disclosure herein, one skilled in the art can easily select the third portion of the second zinc finger domain from a third wild-type zinc finger and / or the fourth portion of the second zinc finger domain from a fourth wild-type zinc finger. For example, such portions can be appropriately selected from wild-type zinc finger domains (particularly wild-type Cys2-His2(C2H2) zinc finger domains) available from various scientific publications and publicly known gene and protein databases (e.g., Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572), where such portions are known or predicted to destabilize proteins (e.g., wild-type proteins comprising such wild-type zinc finger domains) in the presence of an IMiD such as thalidomide, thereby controlling the degradation of such proteins.

[0260] In certain embodiments, the third portion of the second zinc finger domain has the sequence (Z)C(Z)C(Z) 5-6 In the formula, Z represents any amino acid. Therefore, (Z)2 represents two amino acids, but (Z) 5-6represents 5 or 6 amino acids. In one example, the third portion of the second zinc finger domain may have the amino acid sequence Z1Z2C3Z4Z5C6Z7Z8Z9Z 10 Z 11 or Z1Z2C3Z4Z5C6Z7Z8Z9Z 10 Z 11 Z 12 wherein each Z (Z1, Z2, etc.) independently represents any amino acid; more specifically, each Z represents any natural or proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). Those skilled in the art will appreciate that, for example, Z1 may represent a different amino acid than, or the same amino acid as, for example, Z2. Those skilled in the art will appreciate that C3 indicates the presence of a cysteine ​​at the third position of this amino acid sequence in the third portion of the second zinc finger domain. Similarly, C6 indicates the presence of a cysteine ​​at the sixth position. In a preferred embodiment, the third portion of the second zinc finger domain has the amino acid sequence (Z)C(Z)C(Z). 5-6, and even more preferably (Z)2C(Z)2C(Z)6, ​​the amino acid sequence of a third Cys2-His2 zinc finger domain (e.g., a wild-type Cys2-His2 zinc finger domain). In some embodiments, the indicated amino acid sequence of the third portion of the second zinc finger domain can include an additional short stretch of amino acids at its N-terminus, e.g., 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 amino acids, such as those of the third or fourth (wild-type) Cys2-His2 zinc finger domain. For example, FIG. 11 shows an example of a zinc finger degron according to the invention (positions of amino acid substitutions indicated), in which the N-terminus of the second zinc finger domain contains a short stretch of amino acids, SGEKP, as described above. Experiments have confirmed that such a short stretch can be included but does not substantially affect the results obtained with the zinc finger degron.

[0261] As described in detail herein and corresponding to the first hybrid zinc finger domain comprised in the zinc finger degron of the present invention, the second zinc finger domain is also, in a preferred embodiment, a hybrid zinc finger comprising a Cys2-His2 (C2H2) zinc finger domain, which comprises at least two subdomains, i.e., a third portion and a fourth portion, and the amino acid sequence of the third portion is derived from a wild-type zinc finger, more specifically, a wild-type Cys2-His2 (C2H2) zinc finger.

[0262] In certain embodiments, the fourth portion of the second zinc finger domain has the sequence (Z)H(Z) 3-4 H. Thus, (Z)6 represents six amino acids, while (Z) 3-4 represents three or four amino acids. For example, the fourth portion of the second zinc finger domain may have, for example, the amino acid sequence Z 13 Z 14 Z 15 Z 16 Z17 Z 18 H 19 Z 20 Z 21 Z 22 H 23 or Z 13 Z 14 Z 15 Z 16 Z 17 Z 18 H 19 Z 20 Z 21 Z 22 Z 23 H 24 Each Z(Z 13 , Z 14 ...etc.) independently represent any amino acid, and more specifically, each Z represents any natural or proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). Those skilled in the art will appreciate that, for example, Z 13 But, for example, Z 14 It will be understood that these terms may refer to different amino acids than, or may refer to the same amino acid as, H. 19 is understood to indicate the presence of a histidine at position 19 of this amino acid sequence contained in the second portion of the first hybrid zinc finger domain. 23 or H 24 indicates the presence of histidine-cysteine ​​at position 23 of this sequence or at position 24 of this sequence ((Z) 3-4 (These differ depending on whether the amino acid sequence is related to three or four amino acids.) For example, Z 13Note that the position of the (Z)6H(Z) does not indicate a position relative to the first hybrid zinc finger domain. This is because, for example, it is explained elsewhere herein that in some embodiments, a linker may be present between the first hybrid zinc finger domain and the second (hybrid) zinc finger domain. It is also explained herein that the length of the third portion of the second (hybrid) zinc finger may vary, for example, be 11 amino acids long. In a preferred embodiment, the fourth portion of the second zinc finger domain has the amino acid sequence (Z)6H(Z) 3-4 H, the amino acid sequence of a second Cys2-His2 zinc finger domain, ie, more specifically, that of a wild-type Cys2-His2 (C2H2) zinc finger.

[0263] In other words, in a preferred embodiment, the second zinc finger domain included in the zinc finger degron of the present invention comprises a Cys2-His2 (C2H2) domain, and the hybrid zinc finger domain comprises at least two subdomains, i.e., a third portion and a fourth portion, and the amino acid sequence of the third portion is derived from a wild-type zinc finger, more specifically, a wild-type Cys2His2 (C2H2) zinc finger (i.e., a third Cys2-His2 zinc finger domain), and The amino acid sequence of the fourth portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2-His2(C2H2) zinc finger (i.e., a fourth Cys2-His2 zinc finger domain), and preferably the third Cys2-His2 zinc finger domain is different from the fourth Cys2-His2 zinc finger domain (e.g., the third Cys2-His2 zinc finger domain is a wild-type zinc finger that is different from the fourth Cys2-His2 zinc finger domain). Preferably, the amino acid sequence of the third portion is (Z)2C(Z)2C(Z) 5-6 or (Z)2C(Z)2C(Z) 5-6and / or the amino acid sequence of the fourth portion is (Z)H(Z) 3-4 Contains H or (Z)6H(Z) 3-4 It's H.

[0264] In a preferred embodiment, the fourth portion is C-terminal to the third portion.

[0265] As will be appreciated by those skilled in the art, in addition to the domains, moieties, and linkers described above, zinc finger degrons of the present invention may include additional amino acids, for example, at the N-terminus of the first hybrid zinc finger domain or the C-terminus of the second zinc finger domain.

[0266] In a further embodiment, there is provided a zinc finger degron and / or hybrid zinc finger polypeptide according to the invention, wherein the third Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, and ZNF692 ZF5, preferably IKZF1 ZF3, and / or the fourth Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, and ZNF692 ZF5, preferably ZFP91 ZF5.

[0267] The present invention is not particularly limited to the specific (wild-type) Cys2-His2 zinc finger domain that provides the first, second, third, and / or fourth portions of the first hybrid zinc finger domain and the second zinc finger domain, respectively. Various wild-type or naturally occurring Cys2-His2 zinc finger domains have been described in the art, which are suitable for degron systems, including the use of IMiDs (see, for example, those described in (Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572)). Those skilled in the art will understand that such Cys2-His2 zinc finger domains, including typical β-hairpin loops and α-helices, can be suitably used in the present invention, for example, to provide the first, second, third, and / or fourth portions of the first hybrid zinc finger domain and the second zinc finger domain, particularly the second zinc finger domain, respectively, where two amino acid substitutions may be present in the first and / or second portions, as detailed herein. Thus, zinc finger degrons according to the present invention that maintain a zinc finger-like fold (β-hairpin loop and α-helix) and exhibit the properties disclosed herein (e.g., susceptibility to IMiDs in terms of proteolysis) can be readily provided by those skilled in the art.

[0268] In other words, the amino acid sequence of the Cys2-His2 zinc finger domain for selection of the third and / or fourth portions (see below) may, in some embodiments, be selected from the group consisting of SEQ ID NO: 128 (IKZF1 ZF3 - FKCHLCNYACRRRDALTGHLRTH), SEQ ID NO: 129 (IKZF3 ZF3-FKCHLCNYACQRRDALTGHLRTH), SEQ ID NO: 130 (ZFP91 ZF5-FSCNICGKKFEKKDSVVAHKAKSH), SEQ ID NO: 131 (ZNF653 ZF5-FTCDRCGKRFEKLDSVKFHTLKSH), SEQ ID NO: 132 (ZNF276 ZF5-FACDQCGRRFEKAHNLNVHMSMVH), SEQ ID NO: 133 (ZNF827 ZF2-HQCPLCPFRCARKDNLKSHMKVH), and / or SEQ ID NO: 134 (ZNF692 ZF5-FPCEFCGKRFEKPDSVAAHRSKSH).

[0269] As discussed herein, the second zinc finger of a zinc finger degron and / or hybrid zinc finger degron of the present invention can, in certain embodiments, be a hybrid zinc finger domain or a non-hybrid zinc finger domain, in which case the second zinc finger can be selected from either the third or fourth Cys2-His2 zinc finger domain (providing both the first and second portions from the same Cys2-His2 zinc finger domain).

[0270] In some embodiments, one of the first and second Cys2-His2 zinc finger domains and one of the third and fourth Cys2-His2 zinc finger domains are from the same zinc finger protein (e.g., a ZF4 domain from ZFP91 and a ZF5 domain from ZFP91). In some embodiments, the first and second Cys2-His2 zinc finger domains are from two different zinc finger proteins, and the third and fourth Cys2-His2 zinc finger domains are from the same two different zinc finger proteins (e.g., the first Cys2-His2 zinc finger domain and the fourth Cys2-His2 zinc finger domain are both from ZFP91 (zinc finger domains ZF4 and ZF5, respectively), and the second Cys2-His2 zinc finger domain). The guard domain and the third Cys2-His2 zinc finger domain are both derived from different zinc finger proteins, such as IKZF1 (zinc finger domains ZF2 and ZF3, respectively). When two different Cys2-His2 zinc finger domains are provided from the same zinc finger protein, the Cys2-His2 zinc finger domain that is oriented N-terminally relative to the other in the wild-type zinc finger protein is preferably also oriented N-terminally relative to the other in the zinc finger degron of the present invention.

[0271] The listed Cys2-His2 zinc finger domains and their sequences and structures are well known to those skilled in the art and are available in various scientific publications and databases.

[0272] Although the present invention does not require the inclusion of a second zinc finger domain, and zinc finger degrons and / or zinc finger polypeptides of the present invention do not necessarily require the inclusion of a second zinc finger domain (as described below), it has surprisingly been found that the sensitivity of zinc finger degrons and / or zinc finger polypeptides of the present invention can be further enhanced or increased by combining the first hybrid zinc finger domain (e.g., having two substitutions) with an additional second zinc finger domain, preferably a second zinc finger domain described herein. While a variety of second zinc finger domains and / or second zinc finger domains can be used (e.g., as described elsewhere herein, IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, ZNF692 ZF2 ... ZF5) (see also Uniprot accession numbers Q13422, Q9UKT9, Q96JP5, Q96CK0, Q8N554, Q17R98, and Q9BU19, respectively). In preferred embodiments, a zinc finger degron and / or hybrid zinc finger degron according to the invention is provided, wherein a third portion of the second zinc finger domain comprises the amino acid sequence set forth in SEQ ID NO: 104 (FKCHLCNYACRR), a fourth portion of the second zinc finger domain comprises the amino acid sequence set forth in SEQ ID NO: 105 (KDSVVAHKAKSH), and / or the second zinc finger comprises the amino acid sequence set forth in SEQ ID NO: 106 (FKCHLCNYACRRKDSVVAHKAKSH). In a preferred embodiment of the second zinc finger, the second zinc finger is composed of a β-hairpin portion of IKZF1 ZF3 and an α-helical portion of ZFP91 ZF5.

[0273] Thus, in embodiments, there is provided a non-natural hybrid zinc finger polypeptide according to the invention, said non-natural hybrid zinc finger comprising a first hybrid zinc finger domain as defined herein.

[0274] Additionally, non-natural hybrid zinc finger polypeptides of the present invention may further comprise a second zinc finger domain, wherein the second zinc finger domain is a non-hybrid zinc finger domain. As described herein, while inclusion of a second zinc finger domain in a hybrid zinc finger polypeptide of the present invention may be beneficial, it has been established that such a second zinc finger domain is not required to provide the enhanced or increased sensitivity observed with IMiDs. However, in some embodiments, non-natural hybrid zinc finger polypeptides of the present invention may further comprise a second zinc finger domain, wherein the second zinc finger domain is a non-hybrid zinc finger domain. For example, the second zinc finger domain may be a wild-type Cys2-His2 zinc finger domain, such as those disclosed herein, e.g., IKZF1 ZF3, IKZF3 ZF3, but may also be ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, and / or ZNF827 ZF2, ZNF692 ZF5.

[0275] The second zinc finger domain is preferably oriented C-terminal to the first hybrid zinc finger domain and may be directly adjacent to or linked to the first hybrid zinc finger domain, as described elsewhere herein.

[0276] In some embodiments, the second zinc finger domain is a second zinc finger domain, such as those described elsewhere herein. In embodiments in which the second zinc finger domain in a hybrid zinc finger domain of the invention is a non-hybrid, e.g., wild-type or naturally occurring zinc finger domain, or a mutant thereof, the second zinc finger domain is preferably selected from, e.g., IKZF1 ZF3 (SEQ ID NO: 109 (FKCHLCNYACRRRDALTGHLRTH)) or IKZF3 ZF3 (SEQ ID NO: 110 (FKCHLCNYACQRRDALTGHLRTH)), or comprises the amino acid sequence according to SEQ ID NO: 109 or SEQ ID NO: 110. Further examples of suitable second zinc finger domains include, for example, IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, ZNF692 ZF5, SEQ ID NOs: 109-115, and the like.

[0277] According to some embodiments of the present invention, the agent is an immunomodulatory imid drug (IMiD), preferably selected from thalidomide, lenalidomide, pomalidomide, avadomide, iverdimide, CC-885, salts and analogs thereof. In some embodiments, a concentration of the IMiD (e.g., lenalidomide) is used between 0.01 nM and 1000 nM, with increasing priority being 0.05 to 1000 nM, 0.1 to 750 nM, and 0.5 nM to 500 nM. In some embodiments, the daily dose of the IMiD provided to the patient may be between 0.005 and 50 mg / day, e.g., 0.005 to 50 mg / day of lenalidomide.

[0278] Thus, in some embodiments, the cell of the present invention is provided wherein the agent is an immunomodulatory imide drug (IMiD), preferably wherein the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iverdomide, CC-885, salts and analogs thereof. Alternatively, the cell of the present invention is provided wherein the cell further comprises an immunomodulatory imide drug (IMiD), preferably wherein the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iverdomide, CC-885, salts and analogs thereof.

[0279] The protein of interest can be any protein of interest. For example, the protein of interest can be a protein that can contribute to the treatment of a condition or disease, such as the treatment of cancer, for example, for the treatment of tumors. Thus, in some embodiments, it is provided that the nucleic acid encoding the protein of interest encodes a cytokine, interleukin, interferon, chemokine, receptor, ligand, antibody or antibody fragment, bispecific antibody, T cell engager, bispecific T cell engager, checkpoint inhibitor antagonist, agonist, enzyme, regulatory element, transcription factor, or DNA-binding domain of a transcription factor. Those skilled in the art will understand how to provide such a nucleic acid encoding a protein of interest, where the nucleic acid encoding the protein of interest is operably linked to an inducible promoter according to the present invention.

[0280] In some embodiments, the cytokine is a naturally occurring cytokine, and in some embodiments, the cytokine is a single-chain cytokine (e.g., single-chain (sc) interleukin-12). In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, or IL-21. In some embodiments, the interferon is IFNα or IFNβ or IFNγ.

[0281] In some embodiments, the antibody or antibody fragment or bispecific antibody is anti-IL-6, anti-IL-6R, anti-IL-6Ra, anti-TNFα, anti-IL-1, anti-PD1, anti-CD25, anti-CD3, anti-CD20, anti-CD40 agonist antibody, anti-IL-8, anti-MCP1, anti-MIP-1, anti-TGFβ, anti-CD47, anti-CSF1R, anti-CD28, anti-TIGIT, anti-VEGFR, or anti-FAP.

[0282] In some embodiments, the checkpoint inhibitor antagonist is anti-PD-L1, anti-PD-1, anti-CTLA4, anti-LAG3, anti-TIM3, anti-2B4, or anti-CD160, or anti-CD5.

[0283] In some embodiments, the bispecific antibody comprises, but is not limited to, any functional domain of anti-IL-6, anti-IL-6R, anti-IL-6Ra, anti-TNFα, anti-IL-1, anti-PD1, anti-CD25, anti-CD3, anti-CD20, anti-CD40 agonist antibody, anti-IL-8, anti-MCP1, anti-MIP-1, anti-TGFβ, anti-CD47, anti-CSF1R, anti-CD28, anti-TIGIT, anti-VEGFR, or anti-FAP.

[0284] In some embodiments, the chemokine is CCL5, XCL-1, XCL-2, CCR-7, CCL-19, or CCL-21.

[0285] In some embodiments, the transcription factor or regulatory element is T-bet, TCF7, EOMES, a member of the Runx family, BLIMP1, Bcl2, Bcl6, FoxP3, FoxO1, or FoxO1-3A.

[0286] In an embodiment of the present invention, the nucleic acid encoding the protein of interest encodes a chemokine. In an embodiment of the present invention, the chemokine is CCL5, CCL19, or CCL21. In an embodiment of the present invention, the nucleic acid encoding the protein of interest encodes a cytokine receptor. In an embodiment of the present invention, the cytokine receptor is TGFBR (TGF-β receptor) or TGFBR2.

[0287] In some embodiments, cells of the present invention are also provided, wherein the cells are provided with a nucleic acid encoding a receptor, a nucleic acid encoding a chimeric protein, and / or (preferably, a nucleic acid encoding a protein of interest) (and wherein the nucleic acid encoding the protein of interest is operably linked to an inducible promoter, e.g., the nucleic acid provided to the cell includes both an inducible promoter and a gene encoding the protein of interest). In such embodiments, the cells of the present invention are provided with the receptor and chimeric protein of the present invention, and also with the protein of interest (which is subsequently expressed) upon induction of the inducible promoter of the present invention, via transcription and translation.

[0288] In some embodiments, a cell of the present invention already expresses the receptor, e.g., a TCR, NKR, or CAR, and the cell is provided with nucleic acid encoding a chimeric protein and encoding the protein of interest (wherein the nucleic acid encoding the protein of interest is operably linked to the inducible promoter, e.g., the nucleic acid provided to the cell includes both the inducible promoter and the gene encoding the protein of interest).

[0289] In a preferred embodiment, the nucleic acid encoding the receptor disclosed in the present invention and / or encoding the chimeric protein and / or encoding the protein of interest is provided to a cell using a vector that is provided to the cell by exogenously introducing the nucleic acid into the cell. In another preferred embodiment, the nucleic acid encoding the chimeric protein disclosed in the present invention and / or encoding the protein of interest is provided to a cell using a vector that is provided to the cell by exogenously introducing the nucleic acid into the cell. In a preferred embodiment, the nucleic acid encoding the receptor disclosed in the present invention and / or encoding the chimeric protein and / or encoding the protein of interest is prepared or has been prepared using recombinant DNA technology.

[0290] In some embodiments, the nucleic acid encoding the receptor disclosed herein and / or encoding the chimeric protein and / or encoding the protein of interest is not integrated into the genomic DNA of the cell and / or the nucleic acid is extrachromosomal. In some embodiments, the nucleic acid encoding the receptor disclosed herein and / or encoding the chimeric protein and / or encoding the protein of interest is integrated into the genomic DNA of the cell and / or the nucleic acid is not extrachromosomal.

[0291] In some embodiments, the nucleic acid encoding the receptor, the chimeric protein, and / or the protein of interest (and the nucleic acid encoding the protein of interest is operably linked to an inducible promoter, e.g., the nucleic acid provided to the cell includes both an inducible promoter and a gene encoding the protein of interest) are on the same vector. In these embodiments, the cell is provided with a vector comprising the nucleic acid encoding the receptor, the nucleic acid encoding the chimeric protein, and / or the nucleic acid encoding the protein of interest (under the control of an inducible promoter).

[0292] In other embodiments of the present invention, multiple types of vectors may be used. For example, according to the present invention, a first vector comprises a nucleic acid encoding the receptor and a second vector encoding the chimeric protein. In such embodiments, the nucleic acid encoding the protein of interest may be contained in the first vector, the second vector, or both. As will be understood by those skilled in the art, any combination of the nucleic acid encoding the receptor, the nucleic acid encoding the chimeric protein, and the nucleic acid encoding the protein of interest may be contained in a vector, including combinations in which one of the nucleic acids (e.g., the nucleic acid encoding the receptor, the nucleic acid encoding the chimeric protein, or the nucleic acid encoding the protein of interest) is not contained in a vector. In such latter embodiments, an additional vector containing a nucleic acid not contained in the first vector or an additional vector in which the cell already expresses the protein (e.g., a receptor) may be used. In some embodiments, the nucleic acid encoding the receptor and the nucleic acid encoding the chimeric protein are contained in the same vector. In some embodiments, the nucleic acid encoding the receptor and the nucleic acid encoding the protein of interest are contained in the same vector. In some embodiments, the nucleic acid encoding the chimeric protein and the nucleic acid encoding the protein of interest are contained in the same vector. In some embodiments, the nucleic acid encoding the receptor is contained in multiple types of vectors. In some embodiments, the nucleic acid encoding the chimeric protein is contained in more than one type of vector. In some embodiments, the nucleic acid encoding the protein of interest is present in more than one type of vector. In some embodiments, the nucleic acid encoding the receptor, the nucleic acid encoding the chimeric protein, and / or the nucleic acid encoding the protein of interest may be in the same orientation or in opposite, reverse orientations.

[0293] Therefore, in a preferred embodiment of the invention, a (genetically engineered) cell according to the invention is provided, (a) a receptor capable of receiving an activating signal; (b) operably linked to a promoter; i. a docking domain capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives the activating signal; ii. a drug-regulated protein stability domain; and a nucleic acid encoding a chimeric protein comprising: (c) an inducible promoter operably linked to a nucleic acid encoding a protein of interest, the inducible promoter being induced when the receptor receives the activation signal in the absence of the chimeric protein; A cell comprising:

[0294] Those skilled in the art will appreciate that the preferences, features, and embodiments of the invention discussed herein apply equally to this aspect of the invention.

[0295] As will be appreciated by those skilled in the art, in a preferred embodiment, a cell of the present invention comprises a (cytoplasmic) chimeric protein-encoding nucleic acid operably linked to a promoter, and a nucleic acid encoding a protein of interest (operably linked to an inducible promoter), wherein the chimeric protein is expressed in the cell. In another embodiment, a cell of the present invention comprises a (cytoplasmic) chimeric protein-encoding nucleic acid operably linked to a promoter, and a nucleic acid encoding a protein of interest (operably linked to an inducible promoter), wherein both the chimeric protein and the protein of interest are expressed as described herein (e.g., in the presence of a drug that modulates a drug-regulated stability domain in the chimeric protein, e.g., an IMiD such as lenalidomide that modulates a zinc finger degron as a drug-associated stability domain), and under conditions where a receptor in the cell of the present invention receives an activating signal (e.g., by ligand binding). As will be appreciated by those skilled in the art, in such embodiments, the cell also expresses a receptor capable of receiving an activating signal.

[0296] Thus, in these embodiments of the invention, a cell according to the invention is provided, in which said chimeric protein is expressed and / or in which said protein of interest is expressed.

[0297] Given the disclosure herein, one of skill in the art will understand that, using the cells of the present invention, it is possible to modulate expression of the protein of interest by providing an activation signal to the cell or the receptor expressed by the cell, or upon receipt of an activation signal by the cell or the receptor expressed by the cell, using an agent capable of modulating the drug-regulated stability domain contained in the chimeric protein. Modulation or regulation of expression of the protein of interest can be achieved by providing the cell with an effective amount of an agent capable of modulating the drug-regulated protein stability domain. For example, in some embodiments, the amount of agent provided is sufficient to express the protein of interest at a level at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or less of the maximum expression of the protein of interest in the cells of the present invention, where the protein of interest is expressed from a nucleic acid encoding the protein of interest operably linked to an inducible promoter. Maximum expression can be established, for example, in the absence (no expression) of the chimeric protein of the present invention and under conditions in which the receptor of the present invention is activated by an activation signal.

[0298] Thus, in such an embodiment of the invention, there is provided a method of modulating expression of a protein of interest, comprising providing an activating signal to a cell of the invention in vitro or in vivo in the absence of an effective amount of an agent capable of modulating a drug-regulated protein stability domain. As will be appreciated by those skilled in the art, in the context of such an embodiment, an effective amount of an agent refers to a concentration of an agent that allows expression of said protein of interest, e.g., at expression levels as set forth above. As will be appreciated by those skilled in the art, the preferences, features, and embodiments of the invention discussed herein apply equally to this aspect of the invention.

[0299] As will be appreciated by those skilled in the art, the present invention makes it possible to regulate the expression (level) of the protein of interest in a subject, e.g., a patient, in need of such regulation. At the same time, the present invention also makes it possible to regulate the activity of various cells in response to activation signals, e.g., when a TCR recognizes an antigen on a tumor cell and is activated, it regulates the activity of T cells against the tumor cell, resulting in ITAM-mediated signal transduction through the cell. Because the present invention can regulate the expression of the protein of interest and the activity of the cell in response to activation signals, it allows for robust treatment of the condition to be treated, e.g., cancer / tumor, while also making it possible to precisely regulate the activity level of the cell and the expression level of the protein of interest to desired levels in the subject to be treated, e.g., a patient.

[0300] Accordingly, there is also provided a method of modulating expression of a protein of interest in a subject, the method comprising administering to the subject an effective amount of an agent capable of modulating a drug-regulated protein stability domain, wherein the subject comprises a cell according to the invention.

[0301] In such embodiments, the patient is provided with or includes the cells of the present invention and an effective amount of an agent, such as an IMiD (e.g., lenalidomide) when a zinc finger degron is used as the drug-regulatory protein stability domain. As described above, an effective amount of the agent refers to the concentration of the agent that can cause the protein of interest to be expressed at a desired level, e.g., the expression levels set forth above. Using the cells of the present invention, the subject can regulate expression of the protein of interest in response to an activation signal by providing the subject with an agent that modulates the drug-regulatory protein stability domain. For example, if the cell is an immune cell such as a T cell, NK cell, tumor-infiltrating lymphocyte, or macrophage, the activity of the cell can be regulated in response to receiving an activation signal, e.g., binding to an antigen present on a cancer / tumor cell. In some embodiments, the subject is administered a first effective amount of an agent, and then, at a later time, a second or more effective amounts of an agent are administered to the patient, and the first and second or more effective amounts of an agent can be the same or different. In other embodiments, there is a period between the first effective amount and the second or higher effective amount during which the subject is not treated with an effective amount of the drug; e.g., during the period between the two effective amounts of the drug, signal transduction from the receptor that received the activating signal, and / or induction of the inducible promoter, and / or expression of the protein of interest is inhibited or suppressed to some extent, e.g., to the maximum extent possible in the cells of the present invention. Those skilled in the art will also understand that multiple types of drugs capable of modulating a drug-regulated protein stability domain can be used simultaneously or sequentially. For example, a first drug may be more efficient at modulating a drug-regulated protein stability domain than a second drug, and by achieving a specific ratio between the two drugs, more precise modulation of the expression of the protein of interest (and / or the activity of cells, e.g., immune cells, e.g., T cells, tumor-infiltrating lymphocytes, or NK cells) may be achieved.

[0302] Those skilled in the art will appreciate that the preferences, features, and embodiments of the invention discussed herein apply equally to this aspect of the invention.

[0303] In another embodiment of the invention, there is provided a method of modulating expression of a protein of interest in a subject, the method further comprising administering to the subject a cell according to the invention.

[0304] As will be appreciated by those skilled in the art, the cells may be of any cell type suitable for provision to a subject, e.g., a human patient. The cells may be autologous cells, i.e., cells obtained from the subject, modified (engineered), and then genetically engineered to obtain the cells of the present invention. The cells may also be allogeneic cells. The cells are preferably, for example, T cells expressing a TCR and / or CAR T cells, NK cells, including CAR NK cells, tumor-infiltrating lymphocytes, or macrophages, including CAR macrophages.

[0305] In some embodiments, the cells of the present invention are provided to the subject only once. In other embodiments, the cells of the present invention are provided to the subject on multiple, temporally separated occasions. Determining an effective amount of the cells of the present invention to be provided to the patient is within the knowledge of one of ordinary skill in the art. In one example, for example, when using T cells or CAR T cells, the amount of cells of the present invention provided to the subject can be an amount corresponding to the amount or number of cells typically provided to a subject during, for example, T cell therapy and / or CAR T cell therapy.

[0306] In some embodiments, the cells of the present invention can also be administered to a subject in combination with other agents, so long as the additional agents do not adversely affect the ability of the cells of the present invention to perform their intended therapeutic function.

[0307] As demonstrated herein, the present invention can be used to treat various conditions or diseases, e.g., in human patients, where regulated production of a protein of interest in response to an activating signal may be desirable. For example, the cells of the present invention are believed to be useful in treating various immune diseases, including autoimmune diseases. At the same time, as exemplified herein, the cells of the present invention, particularly immune cells of the present invention, such as T cells, NK cells, tumor-infiltrating lymphocytes, or macrophages, can be suitably used in treating subjects with cancer, i.e., tumors, by enabling the production of a protein of interest temporally and spatially and at a desired level (which can be varied by using different effective doses of drugs and / or different combinations of drugs capable of modulating the drug-regulated protein stability domain). Accordingly, a method for modulating the expression of a protein of interest in a subject is also provided, the method being for treating cancer and / or tumors in a subject.

[0308] In view of the disclosure herein, there is also provided a cell according to the invention for use as a medicament, preferably for use in the treatment of cancer and / or the treatment of a tumor in a subject, preferably wherein said treatment comprises administering cells to said subject and, optionally, administering a drug capable of modulating said drug regulatory protein stability domain.

[0309] Those skilled in the art will appreciate that the preferences, features, and embodiments of the invention discussed herein apply equally to this aspect of the invention.

[0310] In view of the disclosure herein, there is also provided a cell of the present invention for use as a pharmaceutical, preferably for use in treating cancer and / or tumors in a subject, wherein administering a drug capable of modulating a drug-regulated protein stability domain comprises varying the dose of the drug capable of modulating the drug-regulated protein stability domain. Varying the dose can adjust the expression level of a protein of interest. Such levels can be monitored, for example, from a blood sample using techniques such as ELISA.

[0311] In yet another embodiment of the present invention there is provided a drug for use as a pharmaceutical, preferably for use in the treatment of cancer and / or the treatment of a tumor in a subject, said treatment comprising administering to a subject a cell according to the present invention and administering said drug, wherein said drug is capable of modulating said drug regulatory protein stability domain.

[0312] Those skilled in the art will appreciate that the preferences, features, and embodiments of the invention discussed herein apply equally to this aspect of the invention.

[0313] Preferably, said agent is at least one IMiD, preferably selected from the group disclosed herein, preferably lenalidomide.

[0314] In some embodiments, a vector, i.e., a DNA vector, is provided that includes any of the designs shown in FIG. 6 , and as will be understood by one of skill in the art, in the context of these embodiments, the specific indicated polyadenylation signal (e.g., SV40pA), constitutive transport element (CTE) (e.g., CTE), specific gene encoding a receptor capable of receiving an activation signal (e.g., CD19 CAR), specific promoter (e.g., pCMV or pMSCV), specific gene encoding a chimeric protein of the present invention (e.g., RheoBrick), specific post-transcriptional regulatory element (e.g., WPRE), specific 3′ UTR (e.g., IL2 3′ UTR), and / or specific inducible promoter (in the context of the present invention, for example, pNFAT) may be any suitable polyadenylation signal, constitutive transport element, gene encoding a receptor capable of receiving an activation signal, promoter, or gene encoding a chimeric protein of the present invention, post-transcriptional regulatory element, 3′ UTR, and / or specific inducible promoter (in the context of the present invention).In some embodiments, a vector or a combination of vectors is provided that includes at least one or more genes encoding a receptor capable of receiving an activation signal of the present invention, and / or one or more genes encoding a chimeric protein of the present invention, and / or one or more inducible promoters of the present invention, and a gene encoding a protein of interest of the present invention operably linked to the inducible promoter. In the case of a combination of vectors, a first vector and a second vector are provided, each of which independently includes at least one of a gene encoding a receptor capable of receiving an activation signal of the present invention, a gene encoding a chimeric protein of the present invention, or an inducible promoter of the present invention and a gene encoding a protein of interest of the present invention operably linked to the inducible promoter. Preferably, the vector or combination of vectors includes one or more RNA degradation elements of the present invention. Even more preferably, the vector or combination of vectors includes one or more genes encoding a receptor capable of receiving an activation signal of the present invention, one or more genes encoding a chimeric protein of the present invention, one or more inducible promoters of the present invention, and a gene encoding a protein of interest of the present invention operably linked to the inducible promoter, optionally with a configuration as shown in FIG. 6 (and with or without other elements as shown in FIG. 6).

[0315] In certain embodiments, one or more vectors of the present invention comprise (alone or in combination) at least one gene encoding a receptor capable of receiving an activation signal, at least one gene encoding a chimeric protein of the present invention, and at least one inducible promoter and a nucleic acid encoding a protein of interest operably linked to said inducible promoter.

[0316] In certain embodiments, one or more vectors of the present invention comprise at least one gene encoding a chimeric protein of the present invention, and at least one inducible promoter and a nucleic acid encoding a protein of interest operably linked to the inducible promoter (alone or in combination). In certain embodiments, one or more vectors of the present invention comprise at least one gene encoding a chimeric protein of the present invention (alone or in combination) (e.g., embodiments in which a cell containing the vector endogenously expresses a receptor capable of receiving an activating signal and comprises an endogenous locus into which a gene encoding a protein of interest has been integrated, the locus comprising a promoter under the control of an inducible promoter of the present invention). In some embodiments, the vector may comprise additional elements as disclosed herein, particularly an RNA degradation element (RDE) as disclosed and described herein.

[0317] In some embodiments, nucleic acids (or vectors) comprising a nucleic acid operably linked to a promoter and encoding a chimeric protein of the invention, and / or a nucleic acid operably linked to an inducible promoter and encoding a protein of interest, alone or in combination, are provided and used to form genetically engineered cells of the invention. In some embodiments, vectors are provided that additionally comprise, or further comprise, a nucleic acid operably linked to a promoter and encoding a receptor of the invention.

[0318] A particularly preferred vector provided to a cell to provide the cell of the present invention is a vector that combines a nucleic acid encoding a chimeric protein of the present invention, a nucleic acid encoding a receptor (e.g., an antigen receptor), and a nucleic acid encoding a protein of interest operably linked to an inducible promoter (also referred to as a cargo cassette, i.e., comprising an inducible promoter and a nucleic acid encoding a protein of interest operably linked). An example of such a design is shown in Figure 6 as CC30. As mentioned above, the different nucleic acids can be in the same orientation or in opposite, reverse orientations. Preferably, the orientations are as shown in CC30.

[0319] In another embodiment, a combination of at least two vectors is provided to provide a cell of the present invention, where both vectors comprise a nucleic acid encoding a chimeric protein of the present invention. One of the vectors may further comprise a nucleic acid encoding a receptor, and the other of the vectors may further comprise a nucleic acid encoding a protein of interest operably linked to an inducible promoter (also referred to as a cargo cassette, i.e., comprising an inducible promoter and a nucleic acid encoding a protein of interest operably linked). An example of a combination of at least two vectors is the combination of the CC39 vector and the CC1 vector shown in Figure 6. As mentioned above, the different nucleic acids may be in the same orientation or in opposite, opposite orientations. Preferably, the orientations are as shown for CC39 and CC1.

[0320] Furthermore, it is preferred that the above vector or combination of vectors comprises at least one 3'UTR comprising at least one ARE, preferably multiple AREs.

[0321] For the various embodiments disclosed herein, the protein of interest is preferably a protein that is secreted by the cells of the invention and / or modified to be secreted by the cells of the invention, or a cell surface protein, such as a transmembrane cell surface protein. In some embodiments, the protein of interest itself is an additional receptor that can receive additional activation signals.

[0322] As will be understood by those skilled in the art, due to the common mechanism as shown herein, the present invention is not particularly limited to a specific combination of cell type, receptor capable of receiving an activation signal, chimeric protein, docking domain, drug-regulatory protein stability domain, inducible promoter, and nucleic acid encoding a protein of interest. As will be understood by those skilled in the art, the teaching of the present application is that any suitable combination thereof can be used. Therefore, for those skilled in the art, any combination thereof is disclosed based on the disclosure of various embodiments herein. In particular, the present application teaches those skilled in the art that the present invention is not particularly limited to a specific Cys2-His2 zinc finger domain capable of drug-induced binding to a CRBN polypeptide, particularly any hybrid zinc finger domain disclosed herein. In particular, the present application teaches those skilled in the art that the present invention is not particularly limited to a specific protein of interest. In particular, the present application teaches those skilled in the art that the present invention is not particularly limited to a specific suitable cell type. In particular, the present application teaches those skilled in the art that the present invention is not particularly limited to a specific immunomodulatory imid drug. Those skilled in the art will appreciate that, in embodiments of the present invention, any Cys2-His2 zinc finger domain capable of drug-induced binding to a CRBN polypeptide, particularly any hybrid zinc finger domain, disclosed herein, can be substituted with any other Cys2-His2 zinc finger domain capable of drug-induced binding to a CRBN polypeptide, particularly any hybrid zinc finger domain disclosed herein. Those skilled in the art will appreciate that, in embodiments of the present invention, any protein of interest disclosed herein can be substituted with any other protein of interest disclosed herein. Those skilled in the art will appreciate that, in embodiments of the present invention, any cell type disclosed herein can be substituted with any cell type disclosed herein. Those skilled in the art will appreciate that, in embodiments of the present invention, any immunomodulatory imide drug disclosed herein can be substituted with any immunomodulatory imide drug disclosed herein.To those skilled in the art, such permutations, and their resulting combinations, are likewise directly and clearly disclosed.

[0323] All references cited herein, including publication articles or abstracts, published patent applications or corresponding patent applications, patents, or other references, are incorporated herein by reference in their entirety, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also incorporated herein by reference in their entirety.

[0324] It is to be understood that the phraseology or terminology used herein is for the purpose of description and not of limitation, and therefore, the terminology or terminology used herein will be interpreted by one of ordinary skill in the art in light of the teaching and guidance presented herein, in combination with the knowledge of those skilled in the art.

[0325] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of an embodiment of the present invention are equally applicable to other aspects or embodiments of the present invention, and therefore it is not necessary to separately detail all such details, embodiments, and preferences for every aspect.

[0326] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided for illustrative purposes and are not intended to limit the invention. Further aspects and embodiments will be apparent to those skilled in the art. [Example]

[0327] Example 1 Introduction, Results, and Discussion As discussed elsewhere herein, there is a need to improve the therapeutic window of cell therapies, particularly immune cell therapies such as T cell-based therapies, in the treatment of tumors, e.g., solid tumors. The inventors have discovered that the invention disclosed herein provides new methods for efficiently delivering engineered cells, genetic constructs, and biologics, such as cytokines, and other proteins of interest, such as antibodies, T cell engagers, and therapeutic proteins, to tumor cells, particularly solid tumors, thereby improving the therapeutic window of therapies, e.g., cancer treatments.

[0328] Local production of proteins of interest, such as biological compounds with antitumor activity, within a defined environment within the body, e.g., the tumor microenvironment, can be achieved by producing these molecules from transplanted adoptive cells. This approach is known in the literature as "armed CAR-T cells," "TRUCK," or "fourth-generation CAR-T cells" (Chmielewski et al. Expert Opin Biol Ther. 2015;15(8):1145-54, Hawkins et al. Biologics. 2021 Apr 14;15:95-105), in which a biologic payload with antitumor properties is released from the transplanted adoptive cells upon antigen receptor activation. Although this method ensures localized delivery of the desired payload in the tumor microenvironment, the timing and dosage of biologic production from CAR-T cells is not controlled, potentially leading to excessive payload production and fatal toxicity (Zhang et al. Clin Cancer Res. 2015 May 15; 21(10): 2278-2288).

[0329] Herein, the present invention discloses a novel approach that allows the production of biologics from genetically engineered cells, in particular immune cells (CAR-T cells, TCR-T cells, TIL cells, NK cells, and other suitable cells known to those skilled in the art) in a manner that is strictly dependent on an activating signal, such as an antigen, to activate receptors that receive such signals and that can be controlled using drugs (e.g., small molecules).

[0330] The present invention, as broadly illustrated in the Examples, makes it possible to use genetically modified cells, particularly immune cells, to precisely control both the timing and dosage of production of a biologic (a protein of interest), thereby enabling the production of appropriate amounts of a biologic at the site of disease (i.e., when such cells are provided to a patient in need thereof) to optimize the therapeutic window and to prevent overproduction of such a biologic to avoid side effects.

[0331] To achieve this, the inventors have created a transgene design in which production of a biologic payload (biologic, protein of interest) is operationally linked to the level of ITAM signaling in immune cells, and the level of ITAM signaling is controlled by a rheostat switch. Examples of such immune cell rheostat switches (also known as CRASH-IT switches) are described in WO 2021 / 080427, and such rheostat switches are preferred in some embodiments. In this example, the rheostat switch is also referred to as RheoBrick®, a registered trademark.

[0332] The rheostat switches used in the present invention, particularly RheoBrick®, tightly control immune cell activation using orally available, clinically approved drugs or small molecules such as lenalidomide (WO 2021 / 080427). In the absence of the drug, immune cell function, e.g., immune cell activation, is severely suppressed, but when the drug is provided, immune cell function is restored, providing remote control (Figure 1).

[0333] Structurally, the rheostat switch (e.g., RheoBrick®) platform consists of three functional domains: a docking domain that forms a reversible interaction with an antigen receptor, an inhibitory domain that suppresses the TCR / CAR / NKR (NK cell receptor) signaling pathway, and a degron domain that controls the stability of the rheostat switch (e.g., RheoBrick® switch) in a drug-dependent (e.g., small molecule-dependent) manner.

[0334] In some embodiments, the rheostat switch used in the examples, e.g., the RheoBrick® switch, comprises a docking domain derived from a Zap70 (2xSH2) domain, an inhibitory signaling domain derived from an ITIM / ITSM containing the signaling domain of the Siglec11 protein, and a synthetic zinc finger (SynFinger) degron sequence optimized to increase sensitivity to lenalidomide several-fold (as described herein; see also Dutch Patent Application Publication No. 2031325). However, as will be appreciated by those skilled in the art, the present invention is not limited to such optimized variable resistor switches. In other embodiments of rheostat switches, e.g., RheoBrick® switches, the docking domain is derived from the SH2 domain of a Syk or Lck protein, the inhibitory signaling domain is derived from an ITIM / ITSM, including the signaling domains of PD1, BTLA, SIRPa, SIGLEC5, SIGLEC9, PECAM1, and Ly9, and the drug-regulated protein stability domain comprises a self-cleaving degron such as a SMASh tag, a PROTAC binding domain such as FKBP12F36V (SEQ ID NO: 135), or other zinc finger degron. Examples of preferred rheostat switches for use in the present invention, e.g., RheoBrick® switches, are described herein as well as in WO 2021 / 080427 and Dutch Patent Application Publication No. 2031325.

[0335] The present inventors have not only developed a technology that allows for tight control of T cell activity in an antigen-dependent and small molecule-controlled manner, but have also surprisingly found that this technology is particularly useful and beneficial for the controlled production of potent payloads (proteins of interest, such as biologics, including cytokines, including IL-12) in the immunosuppressive tumor microenvironment (TME) and T cell engagers.

[0336] This was surprisingly achieved by creating a transgene design in which the production of such a payload (protein, biological substance of interest) was directly controlled by the level of T cell activation. In T cell activation in a rheostat switch, e.g., a RheoBrick® switch, according to the present invention, engineered immune cells, such as T cells, are tightly controlled by both signals that activate receptors (e.g., antigens expressed by tumor cells) and drugs, such as small molecules (Figures 1-2).

[0337] Taking advantage of this, the present invention provides such transgenes that are (specifically) produced at the site where an activation signal (e.g., an antigen) is present, for example, at a tumor site, and where a drug (small molecule) is used to control T cell activity (or signaling) to a desired extent.

[0338] Importantly, we have surprisingly demonstrated that by using a transgene under the control of a promoter that is induced upon cell activation (via a receptor capable of receiving an activation signal) in the absence of a rheostat switch (e.g., RheoBrick®), it is possible to tightly control the production of a payload (biological drug or protein of interest). We demonstrate this here using an NFAT-regulated EGFP transgene, establishing evidence of the feasibility of controlled payload production; feeding CD4 and CD8 cells with lenalidomide increased the total EGFP produced by approximately 20-fold (Figure 2). This is also exemplified by the IL-12 data, discussed below and shown in Figure 7. However, in this example, T cells engineered with two lentiviral vectors (CC3+CC1) encoding a rheostat switch (e.g., RheoBrick® switch, CD19 CAR, and NFAT-EGFP) only faintly expressed EGFP in the absence of lenalidomide, but significantly increased EGFP expression upon lenalidomide (drug) and CD19. +In the presence of Nalm6 cells (activation signal), EGFP expression was significantly upregulated. In contrast, T cells engineered with only a vector encoding CD19 CAR and NFAT-EGFP (CC23+CC22) showed comparable EGFP expression in Nalm6 cocultures, regardless of the presence or absence of lenalidomide. However, a rheostat switch (e.g., RheoBrick®) may be included in one or more vectors used to engineer immune cells. In this example, the presence of a rheostat switch (e.g., RheoBrick® switch) in both lentiviral vectors (CC3+CC1) resulted in improved induction folds compared to vector combinations in which RheoBrick® was present in one vector (CC23+CC1 or CC3+CC22). Thus, in some vector embodiments, a rheostat switch, e.g., a RheoBrick® switch, is included in at least one vector, preferably multiple vectors, e.g., two vectors, used to engineer cells, e.g., immune cells, according to the present invention.

[0339] INTRODUCTION NFAT NF-kB AP-1;Jutz et al. J Immunol Methods. 2016 Mar; 430:10-20) See also Book of Records (IL-2(Publication ID:3558) IL -4(3565)、IL-6(ID:3569)、IL-8(3576)、IL-13(35 96) 、IL-17(3605)、IFNγ(ID:3458)、TNFα(ID:7124)、CD69(ID:969)、CD137(USA) et al.BMC Genomics 2019 Jul 19;20(1):593、Glinos Immun 21(6): 390-408、Redondo-Anton et al.2020 Oct. 1995 Sep 22;270(38):22500-6、Goldfeld et al. J Immunol 1995 Dec 1;155(11):5273-9、Macian et al. Biophys Acta.2004 May 28;1692(1):17-24、Kim et al. Chem 2004 Dec 10;279(50):52762-71、Okamoto et al. J Biol Chem.1994 Mar 18;269(11):8582-9) are known to those skilled in the art and respond to ITAM signaling by increasing transcription. At the same time, proteins such as IL-2 (NM_000586.4), IL-3 (NM_000588.4), IL-4 (NM_000589.4), IL-6 (NM_000600.5), IL-13 (NM_002188.3), IL-17 (NM_002190.3), GATA-3 (NM_001002295.2), IFNγ (NM_000619.3), TNFα (NM The 3' untranslated regions (UTRs) of genes such as CSF2 (NM_000758.4), FasL (NM_000639.3), c-fos (NM_005252.4), and others known to those skilled in the art contain AU-rich elements (AREs), which also respond to ITAM signaling by increasing mRNA stability and nuclear transport (Salerno et al., 2004). et al. Nat Immunol. 2018 Aug; 19(8): 828-837, Casolara et al. J Allergy Clin Immunol. 2008 Apr;121(4):853-9.e4, Dean et al. Mol Cell Biol. 2001 Feb;21(3):721-30, Ouhara et al. Clin Exp Immunol. 2018 Jun;192(3):325-336, Fan et al. EMBO J. 1998 Jun 15;17(12):3448-60, Karginov et al. RNA Biol. 2019 May;16(5):686-695, Drury et al. J Biol Chem. 2010 Oct 8;285(41):31130-8, Chen et al. J Immunol. 2013 Dec 1;191(11):5441-50, Stellato et al. J Immunol. 2011 Jul 1;187(1):441-9). .

[0340] Specifically, induction of ITAM signaling increases the nuclear export of mRNA stabilizing factors, such as HuR and NF90, which bind to AREs in the 3'UTR and displace previously bound mRNA destabilizing factors (Shim et al. Mol Cell. 2002 Dec;10(6):1331-44, Wang et al. J Immunol. 2006 Feb 15;176(4):2105-13, Nicolet et al. Immunol Rev. 2021 Nov;304(1):10-29). Displacement of mRNA destabilizing factors improves mRNA stability and protein expression levels. Importantly, control of payload production via the rheostat (e.g., RheoBrick®) of the present invention surprisingly works for different classes of regulatory gene elements (e.g., promoters and / or 3'UTRs) that share the property of responding to ITAM signaling.

[0341] Activation of ITAM receptor complexes in lymphoid and myeloid cells can activate NFAT signaling (Aramburu et al. J Exp Med. 1995 Sep 1;182(3):801-10, Kulemzin et al. BMC Med Genomics. 2019 Mar 13;12(Suppl 2):44, Fric et al. Blood. 2012 Aug 16;120(7):1380-9). Furthermore, RNA-binding proteins (RBPs) such as HuR regulate mRNA stability in T cells, NK cells, and macrophages (Kim et al. Immune Netw. 2009 Aug;9(4):115-121, Diaz-Munoz et al. Front Immunol. 2018 May 23;9:1094). Therefore, modulation of ITAM signaling and / or subsequent production of a protein of interest by the approach of the present invention is not limited to T cells but is also applicable to other cells, including immune cell subsets including NK cells and macrophages.

[0342] While some degree of leaky expression (of the protein of interest) may be tolerated, it is preferable to limit such expression. By combining multiple control mechanisms that respond to the activation state of immune cells, it may be possible to achieve a higher fold induction while reducing expression leakage. We hypothesize that if using the NFAT promoter or 3'UTR sequence elements individually increases antigen-dependent cargo production from either one by approximately 10-fold, an expression cassette combining both elements in the same expression cassette could reach induction levels of up to approximately 100-fold. Furthermore, we hypothesize that accurate cargo (payload, protein of interest, biologic) expression levels within this expanded dynamic range may be significantly further improved through small molecule-based control of a rheostat (e.g., RheoBrick®) (Figure 3).

[0343] We surprisingly confirmed this by replacing the constitutive mRNA transport element (CTE) and SV40 polyA downstream of the cargo expression cassette with 3'UTR elements from the IL2 or IFNg gene (Figure 4A). In Nalm6 coculture experiments using rheostat-engineered T cells such as RheoBrick®, CD19 CAR, and NFAT-scIL-12 cargo cassettes containing the downstream CTE SV40pA, IL2 3'UTR, or IFNg 3'UTR, respectively, we observed a 24-fold, 112-fold, and 78-fold induction of IL-12 production in the presence of lenalidomide compared to vehicle controls (Figure 4B).

[0344] Because combining ITAM-responsive regulatory gene elements of different classes (e.g., NFAT promoter and regulatory 3'UTR) significantly improved induction folds, we tested whether combining multiple regulatory elements of the same class (IL2 3'UTR and IFNg 3'UTR) might further improve induction folds. Indeed, rheostat (e.g., RheoBrick®)-engineered CAR-T cells and an IL-12-encoding vector combining IL2 and IFNg 3'UTRs showed higher induction folds compared to single-type 3'UTR-encoding vectors in a direct comparison (Figure 5).

[0345] The versatility of the RheoBrick-mediated antigen-dependent and small-molecule-regulated cargo production concept has been demonstrated using a panel of medium- to high-affinity TCRs and CARs. As shown in Figure 16A, primary human T cells were co-transfected with a RheoBrick switch and ITAM signaling-responsive scIL12 expression cassette along with lentiviral vectors encoding either the CDK4 TCR, NY-ESO-1 TCR, CD19 CAR, PSMA CAR, or fibronectin EDB CAR, or no antigen receptor. Analysis of IL12 cargo production in cocultures with antigen-positive Nalm6, PC3-PSMA, NKIRTIL006, A549, A375, or Mel624 cells with or without 500 nM lenalidomide revealed a 42- to 684-fold upregulation of IL12 cargo production in the presence of lenalidomide compared to vehicle controls, whereas minimal IL12 cargo production was detected in cultures without target cells (Figure 16B).

[0346] We also generated a large vector panel to determine optimal vector designs for small molecule and antigen-dependent cargo production applications. Thus, while the various vectors described herein, e.g., provided in the genetically engineered cells of the present invention, are all suitable for use in the present invention, we surprisingly found that certain vectors are more suitable than others. A single-vector design (e.g., a single lentiviral design) combining all three components (rheostat, e.g., RheoBrick®, antigen receptor, and cargo expression cassette) was compared side-by-side in a coculture experiment with a two-vector design, such as a two-vector lentiviral expression system (Figure 6A). Induction of EGFP cargo production was observed with all designs (Figure 6B). However, the two-vector expression system in which T cells were engineered with two lentiviral vectors (e.g., CC39 and CC1), the first vector encoding the cargo expression cassette and the second vector encoding the antigen receptor, and both vectors encoding one copy of a rheostat (e.g., RheoBrick®), showed the highest fold induction (Figure 6C). Higher fold induction was observed with vector designs containing the IL2 3'UTR or IFNg 3'UTR (CC39+CC1 and CC40+CC1, respectively) compared to vectors containing CTE SV40 pA in either the two-vector or single-vector configurations (Figure 6C).

[0347] The ability to precisely control the expression levels of potent payloads, such as cytokines, chemokines, costimulatory or opsonic antibodies, T cell engagers, and NK cell engagers, in transplanted adoptive cells in a small-molecule concentration-sensitive and antigen-dependent manner opens up new possibilities. To demonstrate that CAR-T cells engineered according to the present invention can modulate IL-12 (POI) secretion levels as a function of drug concentration, lenalidomide was titrated in CAR-T cell / Nalm6 cocultures. Robust, small-molecule-dependent IL-12 secretion was observed even at low nanomolar drug concentrations, with IL-12 secretion levels induced between 0.5 nM and 500 nM lenalidomide. At the upper end of the small-molecule titration range, only minimal IL-12 secretion was observed in the absence of Nalm6 cells (Figure 7). Surprisingly, the invention disclosed herein enables precise control of the expression levels of a protein of interest according to the present invention by adjusting the level or concentration of a drug (e.g., a small molecule) that modulates a drug-regulated protein stability domain.

[0348] Of note, although the concept of small molecule-regulated IL-12 cargo secretion has been reported previously (e.g., control of IL-12 expression by a doxycycline-inducible promoter; Alsaieedi et al. Oncoimmunology. 2019; 8(3): 1542917), such studies did not clarify the mechanism linking such small molecule-regulated cargo secretion to signaling through activating receptors to control the site of cargo production. Consequently, in contrast to the present invention, induction of cargo expression by supplying or removing small molecules in such systems leads to cargo expression at both antigen-positive and antigen-negative sites, thereby affecting the therapeutic window.

[0349] To demonstrate that our technology can be used as a building block for the design of AND-gate-based logic circuits with external (small molecule) control, we constructed a proof-of-concept logic circuit that accepts three conditional inputs and produces a tumor-killing output only if all conditional inputs are present (Figure 8).

[0350] The first AND gate receives conditional inputs CD19 antigen and the small molecule lenalidomide, which are detected by a second-generation CD19 CAR and, in this example, a RheoBrick® (i.e., an example of a rheostat system described herein), respectively, to generate as output a PSMA and CD3 bispecific T cell engager (BiTE), which is conditionally expressed from an NFAT-PSMA BiTE-IL2 3'UTR expression cassette (Figure 8A). The second AND gate receives as input the output of the first AND gate (PSMA BiTE generation), detects a third conditional input (PSMA antigen), and generates anti-PSMA+ tumor killing as the final output (Figure 8A).

[0351] To demonstrate conditional secretion of PSMA BiTEs as a function of two conditional inputs, RheoBrick® AND-gated CAR-T cells (i.e., examples of cells engineered as described herein) were co-cultured with or without CD19-positive Nalm6 cells in the presence or absence of lenalidomide (Figures 8B-8C). The supernatant was then collected, filtered, and added to a second co-culture containing PSMA-negative HCT116 PSMA cells or HCT116 WT cells with unmodified PMBCs (Figure 8C).

[0352] At the end of the second coculture, cytokine production and T cell degranulation were examined and found to be positive only in HCT116 PSMA cocultures prepared using conditioned medium from Nalm6 / lenalidomide / RheoBrick® AND gated CAR-T cocultures (Figures 8D-8I).

[0353] To demonstrate the generalizability of the RheoBrick-mediated antigen-dependent and small-molecule-regulated BiTE secretion concept, we designed another AND-gate-based logic circuit. In this circuit, RheoBricks expressing PSMA CAR-T cells secreted blinatumomab, an FDA-approved CD19xCD3 bispecific T cell engager, upon exposure to PSMA antigen-positive target cells in the presence of lenalidomide. Briefly, primary human T cells were transduced with a lentiviral vector encoding either the PSMA CAR, blinatumomab, or a mimic cargo (luciferase) encoding an ITAM signaling-responsive expression cassette, along with RheoBrick (Figure 17A). Transduced cells were cocultured with antigen-positive PC3-PSMA cells or antigen-negative PC3 cells in the presence or absence of lenalidomide (Figure 17B). The supernatant was then collected, filtered, serially diluted, and added to a second coculture containing a 1:1 mixture of CTFR-labeled CD19 antigen-positive Nalm6 cells and CTV-labeled CD19 antigen-negative K562 cells along with naive T cells. After 24 h, the Nalm6 / K562 cell ratio was measured by FACS analysis to determine specific killing, as described in the Methods section. Specific killing analysis showed that conditioned medium from the first coculture of PC3-PSMA cells and lenalidomide induced the highest level of Nalm6 cytotoxicity in the second coculture compared with conditioned medium from PC3-PSMA / DMSO, PC3 / lenalidomide, and PC3 / DMSO conditions, resulting in an approximately two-order increase in cytotoxic potency (Figure 17C). As a positive control, the Nalm6 / K562 cell mixture was incubated with serially diluted purified blinatumomab (Figure 17D).

[0354] Using the same approach, RheoBrick® AND gated T cells (i.e., examples of cells genetically engineered as described herein) were used to screen for EpCAM (P16422), EGFR (P00533), HER2 (P04626), CEA (P06731), and CAIX (Q16790) (Qin et al. Oncoimmunology. 2020; 9(1): 1806009, Liu et al. Cytotherapy. 2020 Oct;22(10):573-580, Morgan et al. Mol Ther. 2010 Apr; 18(4): 843-851, Parkhurst et al. Mol Ther. 2011 Mar; 19(3): 620-626, Lamers et al. Mol Ther. 2013 Apr; 21(4): 904-912), T cell engagers specific for on-target and off-tumor toxicity risk-associated antigens can be selectively secreted at tumor sites, thereby limiting their exposure to healthy tissues.

[0355] To demonstrate the in vivo applicability of the RheoBrick-mediated antigen-dependent and small molecule-regulated cargo production technology, primary human T cells were engineered with a vector encoding a PSMA CAR, RheoBrick, and a luciferase cargo encoding an ITAM signaling-responsive expression cassette (Figure 18A). The luciferase cargo served as a tool to accurately measure cargo expression within solid tumors in living animals. As a control, T cells were engineered with a PSMA CAR, an unrelated protein (huEGFRt), and a luciferase cargo encoding an ITAM signaling-responsive expression cassette, representing conventional TRUCK T cells. A subcutaneous adenocarcinoma model was established by injecting 5 million PC3-PSMA cells into the flank of immunodeficient NSG mice (Figure 18B). Two weeks after tumor injection, mice were intravenously injected with 2.5 million RheoBrick PSMA CAR-T cells encoding an ITAM signaling-induced luciferase cassette or PSMA CAR-T cells without RheoBrick. Immediately after CAR-T cell infusion, mice were administered 1 mg / kg lenalidomide or vehicle control by oral gavage daily. Luciferase cargo production peaked on day 13 after CAR-T cell infusion. Surprisingly, when both groups of animals received daily lenalidomide, RheoBrick PSMA CAR-T cells produced 3.2-fold more cargo in vivo compared with PSMA CAR-T cells without RheoBrick (Figure 18C). Luciferase cargo production density was calculated by normalizing the luciferase signal to tumor size. Surprisingly, when both groups of animals received daily lenalidomide, cargo production density in vivo was also found to be increased in RheoBrick PSMA CAR-T cells compared with PSMA CAR-T cells without RheoBrick (3.7-fold difference on day 13, Figure 18D). Moreover, we were surprised to find that when both groups of animals were administered lenalidomide daily, cargo production from lenalidomide-treated RheoBrick PSMA CAR-T cells in vivo was prolonged compared to PSMA CAR-T cells without RheoBrick (Figure 18D).In the absence of lenalidomide, RheoBrick efficiently inhibited cargo production (a 109-fold difference in cargo production between lenalidomide and vehicle treatment in the group injected with RheoBrick PSMA CAR-T cells on day 13). Furthermore, RheoBrick PSMA CAR-T cells efficiently controlled tumor growth in animals treated with lenalidomide but not in the vehicle control group (Figure 18E). This indicates that luciferase cargo production correlates with T cell activity. This finding was further supported by the increased survival rate of animals injected with RheoBrick PSMA CAR-T cells and treated with lenalidomide compared to the vehicle control group (Figure 18F). Figure 18G schematically illustrates the key results of the animal study, demonstrating that the RheoBrick-mediated antigen-dependent and small molecule-regulated cargo expression platform functions reliably in animal models.

[0356] To our knowledge, there are no other examples of antigen- and small molecule-dependent cargo production platforms in the literature. The low stringency of the CAR-T cell switch systems described in the literature appears to hinder the development of antigen- and small molecule-dependent cargo expression platforms. For example, in other switch platforms, such as the CAR-degron fusion concept, significant residual T cell activation occurs due to incomplete degradation of the CAR (approximately 40% of maximal IFNg production remains in the absence of drug; see Figures 1B and 1D in Weber et al. Science. 2021 Apr 2;372(6537):eaba1786). The low stringency of competing switch platforms precludes the creation of stringent antigen- and small molecule-dependent cargo expression platforms, which is not obvious to those skilled in the art. Due to the unique mechanism of T cell inhibition of the CRASH-IT / RheoBrick switch (competition with endogenous Zap70 and simultaneous recruitment of the inhibitory phosphatases SHP-1 / 2 near the TCR signaling complex), combining RheoBrick with an ITAM signaling-inducible promoter and POI results in near-complete inhibition of T cell signaling (approximately 1% of maximal IFNg production in the absence of drug remains; Figure 19 ), enabling the creation of a stringent antigen- and small molecule-dependent cargo expression platform. Because different embodiments of the RheoBrick switch described herein, e.g., the dependent claims, share the same mechanism of action and can modulate the activity of ITAM signaling-responsive promoters, those skilled in the art will recognize that each RheoBrick / ITAM signaling-inducible promoter-cargo combination disclosed herein represents an inventive contribution over the prior art. The common mechanism of action is independent of the cargo (i.e., the nucleic acid encoding the protein of interest under the control of the inducible promoter). The inducible promoter is induced when the receptor receives an activating signal in the absence of a chimeric protein comprising a docking domain and a drug-associated stability domain as described herein, for example as defined in claim 1 and the dependent claims.Preferably, the nucleic acid encoding the protein of interest (also referred to as cargo), the inducible promoter operably linked to the nucleic acid encoding the protein of interest, or both are independently introduced into a cell according to the present invention, e.g., present in the cell on a vector. In another embodiment, the nucleic acid encoding the protein of interest (also referred to as cargo), the inducible promoter operably linked to the nucleic acid encoding the protein of interest, or both are (or have been) introduced into the genome of the original cell. It is also contemplated that the nucleic acid encoding the protein of interest (also referred to as cargo), the inducible promoter operably linked to the nucleic acid encoding the protein of interest, or both are independently introduced into or present in the original cell on a vector, integrated into the genome (e.g., chromosome), or both. Furthermore, it is unclear whether RheoBrick / ITAM signal-inducible promoter-cargo / CAR-T cells result in greater cargo expression in vivo compared to ITAM signal-inducible promoter-cargo / CAR-T cells without a RheoBrick switch. Surprisingly, in vivo, we found that PSMA CAR-T cells containing a RheoBrick switch and an NFAT-luciferase cargo resulted in higher peak luciferase production and increased persistence of luciferase production compared to PSMA CAR-T cells containing an NFAT-luciferase cargo without a RheoBrick switch (Figure 18).

[0357] To demonstrate that the RheoBrick-mediated antigen- and small molecule-dependent cargo production technology can also be used for cell therapy products that detect antigens using endogenous antigen receptors, tumor-infiltrating lymphocytes (TILs) derived from an ovarian cancer patient were engineered with a lentiviral vector containing a RheoBrick- and ITAM signaling-responsive IL-12 expression cassette (Figure 20A). When engineered TILs were cocultured with ovarian tumor cells derived from the same patient, IL-12 cargo production was strongly induced in the presence of lenalidomide compared to vehicle control (10.7-fold induction, Figure 20B). When engineered TILs were cocultured with the antigen-negative, unrelated tumor cell line PC3, IL-12 cargo production was minimal in the presence of lenalidomide (3.9-fold less cargo production in PC3 cocultures compared to ovarian tumor cell cocultures).

[0358] The flexibility of the RheoBrick® AND gate technology, including the cells, systems, methods, and features of the present invention, as described herein, allows for the placement of numerous therapeutically important biologic modalities downstream of an inducible promoter, such as a cytokine, interleukin, interferon, chemokine, immunocytokine, receptor, ligand, antibody or antibody fragment, bispecific antibody, T cell engager, bispecific T cell engager, checkpoint inhibitor, antagonist, agonist, enzyme, regulatory element, transcription factor, or DNA-binding domain of a transcription factor, as well as combinations of two or more biologic cargo molecules, to be produced at a site of interest defined by the presence of a ligand for the activating receptor and at a desired level controlled, for example, by the amount of a provided small molecule.

[0359] In the present invention, production of cargo molecules can be restricted to cancer sites by using conditional input antigens that are strictly restricted to the tumor microenvironment and / or tumor cells, including, for example, antigens that show heterogeneous expression in tumor tissue.

[0360] Examples of antigens restricted to the tumor microenvironment include fibronectin EDB (EDB-FN) and FAP, which can be detected by CAR-T cells (Wagner et al. Cancer Immunol Res 2021;9:279-290, Bughda et al. Immunotargets Ther. 2021 Aug 5;10:313-323). TCR-T cells targeting the cancer-associated viral antigen HPV E7 are highly specific to tumor cells, and clinical trials have demonstrated that high cell doses (10 11 (Nagarsheth et al. Nat Med. 2021 Mar;27(3):419-425). Furthermore, TCR-T cells specific for the cancer-testis antigen NY-ESO-1 have been tested in clinical trials, and no toxicity due to the transplanted T cells was detected (Robbins et al. Clin Cancer Res. 2015 Mar 1;21(5):1019-27). Other notable target antigens for CAR-T cell therapy include PSMA (Slovin et al. Journal of Clinical Oncology 2013 31:15_suppl, TPS3115-TPS3115), claudin-6 (Mackensen et al. Journal for ImmunoTherapy of Cancer 2021;9), mesothelin (Castelletti et al. Biomark Res. 2021 Feb 15;9(1):11), and GD2 (Richards et al. Front Immunol. 2018;9: 2380), which primarily show tumor-specific expression.

[0361] The controlled cargo release approach of the present invention can also be used to treat non-oncology clinical indications, for example, in the case of autoimmune diseases, as will be appreciated by those skilled in the art, the local immune response can be reversibly suppressed using controlled small molecule local production of immunosuppressive compounds such as IL-10 (P22301) or TGFB (P36897) or anti-inflammatory antibodies at the disease site.

[0362] Materials and Methods Preparation of vector DNA Self-inactivating lentiviral vectors were generated using gene synthesis. Briefly, the expression cassettes shown in Figures 2A, 4A, 5A, 6A, 7A, and 8B were ordered as gene synthesis products flanked by upstream (RSV promoter, 5' LTR (truncated), HIV-1 (psi), RRE, cPPT / CTS) and downstream (3' LTR δU3, SV40 polyA, SV40 ori) lentiviral vector elements derived from the pRRLSIN vector (product no. 12252, Addgene) and cloned into the EcoRV site of the pUC-AMP-GW vector backbone (Genewiz, Azenta). Table 10 summarizes the nucleotide sequences of the complete lentiviral vectors described in the Examples. Table 11 summarizes the nucleotide sequences of the open reading frames, regulatory elements, and structural elements within these vectors. A retroviral vector encoding MP71 PSMA IRESpuro was generated by cloning the synthetic PSMA (UniProt reference number: Q04609) coding sequence, IRES, and puromycin resistance gene into the MP71 vector (Engels et al. Hum Gene Ther 2003;14(12):1155-68). [Table 8-1] [Table 8-2] Rheobrick (2 mutations): two amino acid substitutions within a hybrid zinc finger domain disclosed herein; Rheobrick (4 mutations): four amino acid substitutions within a hybrid zinc finger domain disclosed herein. [Table 9-1] [Table 9-2]

[0363] Cell lines and cell culture K562 cells (ATCC), FLYRD18 cells (Sigma), and NKIRTIL006 cells (Kvistborg et al. Oncoimmunology. 2012 Jul 1;1(4):409-418) were cultured in IMDM / 8% FCS / penicillin-streptomycin (Gibco). HEK293T cells (ATCC), A549 cells (ATCC), and A375 cells (ATCC) were cultured in DMEM / 8% FCS / penicillin-streptomycin (Gibco). HCT116 cells (ATCC) and PC3 cells (ATCC) were cultured in Advanced DMEM / F-12 / 8% FCS / penicillin-streptomycin (Gibco). Nalm6 cells (ATCC) were cultured in RPMI / 8% FCS / penicillin-streptomycin (Gibco). HCT116, HEK293T, and FLYRD18 cells were passaged every 3 days using trypsin-EDTA (Gibco). All cell lines tested negative for mycoplasma using PCR-based screening.

[0364] Lentivirus production Lentiviral particles were produced in HEK293T packaging cells. Briefly, 4.5 million HEK293T packaging cells were seeded per 10-cm dish one day before transfection. The next day, the cell culture medium was refreshed with DMEM supplemented with 8% FCS without antibiotics. 25 μL of X-tremeGENE 9 (Gibco) was mixed with 800 μL of Opti-MEM (ThermoFisher) and incubated for 5 minutes. Next, the Optimem-X-tremeGENE 9 mixture was added to 10 μg of lentiviral plasmid DNA mixture dissolved in water (the plasmid mixture contained 3.5 μg of the transfer vector shown in Figures 2A, 4A, 5A, 6A, 7A, and 8B, and 3.5 μg of pCMVδR8.74 and 3 μg of pMD2.G as packaging plasmids (Zhang et al. Nat Protoc. 2010 Mar;5(3):439-456)). The mixture was incubated for 15 minutes, and the resulting transfection mixture was added dropwise to the packaging cells. The lentivirus-containing supernatant was collected 48 hours post-transfection and used immediately after syringe filtration.

[0365] Retrovirus production Retroviral particles were produced in FLYRD18 packaging cells. One day before transfection, 700,000 FLYRD18 packaging cells were seeded per 10 cm dish. The next day, the cell culture medium was refreshed with IMDM supplemented with 8% FCS without antibiotics. 25 μL of X-tremeGENE 9 was mixed with 800 μL of Opti-MEM and incubated for 5 minutes. The Optimem-X-tremeGENE 9 mixture was then added to 10 μg of retroviral plasmid DNA dissolved in water, incubated for 15 minutes, and the resulting transfection mixture was added dropwise to the packaging cells. The retrovirus-containing supernatant was collected 48 hours post-transfection and used immediately.

[0366] T cell isolation and activation Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor buffy coats (Sanquin, Amsterdam, The Netherlands) by Ficoll-Isopaque density centrifugation (Hokland et al. J Immunol Methods . 1980;32(1):31-39) and cryopreserved until further use. To generate activated T cell populations, PBMCs were thawed in PBS containing 5% FCS, counted, and mixed with CD3 / CD28 Dynabeads (CTS) at a 1:1 cell-to-bead ratio and diluted to a density of 10. 7 After incubation on a tumbler for 30 minutes at room temperature, the mixture was placed on a magnet to remove unbound cells. The bead-bound T cells were then resuspended in RPMI / 10% human serum / penicillin-streptomycin containing 5 ng / ml IL-7 (Peprotech) and 5 ng / ml IL-15 (Peprotech) and diluted to 0.75 × 10 6 Cells were seeded at a density of 1000 cells / mL.

[0367] T cell spin transfer Untreated 6-well cell culture plates were coated with 10 μg / mL Retronectin (Takara) and cultured overnight at 4°C. The next day, the Retronectin solution was removed, and the wells were blocked with 2% BSA (Sigma-Aldrich) in PBS for 30 minutes. Next, 2 million activated T cells (1 × 10 cells / well) in RPMI / 10% human serum / penicillin-streptomycin / 12.5 ng / mL IL-7 and 12.5 ng / mL IL-15 were cultured. 61.5 mL of lentiviral supernatant from two vectors (e.g., CC41 and CC1) was mixed with 3 mL of viral supernatant in a retronectin-coated 6-well plate. For lentiviral co-transduction experiments, 1.5 mL of lentiviral supernatant from two vectors (e.g., CC41 and CC1) was used. The plate was centrifuged at 2,000 rpm for 90 minutes at room temperature (brake off). No selection method was used to enrich for T cells modified with the lentiviral vectors listed in Table 2. HCT116 and PC3 tumor cells transduced with the retroviral MP71 PSMA IRESpuro vector were selected for 3 days with 0.5 μg / mL and 2 μg / mL puromycin, respectively.

[0368] co-culture Six days after transduction, 100,000 T cells were mixed with 100,000 of the indicated target cells (or T cells alone without target cell control) in T cell media in the presence of the indicated concentrations of lenalidomide control or DMSO control in round-bottom 96-well plates and incubated for 24 hours at 37°C. For PSMA BiTE-induced cytokine production and degranulation assays, conditioned medium from the initial coculture (see Figure 8 for details) was collected, filtered, and frozen at -80°C until use. A fresh vial of PBMCs was opened and cultured overnight in standard T cell media containing 5 ng / mL IL-7 and 5 ng / mL IL-15 for cytokine release and degranulation assays. 100 μL of conditioned medium was mixed with 100,000 PBMC cells, 100,000 HCT116 WT cells, or HCT116 PSMA cells in T cell medium supplemented with Golgi Plug (1:1000 dilution, BD, product no. 51-2301KZ) and anti-LAMP1-APC (1:100 dilution, Biolegend, product no. 328620) to prepare 200 μL co-cultures in a round-bottom 96-well plate. The co-cultures were incubated at 37°C for 5 hours.

[0369] For the blinatumomab BiTE-induced cytotoxicity assay, conditioned medium from the first co-culture (see Figure 17 for details) was collected, filtered, and frozen at -80°C until use. A fresh vial of PBMCs was opened, and T cells were enriched using the Dynabeads™ Untouched™ Human T Cells Kit (Thermofisher) and cultured overnight in standard T cell medium containing 5 ng / mL IL-7 and 5 ng / mL IL-15. For the second co-culture, 100,000 naive T cells were cultured with 25,000 CTFR-labeled Nalm6 cells (CD19) in the presence of serially diluted conditioned medium from the first co-culture. + ) and 25,000 CTV-labeled K562 cells (CD19 - After 24 hours, cells were stained with a live / dead cell marker near-infrared dye (ThermoFisher), fixed, and analyzed by FACS to determine the Nalm6 / K562 cell ratio. Specific killing was calculated according to the formula modified from Notoetal. J Vis Exp. 2013; (82): 51105: 100-((antigen) + / antigen - ) Mean of triplicate wells containing target cells and effector cells with / without effector cells and conditioned medium × 100. Antigen-specific cell killing induced by blinatumomab-producing T cell supernatants was normalized by irrelevant cargo (luciferase)-producing T cell supernatants. As a positive control, target cells were cocultured with naive T cells in the presence of the indicated concentrations of purified blinatumomab (product number 100441-2, BPS Bioscience).

[0370] IL-12 ELISA assay Co-culture supernatants were analyzed using ELISA MAX™ Deluxe Set Human IL-12 p70 (Biolegend) according to the manufacturer's instructions. ELISA assays were performed using both undiluted and 1:10 diluted supernatants to fall within the linear range of the standard curve.

[0371] IFNγ ELISA assay Co-culture supernatants were analyzed using ELISA MAX™ Deluxe Set Human IFNγ (Biolegend) according to the manufacturer's instructions. ELISA assays were performed using supernatants diluted 1:10 and 1:1000 to fall within the linear range of the standard curve.

[0372] Flow cytometry Co-cultured cells were washed once with PBS and stained with IR dye (Molecular Probes) at a 1:400 dilution for 5 minutes at 4°C. Subsequently, cells were washed once with FACS buffer (PBS containing 0.5% BSA) and stained with anti-CD8-PerCP Cy5.5 (1:20 dilution, BD, product no. 341050) and anti-CD4 BV711 (1:50 dilution, Biolegend, product no. 317440) for 20 minutes at 4°C. Cells were washed once with FACS buffer and fixed with BD Fixation and Permeabilization Solution for 20 minutes at 4°C. After fixation, cells were washed twice with BD Perm / Wash Buffer. For EGFP expression analysis assays, samples were resuspended in 100 μL of FACS buffer. For PSMA BiTE-induced cytokine production experiments, samples were further stained intracellularly with anti-IFNg-BV421 (1:100 dilution, BD, #564791) and anti-TNFa-BV650 (1:100 dilution, Biolegend, #502938) diluted in permeabilization / wash buffer for 20 minutes at 4°C. Cells were then washed twice and resuspended in 100 μL of FACS buffer. Samples were analyzed directly on a Fortessa Special Order analyzer. Data were analyzed using FlowJo and Prism 9 software.

[0373] Animal experiments All animal experiments were approved by the Animal Welfare Committee of the Netherlands Cancer Institute (NKI) in accordance with national guidelines. Ten-week-old NOD-Scid IL2Rgnull (NSG) mice were subcutaneously injected with PC3-PSMA cells. Two weeks later, mice were randomized according to tumor size. Two-and-a-half million CAR-T cells were injected via the tail vein, and mice were administered 1 mg / kg lenalidomide or vehicle control by oral gavage daily, as shown in Figure 18. The lenalidomide and vehicle control groups were housed in separate cages to prevent exposure of the vehicle control group to lenalidomide in the urine and feces of the treatment groups. Six hours after oral gavage, mice were intraperitoneally injected with 10 μL / g D-luciferin, and luciferase production from CAR-T cells in solid tumors was measured at the indicated time points using a PerkinElmer IVIS Spectrum. Tumor size was measured three times weekly using calipers.

[0374] Tumor-infiltrating lymphocyte (TIL) experiments Ovarian cancer TILs and tumor digests were provided by Dr. John Haanen (NKI). The protocol for expanding ovarian cancer TILs from surgical material and the rapid expansion protocol (REP) have been previously described (van den Berg et al. J Immunother Cancer . 2020 Aug;8(2):e000848). On day 6 of REP, TILs were counted and transfected by spin transfection as described above. On day 14 of REP, 100,000 TILs were cocultured with 100,000 ovarian cancer cells from the same patient or antigen-negative PC3 cells (negative control) in the presence or absence of 500 nM lenalidomide for 24 hours. The supernatants were then analyzed by IL-12 ELISA as described above.

[0375] Example 2 Materials and Methods Cell lines and cell culture FLYRD18 (Sigma-Aldrich), NKIRTIL006 (Kvistborg et al. Oncoimmunology. 2012 Jul 1;1(4):409-418), and Jurkat cells were cultured in IMDM (ThermoFisher) supplemented with 8% FCS (ThermoFisher) and penicillin-streptomycin (100 IU / mL penicillin, 100 μg / mL streptomycin, Sigma-Aldrich). FLYRD18 and NKIRTIL006 cells were passaged every 2–3 days using trypsin-EDTA (ThermoFisher). All cell lines tested negative for mycoplasma using PCR-based screening.

[0376] Retrovirus production Retroviral particles were produced in FLYRD18 packaging cells. Briefly, 700,000 FLYRD18 packaging cells were seeded per 10-cm dish one day before transfection. The following day, cell culture medium was refreshed with IMDM supplemented with 8% FCS without antibiotics. 25 μL of X-tremeGENE 9 (Roche) was mixed with 800 μL of Opti-MEM (ThermoFisher) and incubated for 5 minutes. Subsequently, the Optimem-X-tremeGENE 9 mixture was added to 10 μg of retroviral plasmid DNA dissolved in water, incubated for 15 minutes, and the resulting transfection mixture was added dropwise to the packaging cells. The retrovirus-containing supernatant was harvested 48 hours posttransfection and used immediately or flash-frozen in liquid nitrogen.

[0377] Plasmid We have previously described prior art retroviral vectors encoding CRASH-IT containing a single hybrid, dual zinc finger degron (pMP71-Zap70 2xSH2-PD1-ZFP91 ZF4 β hairpin-IKZF1 ZF2 α helix-IKZF1 ZF3 iresEGFP, WO 2021 / 080427(A1)), a CRASH-IT variant containing a single hybrid, single zinc finger degron (pMP71-Zap70 2xSH2-PD1-(ZFP91 ZF4 β hairpin-IKZF1 ZF2 α helix iresEGFP, WO 2021 / 080427(A1)), and an HLA class I-restricted CDK4 TCR (TCR 17, Stronen et al., Science. 2016 Jun 10;352(6291):1337-41).

[0378] The IKZF1 ZF3 α-helix coding sequence in the pMP71-Zap70 2xSH2-PD1-ZFP91 ZF4 β-hairpin-IKZF1 ZF2 α-helix-IKZF1 ZF3 iresEGFP vector was replaced with a gene synthesis product (IDT, Iowa, USA) encoding the ZFP91 ZF5 α-helix sequence using the Gibson assembly method (Gibson et al. Nat Methods. 2009 May;6(5):343-5). The resulting degron was designated the "double-hybrid degron" (Figures 9 and 11).

[0379] A highly diverse SynFinger library (approximately 50,200 variants) was synthesized by Twist Biosciences (California, USA) using their Combinatorial Variant Library (CVL) service, as shown in Figure 11. pMP71-Zap70-Siglec11-FKBP12F36V iresEGFP was previously described (Sahillioglu et al. Hum Gene Ther. 2021 Oct;32(19-20):1029-1043). The FKBP12F36V coding sequence in this vector was replaced with gene synthesis products encoding double hybrid degrons (FIGS. 9 and 11) or double hybrid degron mutants containing the G14N / K21A, G14M / N15R, L17I / K21L, E4R / Q12L, Q12R / K13T, or Q12R / K13V double substitutions, or the Q12R / K13V / G14N / K21A quadruple substitution, using the Gibson assembly method.

[0380] screening Quality control of the SynFinger library was performed by Twist Biosciences, and as shown in Figure 11, all but two of the approximately 50,200 synthetic sequences were confirmed in the clone library. 150,000 Jurkat cells were transduced with the SynFinger library (SynFingers containing the CRASH-IT switch) at a transduction efficiency of 12.8% and sorted for high EGFP expression to enrich for transduced cells. To quality control the SynFinger-transduced Jurkat cells, SynFinger sequences were amplified and analyzed by next-generation sequencing (NGS). Ten randomly selected SynFinger-encoding DNA sequences, or DNA sequences encoding the same SynFinger amino acid sequence but containing two nucleotide mismatches due to alternative codon usage, were used to assess the ability to distinguish true SynFinger sequences from noise in the NGS data. 10 / 10 SynFingers encoding perfect nucleotide sequence matches were confirmed in the NGS data, while sequences containing two nucleotide mismatches were not detected.

[0381] Jurkat cells expressing the SynFinger library were treated with 50 nM lenalidomide (a suboptimal dose that activates only approximately 50% of cells expressing the parent double hybrid degron-containing CRASH-IT switch in Jurkat cells, compared to the maximally effective 1000 nM lenalidomide dose) or mock-treated for 24 h, followed by activation with anti-CD3 / anti-CD28 antibodies for 5 h in the presence or absence of 50 nM lenalidomide. Cells were then stained with IR dye (1:400) and anti-CD69-PE (1:200). Cells were sorted using an Aria Fusion cytometer as live cells (IR staining negative), EGFP-positive, CD69-high expressing (top 5%), or CD69-low expressing (bottom 5%).

[0382] The cells were then washed with PBS, and the cell pellets were frozen at -20°C until DNA extraction. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions, with 5 × 10 cells per column. 6 DNA was isolated using 100 cells. DNA was eluted in 22 μL of RNase-free water. Deep sequencing adapters and indexes were added to the SynFinger-encoding DNA sequence using PCR amplification. Briefly, 3 μg of genomic DNA (20 μL), indexed forward and reverse primers (10 μM, 2.5 μL each), and 25 μL of NEBNext® High-Fidelity 2X PCR Master Mix were added to the PCR reaction. The optimized PCR reaction (98°C for 30 seconds, 25 cycles of (98°C for 10 seconds, 56°C for 10 seconds, 72°C for 5 seconds) and 72°C for 3 minutes) was performed using an Eppendorf PCR cycler.

[0383] Indexed PCR products were run on an agarose gel and relative DNA concentration...

Claims

1. (a) a receptor capable of receiving an activating signal; (b) a chimeric protein, i. a docking domain capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives the activating signal; ii. a drug-regulated protein stability domain; and a chimeric protein comprising: (c) an inducible promoter operably linked to a nucleic acid encoding a protein of interest, the inducible promoter being induced when the receptor receives an activating signal in the absence of the chimeric protein; Cells containing

2. 2. The cell of claim 1, wherein the inducible promoter is selected from the group consisting of NFAT promoter, NF-κB promoter, AP-1 promoter, CD69 promoter, CD137 promoter, IFNγ promoter, TNFα promoter, GM-CSF promoter, IL-2 promoter, IL-4 promoter, IL-6 promoter, IL-8 promoter, IL-13 promoter, and IL-17 promoter.

3. 3. The cell of claim 1 or 2, wherein the nucleic acid encoding the protein of interest is operably linked to a nucleic acid encoding an RNA degradation element (RDE), preferably the RDE is an AU-rich element (ARE), preferably the ARE is or is derived from IL-2, IL-3, IL-4, IL-6, IL-13, IL-17, GATA-3, IFNγ, TNFα, CSF2, FasL, or c-fos 3′ untranslated region (UTR).

4. 4. The cell of any one of claims 1 to 3, wherein the cell is selected from the group consisting of a T cell, a TCR-expressing T cell, a T cell expressing a modified TCR, a CAR T cell, an NK cell, a CAR NK cell, a tumor-infiltrating lymphocyte, a macrophage, and a CAR macrophage.

5. 5. The cell of claim 1, wherein the receptor is selected from the group consisting of a T cell receptor (TCR), a chimeric antigen receptor (CAR), and an NK cell receptor (NKR), and / or the receptor is directed against an antigen, such as a tumor antigen.

6. 6. A cell described in any one of claims 1 to 5, wherein the docking domain comprises an SH2 domain capable of binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) contained in the receptor, and preferably the SH2 domain is derived from a protein selected from the group consisting of Zap70, Syk, and Lck.

7. The cell of any one of claims 1 to 6, wherein the ITAM present in the receptor is present in a TCR, a CAR, or an NKR, and more preferably the ITAM is derived from or present in a CD3ζ chain, a CD3ε chain, a CD3δ chain, a CD3γ chain, an FceRIγ chain, or a DAP12.

8. 8. The cell of claim 1, wherein the docking domain further comprises an immunoreceptor tyrosine-based switch motif (ITSM), and / or an immunoreceptor tyrosine-based inhibition motif (ITIM), or an ITSM and an immunoreceptor tyrosine-based inhibition motif (ITIM), preferably wherein the ITIM and / or ITSM is derived from an inhibitory receptor protein, preferably an inhibitory immunoreceptor protein, preferably a protein selected from the group consisting of PD1, BTLA, SIRPalpha, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, and LY9.

9. 9. The cell of any one of claims 1-8, wherein the drug-regulated protein stability domain is a CRBN polypeptide substrate domain capable of binding to a CRBN protein in response to a drug, preferably thereby promoting degradation of the chimeric protein via the ubiquitin pathway.

10. the drug regulatory protein stability domain comprises a Cys residue capable of drug-induced binding to a CRBN polypeptide. ２ -His ２ Zinc finger domain, preferably Cys ２ -His ２ The cell of claim 1 , wherein the zinc finger domain is a hybrid zinc finger domain.

11. The Cys ２ -His ２ The zinc finger domain has a first Cys ２ -His ２ A β-hairpin loop derived from the zinc finger domain and a second Cys ２ -His ２ and a zinc finger domain-derived α-helical region. Preferably, the hybrid zinc finger domain is a zinc finger domain having the first Cys ２ -His ２ The β hairpin loop and / or the second Cys derived from a zinc finger domain ２ -His ２ 11. The cell of claim 1, comprising one, two, three, four, or more amino acid substitutions in the alpha helix region derived from a zinc finger domain.

12. The drug regulatory protein stability domain may further comprise an additional, e.g., a second Cys ２ -His ２ 12. The cell of claim 1, comprising a zinc finger domain, preferably a further, e.g., a second, hybrid zinc finger domain.

13. 13. The cell of any one of claims 1 to 12, wherein the agent is an immunomodulatory imid drug (IMiD), preferably the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iveldomide, CC-885, salts and analogues thereof.

14. 14. The cell of any one of claims 1 to 13, wherein the cell further comprises an immunomodulatory imid drug (IMiD), preferably the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iverdomide, CC-885, salts and analogues thereof.

15. 15. The cell of any one of claims 1 to 14, wherein the nucleic acid encoding a protein of interest encodes a cytokine, an interleukin, an interferon, a chemokine, a receptor, a ligand, an antibody or antibody fragment, a bispecific antibody, a T cell engager, a bispecific T cell engager, a checkpoint inhibitor antagonist, an agonist, an enzyme, a regulatory element, a transcription factor, or a DNA-binding domain of a transcription factor.

16. 16. The cell according to any one of claims 1 to 15, wherein the cytokine is an interleukin, preferably selected from the group consisting of IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, and IL-21, and / or the chemokine is CCL5, CCL19, or CCL21, and / or the protein is a cytokine receptor, and / or the cytokine receptor is TGFBR (TGF-β receptor) and / or TGFBR2.

17. (a) a receptor capable of receiving an activating signal; (b) operably linked to a promoter; i. a docking domain capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives the activating signal; ii. a drug-regulated protein stability domain; and a nucleic acid encoding a chimeric protein comprising: (c) an inducible promoter operably linked to a nucleic acid encoding a protein of interest, the inducible promoter being induced when the receptor receives an activating signal in the absence of the chimeric protein; Cells containing

18. The cell of claim 17 , in which the chimeric protein is expressed and / or the protein of interest is expressed.

19. 20. A method for regulating expression of a protein of interest, comprising providing an activation signal in vitro or in vivo to a cell described in any one of claims 1 to 18 in the presence of a drug capable of modulating a drug-regulated protein stability domain.

20. 20. A method for regulating expression of a protein of interest in a subject, comprising administering to the subject a drug capable of regulating the drug-regulated protein stability domain, wherein the subject comprises a cell described in any one of claims 1 to 19.

21. 21. The method of any one of claims 1 to 20, comprising administering to the subject a cell of any one of claims 1 to 20.

22. 22. The method of any one of claims 1 to 21, wherein the method is for the treatment of cancer and / or the treatment of tumors in a subject.

23. 23. A cell according to any one of claims 1 to 22 for use as a medicament, preferably for use in the treatment of cancer and / or the treatment of a tumor in a subject, preferably wherein said treatment comprises administering to said subject a cell according to any one of claims 1 to 22, and optionally administering a drug capable of modulating said drug regulatory protein stability domain.

24. 24. A cell described in any one of claims 1 to 1 to 23 for use as a pharmaceutical agent described in any one of claims 1 to 23, wherein administering a drug capable of modulating the drug-regulated protein stability domain comprises varying the dose of the drug capable of modulating the drug-regulated protein stability domain.

25. 25. A drug for use as a pharmaceutical, preferably for use in the treatment of cancer and / or the treatment of a tumor in a subject, said treatment comprising administering to said subject a cell described in any one of claims 1 to 24 and administering said drug, said drug being capable of regulating said drug regulatory protein stability domain.

26. A vector or a combination of vectors comprising at least (a) one or more genes encoding a receptor capable of receiving an activation signal as defined in any one of claims 1 to 25, and / or one or more genes encoding a chimeric protein as defined in any one of claims 1 to 25, and / or one or more inducible promoters as defined in any one of claims 1 to 25, and a gene encoding a protein of interest as defined in any one of claims 1 to 25 operably linked to said inducible promoters; (b) in the case of a combination of vectors, a first vector and a second vector, each independently comprising at least one of a gene encoding a receptor capable of receiving an activation signal as defined in any one of claims 1 to 25, a gene encoding a chimeric protein as defined in any one of claims 1 to 25, or an inducible promoter as defined in any one of claims 1 to 25 and a gene encoding a protein of interest as defined in any one of claims 1 to 25, operably linked to the inducible promoter; Including, Preferably, the vector or the combination of vectors comprises one or more RNA degradation elements as defined in any one of claims 1 to 25, and more preferably, the vector or the combination of vectors has a configuration comprising one or more genes encoding a receptor capable of receiving an activation signal as defined in any one of claims 1 to 25, one or more genes encoding a chimeric protein as defined in any one of claims 1 to 25, one or more inducible promoters as defined in any one of claims 1 to 25, a gene encoding a protein of interest as defined in any one of claims 1 to 25 operably linked to the inducible promoter, and optionally one or more RNA degradation elements as defined in any one of claims 1 to 25, as shown in Figure 6. A vector or combination of vectors.

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