In-situ production of receptor and drug dependent modulated biologics

Abstract

The present invention relates generally to the production of biologics using genetically modified cells, including genetically modified immune cells. An engineered cell (e.g., a T cell) comprises a receptor (e.g., a chimeric antigen receptor) capable of receiving an activation signal (e.g., binding to an antigen). The cell further comprises a chimeric protein comprising a docking domain capable of binding to a receptor, thereby inhibiting normally induced signal transduction when the receptor receives an activation signal. The chimeric protein further comprises a drug modulated protein stability domain. The cell further comprises an inducible promoter operably linked to a nucleic acid encoding a protein of interest (e.g., a biological product) wherein the inducible promoter is induced when the receptor receives an activation signal in the absence of a chimeric protein. Drug-modulated chimeric proteins capable of modulating the stability and level of drug-modulated protein stability domains, thereby modulating the production of biological products.

Description

FIELD OF THE INVENTION

[0001] The present invention generally relates to the production of biologics using genetically modified cells, including genetically modified immune cells. The modified cells (e.g., T cells) contain receptors capable of receiving activation signals (e.g., activation signals that bind to an antigen), such as chimeric antigen receptors. The cells also contain a chimeric protein containing a docking domain that is capable of binding to the receptor, thereby inhibiting signal transduction that is normally induced when the receptor receives an activation signal. The chimeric protein also contains a drug-regulated protein stability domain. The cells also contain an inducible promoter operably linked to a nucleic acid encoding a protein of interest (e.g., a biologic), wherein the inducible promoter is induced when the receptor receives an activation signal in the absence of the chimeric protein. A drug that can modulate the drug-regulated protein stability domain modulates the stability and level of the chimeric protein, thereby modulating the production of the biologic. BACKGROUND OF THE INVENTION

[0003] The background description includes information that may be helpful in understanding the present invention. This does not mean that any of the information provided herein is prior art or relevant to the present invention, nor that any of the specifically or implicitly cited publications are prior art.

[0004] Adoptive T cell therapy has shown significant activity in several human malignancies. Specifically, chimeric antigen receptor (CAR) T cell therapy has shown great clinical activity in hematologic malignancies, leading to the approval of many drugs. In addition, in melanoma, the clinical activity of tumor-infiltrating lymphocytes (TILs) has been well established. Although not optimal, there is thus increasing evidence that tumor-reactive immune cell products (e.g., T cell products) show activity and can be used to treat cancer, particularly solid cancers of this type.

[0005] At the same time, the impact of current immune cell-based therapies (e.g., T cell receptor (TCR)-T cell therapy, CAR-T cell therapy, and TIL therapy) on other solid malignancies has thus far been modest and remains suboptimal. Based on these data, it is proposed that next-generation immune cell-based therapies (e.g., adoptive T cell therapy) should not only involve the infusion of tumor-reactive T cells. Instead, such cell products may be endowed with the ability to induce intratumoral inflammation. For example, preclinical and clinical data provide evidence that the local production of (potent) cytokines (e.g., IL-12) can drive tumor control.

[0006] However, due to severe toxicity, systemic administration, or the unregulated production of these cytokines from adoptively transferred cells is hindered.

[0007] A second problem in the field of immune cells (e.g., the CAR and TCR T fields) is the lack of sufficient tumor-selective targets. For example, on-target / off-tumor toxicity can be a life-threatening complication of CAR-T cell and bispecific T cell engager (BiTE) therapeutic products (Edeline et al., J Hematol Oncol. 2021;14:65, Bonifant et al., Mol Ther Oncolytics. 2016 Apr 20;3:16011). Thus, there are only a few sufficient tumor-specific antigens (TSAs) that can be safely targeted with cell-based therapies without triggering adverse reactions associated with on-target / off-tumor toxicity.

[0008] To improve the therapeutic index of cell-based therapies and reduce on-target / off-tumor toxicity, attempts have been made to confine the immune response to the tumor microenvironment. Examples of such methods include SynNotch-based CAR-T cells (Morsut et al., Cell. 2016 Feb 11;164(4):780-91), which can use a synthetic Notch receptor to detect the presence of a first antigen and then result in the expression of an antigen receptor that can detect a second antigen. This approach can increase the stringency of tumor detection, but the intensity and duration of the induced immune response are not regulated. This lack of control can be problematic because 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;34:45-55), and can also lead to cell exhaustion (Gumber et al., EBioMedicine. 2022 Mar;77:103941). In addition, the immunogenicity of non-human sequences in engineered cell therapy products is also a concern and can, for example, result in the rejection of adoptively transferred T cells.

[0009] In view of this, there is a great expectation for new products, compositions, methods and uses that allow (preferably simultaneously) spatial (at or near the tumor, such as the tumor microenvironment (TME)) and temporal (i.e., the ability to control the activity level in a timely manner) control of the production of a target protein (such as a biologic like a cytokine), but such are not readily available at present. At the same time, immune cell therapies, including T cell therapies, that induce immune cell activation (such as inducing T cell activation) in a manner that more restrictively confines the immune response to the target (such as the tumor microenvironment) are highly desirable, but are not readily available at present. In particular, there is a clear need in the art for reliable, effective and renewable products, compositions, methods and uses that are useful for immune cell therapies, particularly for treating cancer and tumors such as solid tumors. Thus, the technical problem addressed by the present invention can be seen from providing products, compositions, methods and uses that meet any of the above needs. The said technical problem is solved by the claims and the embodiments described hereinafter. SUMMARY OF THE INVENTION

[0011] 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), for treating tumors (such as solid tumors) in various cancers. As embodied and broadly described herein, the present invention is directed to the surprising discovery that the therapeutic window of such therapies (such as cancer therapies) can be improved by the invention disclosed herein, providing genetically engineered cells, gene constructs and methods that provide for the effective delivery of biologics (such as cytokines) as well as other biologic agents (such as antibodies, T cell engagers) and any other target protein (such as a protein with therapeutic effect) to, for example, tumor cells, particularly solid tumors. The target protein can also be more than one target protein, and / or can be a protein complex (a part of a protein complex). In the present context, the invention is illustrated in the context of various immune cells (including T cells and NK cells), and the production of a target protein in the form of, for example, a cytokine or a bispecific T cell engager, for treating tumors (cancer), particularly solid tumors. Based on the general disclosure herein, it will be readily understood by those skilled in the art that the present invention is not limited to such examples. Based on the general teachings provided herein, those skilled in the art are able to apply the invention disclosed herein to other contexts, such as by other cells, other receptors, other target proteins to be produced and other diseases to be treated.

[0012] Accordingly, the present invention provides a novel and improved method that allows for the production of a protein of interest (or biologic) from genetically modified cells, particularly immune cells (CAR-T cells, TCR-T cells, TIL cells, NK cells, and other suitable cells known to those skilled in the art). The production mode strictly depends on an activation signal (such as an antigen), which activates the receptor receiving such a signal, and on regulation using a drug (such as a small molecule). In some embodiments, the activation signal is specific to (or confined to) a specific microenvironment in a patient's body, such as the tumor microenvironment. By providing a patient with the cells according to the present invention, the receptors present in the cells can receive the activation signal provided in the specific microenvironment (such as an antigen present in a tumor, preferably a tumor-specific antigen). However, due to the expression and / or presence of a chimeric protein in the cells, once the receptor receives an activation signal (e.g., only when the ligand of the receptor binds to it), the chimeric protein can interact with or bind to the receptor. Therefore, the receptor in the cells according to the present invention cannot or can only to a very limited extent transduce the signal received through cell transduction and cannot or can only to a limited extent activate the cells. Since the chimeric protein interacts with or binds to the receptor that has received the activation signal (i.e., has been activated), the chimeric protein according to the present invention can block or inhibit the transduction of the signal received by the receptor. For example, when the (domain in the) chimeric protein binds to the receptor that has been activated (by receiving an activation signal), such as binding to the cytoplasmic portion of a transmembrane cell surface receptor (including a receptor complex), the activated receptor is inhibited or blocked in transducing the signal into the cell, and thus is inhibited or blocked in further activating the cell. For example, it prevents the cell from reacting naturally to the activation signal.

[0013] The chimeric protein according to the present invention further comprises a drug-regulated protein stability domain, which causes the degradation of the chimeric protein in the presence of the drug, thereby releasing the blockage of signal transduction through the receptor. This release, in turn, allows for signal transduction through the cell and will allow the cells according to the present invention to produce a protein of interest (or biologic). By providing a drug to a patient having the modified cells according to the present invention, it is now possible for the first time to strictly regulate, both in a spatial (in the specific microenvironment where the activation signal may be present) and temporal (at the desired moment) manner, the production of a biologic (protein of interest) by the modified cells. In addition, changing the amount of the drug (such as the amount of the drug provided to the patient) will allow for strict regulation of the production level of the biologic. Accordingly, the present invention allows for strict regulation of the activation of cells (such as T cells) in a patient's body and the in situ (such as in the tumor microenvironment) production and delivery of a protein of interest (such as a therapeutic agent) to targeted cells (such as tumor cells).

[0014] Thus, by using genetically modified cells, particularly immune cells, the present invention (illustrating the general teachings in one or more embodiments) enables precise control of the timing and dosage of the production of a protein of interest, as well as local delivery (targeted delivery). This allows for the production of an appropriate amount of a biologic at the disease site for the first time. Accordingly, the present invention allows for the optimization of the therapeutic window and prevention of overproduction of such biologic agents to avoid side effects, while also allowing for the prevention of exhaustion of immune cells upon activation (by receiving an activation signal). Thus, it has surprisingly been found that control over the production level of the protein / biologic of interest is provided by the pharmaceutically regulated chimeric proteins described herein. Additionally, surprisingly, the present invention enables improved synthesis of the protein of interest intracellularly. Further, surprisingly, the present invention has found that in the absence of a drug (particularly an immunomodulatory imide drug such as lenalidomide) that regulates the protein stability domain according to the present invention, the residual activity of cells (activated), such as T cells, according to the present invention is very low compared to prior art systems. In other words, the system disclosed herein allows for control of the amount of protein produced at the target site.

[0015] In an example, the inventors created a transgenic design in which the production of a protein of interest (biologic payload, biologic) is operationally related to the level of ITAM signaling in immune cells, and in which the level of ITAM signaling is regulated by a chimeric protein (also referred to herein as a rheostat switch). The rheostat switch used in the present invention and illustrated in the example, particularly exerts tight control over immune cell activation using an orally administered, clinically approved drug or small molecule (such as lenalidomide) (see also WO2021 / 080427). The RheoBrick concept may also be referred to as "CRASH-IT", as discussed, for example, in Sahillioglu et al., Cancer Immunol Res. 2021 Sep;9(9):999-1007 and Sahillioglu et al., Hum Gene Ther. 2021 Oct;32(19-20):1029-1043.

[0016] Structurally, the rheostat switch contained in the cells according to the present invention (e.g., ) consists of three functional domains; a docking domain that forms a reversible interaction with the antigen receptor and also contains, for example, in T cells (such as tumor-infiltrating lymphocytes (TILs)) and NK cells, an inhibitory domain or portion that inhibits the TCR / CAR / NKR (NK cell receptor) signaling pathway, particularly the ITAM receptor-dependent signaling pathway (such as the TCR / CAR / NKR signaling pathway), and a degron domain (or pharmaceutically regulated protein stability domain) that controls the stability of the rheostat switch.

[0017] In addition, the engineered cells according to the invention comprise a transgene under the control of a promoter which is induced / expressed in the cells according to the invention only or preferentially in the absence of a chimeric protein (such as a rheostat switch), for example after providing a sufficient amount of a drug to the cell / patient, when activating the cell (activating the cell via a receptor capable of receiving an activation signal). The transgene encodes a protein of interest, and thus the protein of interest is (preferably) produced in the presence of a chimeric protein (such as a rheostat switch), for example in the case of providing a drug to the patient that triggers the degradation of the chimeric protein (such as a rheostat switch), thereby releasing the blockade of signal transduction to the receptor activated by the activation signal.

[0018] In some embodiments, there are provided nucleic acids and / or vectors which (1) encode a chimeric protein according to the invention, or (2) comprise an inducible promoter operably linked to a nucleic acid encoding a protein of interest, or (3) encode a receptor according to the invention, or a combination thereof. The nucleic acids and / or vectors may additionally comprise additional elements detailed herein to further improve the expression of the chimeric protein and the protein of interest according to the invention, thereby improving the precise control of the timing and dosage of biologic production ex vivo or in vivo (such as in a patient).

[0019] Thus, according to a first aspect, there are provided engineered cells comprising:

[0020] (a) a receptor (such as a TCR, CAR, and / or NKR and other ITAM receptors) capable of receiving an activation signal;

[0022] (b) a chimeric protein comprising:

[0023] i. a docking domain capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives an activation signal;

[0024] transduction;

[0025] ii. a drug-regulated protein stability domain; and

[0026] (c) an inducible promoter operably linked to a nucleic acid encoding a protein of interest, wherein the inducible promoter is induced when the receptor receives an activation signal in the absence of the chimeric protein.

[0027] In some embodiments according to the invention, the engineered cells can be immune cells. The engineered cells can also be selected from the group consisting of: T cells, CAR T cells, NK cells, CAR NK cells, macrophages, tumor infiltrating lymphocytes (TIL), and CAR macrophages.

[0028] In embodiments of the present invention, the engineered cells are present in an animal. In embodiments of the present invention, the engineered cells are present in a non-human animal. In embodiments of the present invention, the engineered cells are present in a human. In embodiments of the present invention, an animal is provided that comprises at least one engineered cell according to the present invention. In embodiments of the present invention, a non-human animal is provided that comprises at least one engineered cell according to the present invention. In embodiments of the present invention, a human is provided that comprises at least one engineered cell according to the present invention. In embodiments of the present invention, the animal, non-human animal or human further comprises a tumor. In embodiments of the present invention, the animal, non-human animal or human has cancer.

[0029] In some embodiments according to the present invention, the receptor is a transmembrane receptor, such as a cell surface receptor, such as a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a natural killer cell receptor (NKR) and / or wherein the receptor is antigen-specific, such as a tumor antigen.

[0030] In some embodiments according to the present invention, the activating signal is a ligand capable of interacting with the receptor, thereby providing an activating signal that can activate the receptor to conduct a signal throughout the cell. In some embodiments, the ligand is an extracellular ligand. In some embodiments, the ligand is a protein. In a preferred embodiment, the activating signal is an antigen, such as a tumor-specific antigen.

[0031] In some embodiments according to the present invention, the chimeric protein is a cytoplasmic chimeric protein. In some embodiments, the docking domain comprised in the chimeric protein binds to the receptor only (or preferentially) when the receptor has been activated by the activating signal. In some embodiments, the docking domain comprises an SH2 domain capable of binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) comprised in the receptor (wherein the ITAM becomes phosphorylated upon receipt of the activating signal (e.g., upon interaction of the receptor with a ligand (such as an antigen)). In some embodiments according to the present invention, the SH2 domain is from a protein selected from the group consisting of Zap70, Syk and Lck.

[0032] In some embodiments, the ITAM present in the receptor is present in a TCR, CAR or NKR, and more preferably, wherein the ITAM is from or located in the CD3ζ chain, CD3ε chain, CD3δ chain, CD3γ chain, FceRIγ chain or DAP12.

[0033] 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 wherein the ITIM and / or ITSM are from an inhibitory receptor protein (preferably an inhibitory immunoreceptor protein), preferably from a protein selected from the group consisting of PD1, BTLA, SIRPα, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, and LY9. The ITSM, ITIM, or both contained in the docking domain inhibit signal transduction induced when the receptor receives an activating signal. In other words, in these embodiments, when the receptor receives an activating signal (e.g., binds a ligand, such as an antigen), the chimeric protein according to the invention binds to the receptor due to the presence of the docking domain (e.g., comprising an SH2 domain), and subsequently blocks signal transduction through the presence of an inhibitory domain according to the invention (e.g., comprising an ITSM, ITIM, or both). Although the SH2 domain is capable of providing inhibition of ITAM signaling (as an example of signal transduction by the receptor after it has received its activating signal), it has been found that in some embodiments, the mere presence of the SH2 domain provides only partial inhibition of ITAM signaling, particularly when ITAM signaling is strong. Thus, for strict inhibition of ITAM signaling, preferably an ITIM, ITSM, or ITIM and ITSM domains are present.

[0034] In some embodiments according to the invention, the drug-regulated protein stability domain is a CRBN polypeptide substrate domain that is capable of binding to the CRBN protein in response to a drug, preferably thereby promoting ubiquitin pathway-mediated degradation of the chimeric protein. In some embodiments, the drug-regulated protein stability domain comprises a Cys2-His2 zinc finger domain capable of drug-induced binding to the CRBN polypeptide, preferably wherein the Cys2-His2 zinc finger domain is a heterozygous zinc finger domain (e.g., the domain is a fusion protein / domain that comprises portions obtained from at least two different zinc fingers and that is not naturally occurring). It has been found that when used to control the level of the chimeric protein, such a Cys2-His2 zinc finger domain allows for strict control and regulation of cell activation and thus regulation of the production of the protein of interest, particularly when the Cys2-His2 zinc finger domain is combined with other preferred features of the chimeric protein according to the invention and an inducible promoter is operably linked to the nucleic acid encoding the protein of interest.

[0035] In some embodiments, the Cys2-His2 zinc finger domain is a chimeric zinc finger domain, which is 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, preferably where the chimeric zinc finger domain contains one, two or more amino acid substitutions relative to 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 chimeric zinc finger domain contains two amino acid substitutions relative to 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 chimeric zinc finger domain contains three amino acid substitutions relative to 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 chimeric zinc finger domain contains four amino acid substitutions relative to 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 chimeric zinc finger domain is the chimeric zinc finger domain disclosed herein.

[0036] In some embodiments, the drug-regulated protein stability domain contains additional Cys2-His2 zinc finger domains, preferably additional chimeric zinc finger domains.

[0037] In some embodiments, the drug capable of inducing the degradation of the chimeric protein according to the present invention is an immunomodulatory imide drug (IMiD), preferably where the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iberdomide, CC-885, their salts and analogs.

[0038] In some embodiments, the nucleic acid encoding the protein of interest encodes a cytokine, interleukin, interferon (in some embodiments, the interferon is interferon γ), 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 or IL-21. In an embodiment according to the invention, the nucleic acid encoding the protein of interest encodes a chemokine. In an embodiment according to the invention, the chemokine is CCL5, CCL19 or CCL21. In an embodiment according to the invention, the nucleic acid encoding the protein of interest encodes a cytokine receptor. In an embodiment according to the invention, the cytokine receptor is TGFBR (TGF-β receptor) or TGFBR2.

[0039] In some embodiments, the inducible promoter is selected from the group consisting of: NFAT promoter, synthetic NFAT promoter, (synthetic) NF-κB promoter, AP-1 promoter, (native) IL-2 promoter, IFNγ promoter, TNFα promoter, IL-6 promoter, CD69 promoter and CD137 promoter. In some embodiments, the gene encoding the protein of interest (e.g., transgene) is introduced into an endogenous locus that responds to signaling of an activating receptor, such as where the expression of said locus is controlled by an inducible promoter according to the invention. In one such example, the transgene can be introduced, for example, into the IFNγ locus and be under the control of an inducible IFNγ promoter according to the invention. Those skilled in the art will understand that in embodiments, the (trans)gene can be introduced into any position in the cell genome as long as it is under the control of an inducible promoter according to the invention. Similarly, an inducible promoter can be introduced into the genome and operably linked to a gene (e.g., endogenous or intentionally introduced (i.e., as a transgene)).

[0040] 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, both the inducible promoter and the nucleic acid encoding the protein of interest are contained in a vector, such as in an expression vector, or such as in an expression cassette. In some embodiments, the nucleic acid containing the inducible promoter and the nucleic acid encoding the protein of interest are chimeric nucleic acids. In some embodiments, the nucleic acid containing the inducible promoter has been provided to the cell. In some embodiments, the inducible promoter is present on a vector that has been introduced into the cell. In embodiments of the present invention, an inducible promoter has been provided to the cell such that the inducible promoter has been incorporated into the genome of the cell. In some embodiments, the nucleic acid containing the protein of interest has been provided to the cell. In some embodiments, the nucleic acid encoding the protein of interest is present on a vector that has been introduced into the cell. In some embodiments, the nucleic acid encoding the protein of interest has been provided to the cell such that the inducible promoter has been incorporated into the genome of the cell. In embodiments of the present invention, the inducible promoter and / or the nucleic acid encoding the protein of interest are recombinant inducible promoter and / or nucleic acid encoding the protein of interest. In embodiments of the present invention, the inducible promoter and / or the nucleic acid encoding the protein of interest are (for the cell) non-native inducible promoter and / or nucleic acid encoding the protein of interest. In embodiments of the present invention, the inducible promoter and / or the nucleic acid encoding the protein of interest are not in the position in the cell chromosome where such inducible promoter and / or nucleic acid encoding the protein of interest are naturally present in such cells. In other words, in embodiments of the present invention, except for the (transgenic) inducible promoter and / or the (transgenic) nucleic acid encoding the protein of interest according to the present invention, the same inducible promoter and / or nucleic acid encoding the protein can independently occur naturally in the cells according to the present invention.

[0041] In some embodiments, the chimeric nucleic acid containing the inducible promoter and the nucleic acid encoding the protein of interest is not integrated into the genome of the cell. In some embodiments, the chimeric nucleic acid containing the inducible promoter and the nucleic acid encoding the protein of interest has been integrated into the genome of the cell.

[0042] In some embodiments, the nucleic acid (or vector) provided and used to form the engineered cell according to the present invention, the nucleic acid (or vector) alone or in combination contains a nucleic acid operably linked to a promoter and encoding a chimeric protein according to the present invention, and / or a nucleic acid operably linked to an inducible promoter according to the present invention and encoding a protein of interest. In some embodiments, a vector is further provided that contains or further contains a nucleic acid encoding a receptor according to the present invention, the nucleic acid being operably linked to a promoter.

[0043] In some embodiments, a nucleic acid encoding a 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 where if the nucleic acid is transcribed, an mRNA transcript encoding the protein linked to the RDE is obtained, and where the RDE is an RDE that results in RNA stabilization by interacting with an RNA-stabilizing protein). In some embodiments, the ARE is or is from the IL2 or IFNγ 3' untranslated region (UTR).

[0044] In other aspects of the invention, provided are methods of modulating the expression of a protein of interest, the methods comprising providing an activating signal, in vitro or in vivo, to a modified cell according to the invention in the presence of an effective amount of a drug capable of modulating a drug-modulatable protein stability domain.

[0045] In another aspect of the invention, provided are methods of modulating the expression of a protein of interest in a subject, the methods comprising administering to the subject an effective amount of a drug, where the drug is a drug capable of modulating a drug-modulatable protein stability domain (and thereby modulating the level or turnover of a chimeric protein according to the invention), and where the subject comprises a modified cell according to the invention.

[0046] In another aspect of the invention, provided are cells according to the invention for use as a medicament, preferably for treating cancer in a subject and / or for treating a tumor in a subject, preferably where the treatment comprises administering to the subject the cells according to the invention, and optionally administering a drug capable of modulating a drug-modulatable protein stability domain.

[0047] In some embodiments, the invention also provides changing the dose of a drug capable of modulating a drug-modulatable protein stability domain, thereby modulating the expression of a protein of interest.

[0048] In some embodiments, the invention provides a drug for use as a medicament, preferably for treating cancer in a subject and / or for treating a tumor in a subject, preferably where the treatment comprises administering to the subject the cells according to the invention, and administering the drug, where the drug is capable of modulating a drug-modulatable protein stability domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Embodiments of the invention will be further described below in conjunction with the drawings, where:

[0051] Figure 1 A and B: Chimeric proteins according to the invention (e.g., rheostat switches, e.g., Schematic diagram for mediating the control of the expression of a protein of interest (POI). In this example, the POI-encoding cargo gene is placed downstream of an ITAM signaling-responsive promoter (such as the NFAT promoter). ITAM signaling is provided by an antigen receptor, such as in the case of cognate antigen ligation, by a CAR, T cell receptor, Fc receptor, or NK cell receptor. To reversibly and titratably control the expression of the POI, immune cells are modified to express a chimeric protein according to the present invention, such as a rheostat switch, such as switch. The chimeric protein (such as a rheostat switch), such as may have three functional domains: First, an SH2-based docking domain that can engage with the phosphorylated ITAM motif present in the activated antigen receptor. Second, a domain containing an inhibitory ITIM / ITSM motif that can recruit inhibitory SHP1 and / or SHP2 phosphatases, thereby downregulating the ITAM signaling pathway. Third, a drug-regulated protein stability domain (or degron) that can dynamically control the rheostat switch (such as ), protein stability, and thus regulate the degree of ITAM signaling in immune cells. The rheostat switch (such as ) mediates fine control of ITAM signaling, which can regulate the expression level of the POI in an antigen-dependent and drug (small molecule)-regulated manner. Thus, the expression of the POI is controlled by an AND logic gate, which can precisely control the production level of the POI in the antigen-positive tumor microenvironment.

[0052] Figure 2 : The rheostat switch (such as switch) can achieve antigen-dependent and drug (such as lenalidomide)-regulated expression of the protein of interest. A) Schematic diagram of vector design. The rheostat switch (such as switch) in this figure and the following figure refers to the Zap70(2xSH2)-Siglec11 signaling domain-based Switch design (SEQ ID NO: 204), wherein the SynFinger encoding comprises a double hybrid degron (SEQ ID NO: 205) with a Q12R / K13V substitution. B) Primary human T cells were modified with the vector shown in (A). The data shows the total EGFP expression levels of CD4 (B - C) cells and CD8 (D - E) cells after co - culture with CD19 - negative K562 cells or CD19 - positive Nalm6 cells in the absence or presence of 500 nM lenalidomide, calculated by multiplying the percentage of EGFP - positive cells by their mean fluorescence intensity (MFI) of EGFP expression. C and E respectively depict the comparison of fold - induction levels in Nalm6 cell co - cultures (B and D) in the presence and absence of lenalidomide. Error bars represent standard deviation (n = 3).

[0053] Figure 3 : Schematic diagram of an AND logic gate containing an AU - rich element (ARE). By placing a 3'UTR containing an ITAM - signaling - responsive ARE downstream of the cargo gene (encoding the POI), the stringency and fold - induction of POI expression according to the present invention by the chimeric protein (or rheostat switch) (e.g., ) can be further increased. This indicates that the rheostat (e.g., ) is capable of controlling the expression of a POI operably linked to different ITAM - signaling - responsive elements, e.g., the element is not only the NFAT promoter but also, for example, a 3’UTR containing an ARE. In the absence of ITAM signaling, the 3'UTR containing an ARE reduces mRNA stability, while in the presence of ITAM signaling, RNA - binding proteins (RBPs) such as NF90 and HuR are exported from the nucleus, bind to the 3'UTR containing an ARE and increase mRNA stability and mRNA export from the nucleus, thereby producing the POI.

[0054] Figure 4 : An AU - rich element (ARE) in the 3’UTR of the cargo expression cassette can improve the fold - induction and stringency of expression control. A) Schematic diagram of the vector design. B) Primary human T cells were modified with the vector shown in (A). The data depicts the secreted IL12(p70) after co - culture with or without CD19 - positive Nalm6 cells in the presence or absence of 500 nM lenalidomide. The lenalidomide - induced IL - 12 fold - induction is shown in the bar graph. Error bars represent standard deviation (n = 3).

[0055] Figure 5: Combinations of 3'UTRs containing ARE elements can enhance the fold induction and stringency of expression control. A) Schematic of vector design. B) Primary human T cells modified with the vectors shown in (A). Data depict IL12(p70) secreted after co-culture with or without CD19-positive Nalm6 cells in the presence or absence 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).

[0056] Figure 6 : Impact of vector design on fold induction and stringency of cargo production control. A) Schematic of vector design. B) Primary human T cells modified with the vectors shown in (A). Data depict the percentage of EGFP-positive CD4 cells after co-culture with or without CD19-negative K562 cells or CD19-positive Nalm6 cells in the presence or absence of 500 nM lenalidomide. Error bars represent standard deviation (n = 3). Abbreviations: CD19 CAR, second-generation anti-CD19 chimeric antigen receptor containing the CD28 co-stimulatory signaling domain and the CD3ζ domain; rheostat switch containing a switch design based on the Zap70(2xSH2)-Siglec11 signaling domain- where SynFinger encodes a double-heterozygous degron containing the Q12R / K13V substitution; EGFP, enhanced green fluorescent protein; pNFAT, synthetic NFAT promoter containing 4 copies of the NFAT binding sequence and a minimal promoter (Jutz et al., J Immunol Methods. March 2016; 430:10 - 20); pMSCV, murine stem cell virus U3 promoter derived from the 3' long terminal repeat of the MSGV1 vector (Hughes et al., Hum Gene Ther. April 2005; 16(4):457 - 72); pCMV, cytomegalovirus minimal promoter; IL2 3'UTR, 3' untranslated region of interleukin 2; IFNg 3'UTR, 3' untranslated region of interferon γ; SV40 pA, simian virus 40 polyadenylation signal; CTE, constitutive transport element (CTE) of Mason-Pfizer virus (Bray et al., Proc Natl Acad Sci U S A. February 15, 1994; 91(4):1256–1260); WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; BGH pA, bovine growth hormone polyadenylation signal.

[0057] Figure 7: Secreted IL-12 levels can be fine-tuned by adjusting the small molecule dose. A) Schematic diagram of vector design. B) Primary human T cells modified with the vector shown in (A). Data depict IL12 (p70) secreted after co-culture with or without CD19-positive Nalm6 cells at the indicated concentrations of lenalidomide. Error bars represent standard deviations (n=3).

[0058] Figure 8 :The invention disclosed herein enables the secretion of antigen-dependent and small molecule-regulated T cell engagers (also known as BiTEs) to redirect unmodified naive T cells to cells expressing target (tumor) antigens. A) Logic gate circuit diagram. B) Schematic diagram of vector design. C) Illustration of co-culture conditions. Primary human T cells were modified with the vector shown in (B), which contained a second-generation CD19 CAR, and NFAT-PSMABiTE-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). As shown in the left figure, only in the presence of both Nalm6 target cells and lenalidomide, (according to the present invention) to secrete PSMABiTE. The conditioned medium from the first co-culture was harvested, filtered, and used for the second co-culture as shown in the right figure. The presence of PSMA-BiTE in the conditioned medium triggers the release of IFNg and TNFa cytokines and degranulation by unmodified naive T cells only in the presence of PSMA-positive HCT116 tumors, but not in the presence of PSMA-negative HCT116 WT tumors. (D) and (E) depict the first co-culture conditions, a summary of the expected state of BiTE production by modified T cells, the second co-culture conditions, and the expected degranulation / cytokine production of unmodified naive T cells under antigen-dependent and small molecule-regulated TCE activity control, respectively. (FI) Data describe the production of IFNg (F) and TNFa (HI) by CD8+T cells (FH) and CD4+T cells (I) in the second co-culture as described in (E), as well as the expression of cell surface LAMP1 (G). Error bars represent standard deviations (n=3).

[0059] Figure 9: Sensitivity of zinc finger degrader to IMiD can be improved by altering the second zinc finger sequence. (A) Schematic of a set of zinc finger degraders containing the ZFP91 ZF4β hairpin and the IKZF1 ZF2α helix as a heterozygous first zinc finger. In this set, the degrader contains a second zinc finger sequence selected from IKZF1 ZF3 (single heterozygous, dual zinc finger degrader), IKZF1 ZF3β hairpin-ZFP91 ZF5α helix (dual heterozygous degrader), or the degrader does not contain a second zinc finger (single heterozygous, single zinc finger degrader). (B-E) Primary human T cells modified with an HLA class I-restricted CDK4 TCR plus Zap70-PD1-degrader CRASH-IT switch were pretreated with thalidomide at the indicated concentrations, where the degrader sequences were selected from (A). Data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+ cells, EGFP-high cells, and CD8+ T cells after co-culture with NKIRTIL006 melanoma cells in the continued presence of the indicated concentration of thalidomide. Error bars represent standard deviation (n = 2). Data represent two independent experiments.

[0060] Figure 10 : Schematic overview of a non-limiting embodiment according to the present invention.

[0061] Figure 11 : Design of the synthetic zinc finger (SynFinger) library. The SynFinger library contains Zap70-PD1-degrader CRASH-IT switch variants, where the parental zinc finger degrader contains single or double amino acid substitutions, or no substitution. The figure depicts the sequence of the dual heterozygous degrader used as the parental zinc finger sequence in the SynFinger library screening. The amino acids indicated by the arrows are replaced with amino acids encoded by any other gene except cysteine. Two cysteines and a conserved glycine in the ZFP91 ZF4β hairpin, and two histidines in the IKZF1 ZF2α helix remain unchanged. Substitution mutations are numbered starting from the first amino acid of the ZFP91 ZF4β hairpin (marked with an asterisk).

[0062] Figure 12: The top 100 degradation resolvases enrich for certain amino acid substitutions in the stator. After removing SynFingers with low sequence reads, the SynFinger degradation resolvases were ranked according to the enrichment index (EI) value (see Examples). This figure depicts the number of each amino acid substitution found in combination with other amino acid substitutions in the top 100 SynFinger degradation resolvases. For example, in the top 100 SynFingers, the Q12R mutation was observed in 11 double amino acid substitution combinations, as well as any of K13T, L17M, K13V, E4L, L17Y, L22H, C10A, I20R, N15S, E4W, and E4Q.

[0063] Figure 13 : SynFinger fusion proteins enable sensitive control of immune cell function. Subsets of SynFingers identified in the SynFinger library screen were individually validated. (A - H) Primary human T cells were modified with an HLA class I - restricted CDK4 TCR plus a Zap70 - Siglec11 - degradation resolvase CRASH - IT switch, where the degradation resolvase sequence contained the parental zinc - finger degradation resolvase ( Figure 9 and 11 the double - heterozygous degradation resolvase shown therein), or contained the parental zinc - finger degradation resolvase with the following mutations: G14N / K21A, G14M / N15R, L17I / K21L, E4R / Q12L, Q12R / K13T, or Q12R / K13V. As a control, primary human T cells modified with an HLA class I - restricted CDK4 TCR plus a vector control were used. Cells were pretreated with 5 nM lenalidomide or not. The data depict the intracellular IFNγ, IL2, TNFα, and cell - surface LAMP1 expression of CDK4 TCR+ cells, EGFP intermediate cells, and CD8+ T cells after co - culture with NKIRTIL006 melanoma cells in the continuous absence (E - H) or continuous presence of 5 nM thalidomide. The dashed horizontal line represents the cytokine production and degranulation levels of cells modified with the CRASH - IT switch containing the parental zinc - finger degradation resolvase. Error bars represent the standard deviation (n = 2). A one - sided t - test was used to compare the SynFinger and vector control groups with the parental zinc - finger group: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, n.s. = not significant. The data represent two independent experiments.

[0064] Figure 14:Compared to zinc finger degrons that are not of the present invention, the CRASH-IT switch containing SynFinger can restore cytokine production and degranulation at lower IMiD concentrations. (A-D) Primary human T cells modified with an HLA class I-restricted CDK4 TCR plus Zap70-Siglec11-degron CRASH-IT switch were pretreated with the indicated concentrations of lenalidomide or left untreated, where the degron sequence contained a prior art zinc finger degron ( Figure 9 the single heterozygous, dual zinc finger degron shown in Figure 9 ), the parental zinc finger degron ( 11 the dual heterozygous degron shown in

[0065] Figure 15 ), or the parental zinc finger degron with the following mutations: G14N / K21A, L17I / K21L, or Q12R / K13V. Data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+ cells, EGFP intermediate cells, and CD8+ T cells after co-culture with NKIRTIL006 melanoma cells in the continuous presence or absence of the indicated concentrations of lenalidomide (A-D). Error bars represent standard deviation (n = 3). Data represent two independent experiments. (E) The table describes the EC50 lenalidomide (nM) values for the cells in (A-D), as well as the fold change in EC50 between the prior art zinc finger degron and SynFinger containing the Q12R / K13V substitution. Figure 1 and 3 ), or the parental zinc finger degron with the following mutations: G14N / K21A, Q12R / K13V, or Q12R / K13V / G14N / K21A. Data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+ cells, EGFP intermediate cells, and CD8+ T cells after co-culture with NKIRTIL006 melanoma cells in the continuous presence or absence of the indicated concentrations of lenalidomide (A-D). Error bars represent standard deviation (n = 3). Data represent two independent experiments.

[0066] Figure 16: 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 modified with the vector shown in (A). Data depict IL12(p70) secreted after co-culture with antigen-positive Nalm6 cells, PC3-PSMA cells, NKIRTIL006 cells, A549 cells, A375 cells, or Mel624 cells in the presence or absence 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).

[0067] Figure 17 : RheoBrick can be used to control the cytotoxicity of blinatumomab derived from CAR-T cells. A) Schematic of vector design. B) Description of co-culture conditions. Primary human T cells were modified with the CC81+CC16 vector containing the second-generation PSMACAR, RheoBrick, and an ITAM-signaling-responsive expression cassette encoding blinatumomab. To normalize cytotoxicity measurements, T cells were modified with the CC76+CC16 vector containing the second-generation PSMACAR, RheoBrick, and an ITAM-signaling-responsive expression cassette encoding an irrelevant cargo (luciferase). The modified T cells were co-cultured with antigen-positive PC3-PSMA cells or antigen-negative PC3 cells for 24 hours (first co-culture) in the presence or absence of 500 nM lenalidomide. As shown in the left panel, RheoBrick (according to the present invention) can secrete blinatumomab only in the presence of both PC3-PSMA target cells and lenalidomide. The conditioned medium from the first co-culture was harvested, filtered, and used for a second co-culture as shown in the right panel. Specific killing of CD19-positive Nalm6 cells was measured as described in the methods. C) Specific killing of Nalm6 cells after co-culture with naive T cells for 24 hours in the presence of serial dilutions of conditioned medium from the first co-culture. D) Positive control of specific killing of Nalm6 cells after co-culture with naive T cells in the presence of the indicated concentrations of purified blinatumomab. Error bars represent standard deviation (n = 3). Data represent two independent experiments.

[0068] Figure 18 : RheoBrick regulates cargo production in vivo. A) Schematic of vector design. B) Description of the animal study. NSG mice were injected subcutaneously with 5 million PC3-PSMA cells. Two weeks later, according to tumor size (~40 - 50 mm 3) Mice were randomly grouped and injected with human T cells modified with the CC83+CC84 vector encoding RheoBrick&PSMACAR (groups X1 and X2) or the CC130+CC131 vector encoding PSMACAR but not 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 were administered vehicle control daily, while groups X2 and Y2 were administered 1 mg / kg lenalidomide daily (for groups X1, X2, Y1, Y2, n = 6. For the untreated group, n = 12). C) Luciferase production was measured using an IVIS imaging system at the indicated time points. D) The density of luciferase cargo production was calculated by normalizing the IVIS imaging results to tumor size. E) Tumor volume was measured using calipers. F) Survival analysis. G) Graphical summary of the animal study.

[0069] Figure 19 : The production of endogenous IFNγ was tightly regulated using the RheoBrick switch. Primary human T cells were transduced with the CC83 vector encoding PSMACAR and RheoBrick or the CC130 vector encoding PSMACAR and huEGFRt. Data show the production of endogenous IFNγ after 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.

[0070] Figure 20 : Ovarian cancer tumor-infiltrating lymphocytes (TILs) produce RheoBrick-mediated antigen- and small molecule-dependent IL-12 cargo. A) Schematic of vector design. B) TILs derived from ovarian cancer patients were modified with the CC82 vector containing RheoBrick and an ITAM signaling-responsive expression cassette encoding scIL-12. Data depict the secretion of IL-12(p70) after co-culture with ovarian tumor cells or antigen-negative PC3 cells derived from the same patient in the absence or presence of 500 nM lenalidomide. The fold induction of lenalidomide-induced IL12 cargo production is shown in the bar graph. Error bars represent standard deviation (n = 3).

[0071] Description

[0072] Definition

[0073] Part of this disclosure contains copyrighted material (such as, but not limited to, diagrams, device photographs, or any other aspect of this submission that is or may be copyrighted in any jurisdiction). The copyright owner does not object to anyone faxing copies of the patent document or patent disclosure, provided that it appears in the patent office's patent file or records, but reserves all copyrights otherwise.

[0074] The chapter headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter described.

[0075] Throughout the specification and claims, various terms related to the methods, compositions, uses, and other aspects of the present invention are used. Unless otherwise indicated, such terms shall have their ordinary meanings in the art to which the present invention pertains. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein may be used in the practice of testing the present invention, the preferred materials and methods are described herein.

[0076] For the purposes of the present invention, the following terms are defined below.

[0077] As used herein, the singular terms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, etc.

[0078] As used herein, "about" and "approximately" when referring to measurable values such as amounts, duration of time, etc., mean including variations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, still more preferably ±0.1%, as these variations are applicable to practicing the disclosed invention.

[0079] As used herein, the term "and / or" refers to a situation where one or more of the stated circumstances may occur alone, may occur in combination with at least one of the stated circumstances, or may even occur together with all of the stated circumstances.

[0080] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" should be understood to be the same as "5 or less", i.e., 5, 4, 3,... -10, -11, etc.

[0081] As used herein, the term "comprising" shall be interpreted as inclusive and open-ended, rather than exclusive. Specifically, the term and its variants mean including the specified features, steps, or components. These terms should not be construed as excluding the presence of other features, steps, or components. It also encompasses the more restrictive "consisting of".

[0082] As used herein, "conventional techniques" or "methods known to those skilled in the art" refer to situations where the methods of conventional techniques used in the methods according to the present invention would be obvious to a skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology, and related fields is well-known to those skilled in the art and is discussed in various manuals and literature references.

[0083] As used herein, the term "exemplary" means "serving as an example, instance, or illustration" and should not be construed as excluding other configurations disclosed herein.

[0084] As used herein, the term "cancer" refers to a physiological condition in a mammal that is typically characterized by unregulated cell growth. The terms "cancer", "neoplasm", and "tumor" are often used interchangeably to describe cells that have undergone malignant transformation, which results in pathological changes in the host organism. Primary cancer cells can be distinguished from non-cancerous cells by techniques known to those skilled in the art. Cancer cells as used herein include not only primary cancer cells but also cancer cells derived from the primary cancer cell, including metastatic cancer cells and cell lines derived from cancer cells. Examples include solid tumors and non-solid tumors or hematological tumors. Preferably, the tumor is a solid tumor. Examples of cancers 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 lymphoma, colon cancer, (malignant) melanoma, thyroid cancer, papillary thyroid cancer, lung cancer, non-small cell lung cancer, and lung adenocarcinoma). It is well-known that tumors can metastasize from a first site to one or more other body tissues or sites. Treatment of a "neoplasm", "tumor", or "cancer" in a patient includes treatment of the primary cancer and, where appropriate, treatment of metastases.

[0085] As used herein, the term "chimeric gene" or "chimeric nucleic acid" refers to any gene or nucleic acid that does not normally exist in nature in a species, particularly a gene or nucleic acid in which one or more portions of the nucleotide sequence are not related to each other in nature. For example, a promoter or 3'UTR is not related in nature to a portion or all of the transcription region or to another regulatory region, or different portions of the transcription region are not related in nature. The term "chimeric gene" should be understood to include expression constructs in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences. In some embodiments, the chimeric gene of the chimeric nucleic acid can be used to produce a chimeric protein.

[0086] As used herein, the terms "chimeric polypeptide", "chimeric protein", or "fusion protein" refer to any polypeptide that does not normally exist in nature in a species, particularly a polypeptide in which one or more portions of the amino acid sequence are not related to each other in nature. For example, a chimeric protein can comprise an N-terminal portion consisting of a first amino acid sequence and a C-terminal portion consisting of a second amino acid sequence, where the first amino acid sequence and the second amino acid sequence are not related to each other in nature and / or are not related to each other in this order in nature. A chimeric protein can be obtained, for example, by transcription and translation of a chimeric gene of nucleic acid.

[0087] As used herein, the term "immunomodulatory drug", "immunomodulatory imide drug", or "IMiD" refers to compounds known in the art. IMiDs include thalidomide, pomalidomide, lenalidomide, ibalidomide (CC-220), avadomide (CC-122), and CC-885, or pharmaceutically acceptable salts thereof; these compounds may also be referred to as cereblon modulators (CRBN modulators). Thalidomide, lenalidomide, and pomalidomide have all been approved for the treatment of various diseases, while other IMiDs or cereblon modulators are under review. The compound can be in the form of a free acid or free base, or a pharmaceutically acceptable salt.

[0088] As used herein, the term "isolated", when referring to a polynucleotide (nucleic acid) or a polypeptide (protein), refers to a protein or nucleic acid that is present in a non-natural environment, for example, a protein or nucleic acid that is separated from its natural environment. For example, an isolated protein or polypeptide according to the present invention refers to a protein that is no longer in its natural environment, such as a protein in vitro or in a recombinant host cell. The term refers not only to such proteins or nucleic acids isolated from natural sources but also to such proteins or nucleic acids produced artificially or synthetically. In the context of the present invention, it will be clear to the person skilled in the art whether a reference to a protein, polypeptide, nucleic acid, or polynucleotide includes a reference to an "isolated" protein, polypeptide, nucleic acid, or polynucleotide.

[0089] As used herein, the term "immunoreceptor tyrosine-based activation motif (ITAM)" refers to a conserved sequence of four amino acids that is repeated twice and is present in the cytoplasmic tail (i.e., intracellular domain) of certain cell surface proteins of the immune system. A half-ITAM contains a tyrosine residue (Y) that is separated from a leucine residue (L) or isoleucine residue (I) by any two other amino acids. The consensus sequence of a half-ITAM is YxxL / l. Two half-ITAMs are typically separated from each other by 6 to 8 amino acids to form a complete ITAM. The consensus sequence of an ITAM is YxxL / lx(6-8)YxxL / l. ITAMs play an important role in signal transduction in immune cells and, among other things, they are present in the cytoplasmic tails of cell signaling molecules in the T cell receptor complex (CD3ε chain, CD3δ chain, CD3γ chain, and / or CD3ζ chain). In NK cells, ITAMs are present in the NK cell receptor complex, which contains the CD3ζ chain, the γ chain of the immunoglobulin receptor FcεRI, and DAP12 (Lanier et al., Nat Immunol. May 2008;9(5):495–502). ITAMs are also present in chimeric antigen receptor (CAR) complexes, which contain the CD3ζ chain (Abate-Daga et al., Mol Ther Oncolytics. 2016;3:16014), the CD3ε chain (Nolan et al., Clin Cancer Res. Dec 1999;5(12):3928-41), the γ chain of the immunoglobulin receptor FcεRI (Ren-Heidenreich et al., Cancer Immunol Immunother. Oct 2002;51(8):417-23), and DAP12( et al., J Immunol. Apr 1, 2015;194(7):3201-12).

[0090] As used herein, the term "immunoreceptor tyrosine-based inhibitory motif (ITIM)" generally refers to a conserved amino acid sequence found in the cytoplasmic tails of many inhibitory receptors of the immune system. The ITIM motif contains a serine residue (S), an isoleucine residue (I), a valine residue (V), or a leucine residue (L), which is separated from a tyrosine residue (Y) by any other amino acid residue (x), and the tyrosine residue (Y) is separated from an isoleucine residue (I), a valine residue (V), or a leucine residue (L) by any two other amino acids. The consensus tag is S / I / V / LxYxxI / V / L. In vivo, inhibitory receptors with ITIM interact with their ligands, resulting in the enzymatic phosphorylation of the ITIM motif by Src kinases, enabling them to 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), as well as 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). By dephosphorylating ITAMs in TCRs, CARs, and other immunoreceptors, PTPs can reverse the activating effects of ITAM phosphorylation. Lipid phosphatases regulate cell signaling by altering the concentrations of lipid phosphates and their dephosphorylated products.

[0091] As used herein, the term "immunoreceptor tyrosine-based switch motif (ITSM)" refers to a conserved amino acid sequence found in the cytoplasmic tails (or cytoplasmic domains or intracellular domains or intracytoplasmic domains; in other words, the protein portions present in the cytoplasm of a cell (as opposed to in the membrane and / or extracellular space)) of many inhibitory receptors of the immune system. The ITSM motif contains a threonine residue (T) that is separated from a tyrosine residue (Y) by any other amino acid residue, and the tyrosine residue (Y) is separated from a valine residue (V) or an isoleucine residue (I) by any other two amino acid residues. The consensus tag is TxYxxV / I. Similar to inhibitory receptors with ITIMs, inhibitory receptors with ITSMs interact with their ligands, resulting in enzymatic phosphorylation of the ITIM motif by Src kinases, enabling them to recruit SH2-containing phosphatases (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). However, in other studies, the ITSM motif has been shown to be mainly responsible for the inhibitory action of PD1, while the role of the ITIM motif is limited (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).

[0092] As used herein, the term "linker", when referring to a portion of a protein, refers to a stretch of amino acid sequence that joins two portions of a protein, such as in a fusion protein. Generally, such molecules do not have a specific biological activity other than to link or maintain a minimal distance or other spatial relationship between proteins. However, in certain embodiments, a linker can be selected to affect some properties of the linker and / or the protein, such as the folding, net charge, or hydrophobicity of the linker.

[0093] As used herein, the term "non-natural" refers to a polypeptide, polynucleotide, or domain contained within such a polypeptide or polynucleotide, and which does not exist in nature, e.g., does not exist in (human) cells, i.e., where one or more portions of the polypeptide, polynucleotide, or domain are not related to each other in nature. An example is a fusion protein or chimeric protein according to the present invention.

[0094] As used herein, the term "pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable composition form for administration to a cell or a subject. The compositions according to the invention may also be administered in combination with other agents, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. Pharmaceutical compositions typically also contain one or more pharmaceutically acceptable carriers (or excipients) in addition to the pharmaceutically active agent. The pharmaceutical compositions can be specifically formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for: (1) oral administration, e.g., drench (aqueous solution or non-aqueous solution or suspension), tablets, pills, powders, granules, pastes; (2) parenteral administration, e.g., in the form of a sterile solution or suspension, e.g., by subcutaneous injection, intramuscular injection or intravenous injection; (3) topical application, e.g., application to the skin in the form of a cream, ointment or spray; (4) intravaginal or intrarectal, e.g., in the form of a vaginal suppository, cream or foam; or (5) aerosol, e.g., in the form of an aqueous aerosol, liposomal formulation or solid particles containing the compound. The drugs, therapeutic agents, medicaments and pharmaceutical compositions according to the invention can be formulated for administration by a variety of routes, including but not limited to parenteral, intravenous, intraarterial, intramuscular, intratumoral and oral. The drugs, therapeutic agents, medicaments and compositions can be formulated in fluid or solid form. The fluid formulations can be formulated for administration by injection to a selected area of the human or animal body.

[0095] As used herein, the terms "protein" and "polypeptide" refer to molecules composed of chains of amino acids, without regard to a particular mode of action, size, three-dimensional structure or source. 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 in its natural environment, e.g., a protein or polypeptide in vitro or in a recombinant host cell.

[0096] As used herein, the term "SH2 domain" refers to the SRC homology 2 domain. The SH2 domain is a structurally conserved protein domain contained within the Src oncoprotein and many other intracellular signal transduction proteins. The SH2 domain allows proteins containing these domains to dock with phosphorylated tyrosine residues on other proteins. Thus, the SH2 domain is a modular protein domain that can act as an adaptor and mediate protein-protein interactions by binding to phosphorylated peptides in their respective protein binding partners.

[0097] As used herein, "subject" or "patient" refers to an organism to be treated, e.g., an organism to which administration is contemplated. According to the present invention, a subject can be any subject, including but not limited to humans, males, females, infants, children, adolescents, adults, young adults, middle-aged or elderly individuals, and / or other primates or mammals. Preferably, the subject is a human patient. The subject may have been diagnosed with cancer or be suspected of having cancer.

[0098] As used herein, "treatment", "treating", "alleviation", "mitigation", and "amelioration" all refer to methods of obtaining a beneficial or desired result (including but not limited to a therapeutic benefit) in the context of a subject to be treated. A therapeutic benefit refers to eradicating or ameliorating the underlying disorder being treated. Additionally, a therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, although the patient may still be afflicted with the underlying disorder.

[0099] Detailed Description

[0100] The present invention is defined herein, particularly in the appended claims. Subject matter not covered by the claims does not form part of the invention claimed.

[0101] It is contemplated that any method, use, or composition described herein can be practiced relative to any other method, use, or composition described herein. Embodiments discussed in the context of a method, use, and / or composition according to the present invention can be employed relative to any other method, use, or composition described herein. Thus, embodiments relating to a method, use, or composition can also be applied to other methods, uses, and compositions according to the present invention.

[0102] Any reference in the specification to a method of treatment also refers to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method of treating the human (or animal) body by therapy.

[0103] As embodied and broadly described herein, the present invention relates to the surprising discovery that engineered cells can now be provided that can be tightly regulated in a spatial (in a specific microenvironment) and temporal (at a desired moment) manner to produce biologics (target proteins) for the first time.

[0104] Thus, the present invention allows for precise regulation of the activation of cells (such as T cells) in a patient's body, as well as the in situ (e.g., in the tumor microenvironment) production and delivery of a protein of interest (e.g., a therapeutic agent) to targeted cells (e.g., tumor cells). This allows for the production of the required amount of a biological agent at the disease site for the first time. Thus, the present invention allows for optimization of the therapeutic window of cells (especially immune cells) and control of the production of such biological agents to avoid or prevent side effects, while the present invention allows for prevention of the exhaustion of immune cells upon activation (by receiving an activation signal). Furthermore, in a preferred embodiment, the chimeric protein according to the present invention consists of human protein domains. Additionally, in a preferred embodiment, the protein of interest consists of human protein domains.

[0105] According to a first aspect of the present invention, there is provided a cell (such as a modified cell) comprising:

[0106] a receptor capable of receiving an activation signal;

[0107] a chimeric protein comprising:

[0108] a docking domain capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives an activation signal;

[0109] a drug-regulated protein stability domain; and

[0110] an inducible promoter operably linked to a nucleic acid encoding a protein, wherein the inducible promoter is induced when the receptor receives an activation signal in the absence of the chimeric protein.

[0111] The cell can be any cell, such as a modified cell, such as a genetically engineered cell. In other words, the cell according to the present invention is a cell that has been modified to contain a receptor, a chimeric protein, and an inducible promoter operably linked to a nucleic acid encoding a protein of interest, and / or a nucleic acid containing a nucleic acid encoding the receptor according to the present invention, a nucleic acid encoding the chimeric protein according to the present invention, and / or an inducible promoter operably linked to a nucleic acid encoding a protein of interest.

[0112] In some embodiments, the original cell is modified by providing the cell with the chimeric protein according to the present invention or a nucleic acid encoding such a chimeric protein (preferably operably linked to a promoter). In some embodiments, the original cell is (further) modified by providing the cell with a nucleic acid containing an inducible promoter and further containing a gene encoding a protein of interest, wherein the inducible promoter is operably linked to the gene encoding the protein of interest. In some embodiments, the original cell is (further) modified by providing the cell with a receptor or a nucleic acid encoding the receptor (preferably operably linked to a promoter).

[0113] The receptor can be any suitable receptor capable of receiving an activation signal, preferably where the receptor activates an intracellular pathway upon receiving the activation signal, thereby resulting in the transduction of the activation signal through the cell. The receptor can be a cytoplasmic receptor or a transmembrane receptor, preferably a transmembrane receptor. Preferably, the 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, which can interact with the extracellular environment, for example, with a cognate ligand of a receptor expressed on the cell surface of other cells (such as an antigen) or a cognate ligand of a receptor present in the extracellular environment (such as secreted by other cells, such as specific extracellular matrix proteins, cytokines, hormones, or antibodies). In some embodiments, the receptor can interact with or bind to a ligand (which provides an activation signal) administered to the patient during the treatment of the patient's disease.

[0114] The activation signal according to the present invention can be any signal capable of interacting with or binding to the receptor and thereby causing receptor activation. In the context of the present invention, activation of the receptor represents, for example, the induction of signal transduction through the cell after the activation signal interacts with the receptor. Non-limiting examples of such activation signals include any cognate binding partner, which includes antigens, tumor antigens, proteins, antibodies (including agonistic or antagonistic antibodies), other cells, nucleic acids, drugs, and small molecules, which are capable of activating the receptor in the context of the present invention. Preferably, the activation signal is a ligand, i.e., a cognate binding partner of the receptor, which binds to or interacts with 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 extracellular to the cell according to the present invention, such as an antigen present on the cell surface of a tumor cell or an antigen secreted or present in the extracellular environment.

[0115] The chimeric protein according to the present invention is preferably a cytoplasmic chimeric protein. The chimeric protein is expressed in the cell according to the present invention and can be provided to the cell according to the present invention, for example, by providing the cell with a nucleic acid encoding the chimeric protein according to the present invention, and wherein the nucleic acid encoding the chimeric protein according to the present invention is operably linked to any suitable promoter. Preferably, the chimeric protein comprises or consists of human protein domains.

[0116] As will be described in detail below, the chimeric protein comprises a docking domain that is capable of binding to a receptor and inhibiting signal transduction induced when the receptor receives an activation signal. The binding of the docking domain to the receptor is preferably a reversible binding, which means 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, for example, a transmembrane cell surface receptor (such as TCR, CAR or NKR (NK cell receptor)), it preferably interacts with or binds to the cytoplasmic part of the receptor.

[0117] As will be described in detail below, the chimeric protein further comprises a drug-regulated protein stability domain (sometimes also referred to herein as a "degron"). The drug-regulated protein stability domain can be any suitable drug-regulated protein stability domain, and various suitable examples are described herein and / or are known to those skilled in the art. The drug-regulated protein stability domain contained in the chimeric protein according to the present invention allows the regulation of the stability of the chimeric protein in response to a drug. The drug is a drug that is capable of interacting with or binding to the drug-regulated protein stability domain, thereby regulating the stability of the chimeric protein according to the present invention, for example, by regulating the degradation of the chimeric protein according to the present invention. By regulating the stability of the chimeric protein according to the present invention, the degree of binding of the chimeric protein according to the present invention to the receptor can be regulated, and accordingly, the signal transduction of the receptor activated by the activation signal through the cell can be regulated, for example, the signal transduction of the receptor activated by the binding of the receptor to a ligand (such as an antigen recognized by the receptor) through the cell. In a non-limiting example, the chimeric protein comprises 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 sequences according to SEQ ID NO 178–179, SEQ ID NO 180 or SEQ ID NO 181-191 or SEQ ID NO:192-201. Those skilled in the art should understand that those sequences defined above are also included, in which 1, 2, 3, 4, 5, 6, 10, 15 amino acids are deleted, replaced or inserted. The chimeric polypeptide may further comprise additional (multiple) amino acid sequences adjacent to the amino acids defined above, as long as the chimeric polypeptide according to the present invention remains functional within the scope of the present invention. Other preferred chimeric proteins are also as described herein.

[0118] As will be described in detail below, the cells according to the present invention further comprise an inducible promoter operably linked to a nucleic acid encoding a protein of interest, wherein the inducible promoter is induced when the receptor receives an activating signal in the absence of the chimeric protein. Those skilled in the art will understand that the protein of interest can be any suitable protein selected by those skilled in the art, for example, for the purpose of treating a disease, condition or disorder (such as a tumor) of a patient with the cells according to the present invention. The inducible promoter can be any promoter that is induced when the receptor is activated by an activating signal and allows 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 subsequently signal transduction occurs through the cell. In an embodiment of the present invention, in the presence of the chimeric protein of the present invention, the signal transduction of the receptor that has been activated by the activating signal is inhibited, reduced or blocked. In such embodiments, the induction of the inducible promoter is thus also inhibited, reduced or blocked, and the expression of the protein of interest is likewise inhibited, reduced or blocked.

[0119] Preferably, the inducible promoter and the nucleic acid encoding the protein of interest are provided as transgenes; that is, they are purposefully introduced into the cells 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, for example, in a cell in which an inducible promoter operably linked to the nucleic acid encoding the protein of interest is introduced to provide a cell according to the present invention. Preferably, the operably linked inducible promoter and nucleic acid encoding the protein of interest are provided in the form of a chimeric nucleic acid. In some embodiments, the operably linked inducible promoter and nucleic acid encoding the protein of interest are integrated into the genome. In some embodiments, the operably linked inducible promoter and nucleic acid encoding the protein of interest are provided to the cell in a vector and preferably not integrated into the genome of the cell. However, in some other embodiments, the gene encoding the protein of interest can be integrated into the genome, for example, at a locus responsive to the signal transduction of the activated receptor, that is, under the control of the inducible promoter according to the present invention. In some other embodiments, the inducible promoter and / or the protein of interest are introduced / integrated into the genome. In some embodiments, by introducing / integrating the inducible promoter into the genome, an endogenous protein can be under the control of such an inducible promoter according to the present invention. In some embodiments, the protein of interest is operably linked to an inducible promoter according to the present invention, which is an endogenous promoter / an inducible promoter integrated into the genome.

[0120] As will be understood by those skilled in the art, suitable inducible promoters for use in the present invention thus depend on the receptor and one or more signal transduction pathways that are activated after receptor activation. Those skilled in the art know how to identify or select a suitable inducible promoter for the present invention based on the disclosure herein.

[0121] According to the present invention, an inducible promoter is any promoter that is induced upon receptor activation and subsequent signal transduction through the cell. In some embodiments, an inducible promoter operably linked to a nucleic acid encoding a protein of interest is any promoter whose activation is responsive to a transcription factor that increases when an immune cell is specifically activated after receptor activation. In some embodiments, the inducible promoter is a promoter responsive to ITAM-mediated signal transduction (see, e.g., Love et al., Cold Spring Harb Perspect Biol. 2010 Jun; 2(6):a002485. doi:10.1101 / cshperspect.a002485), such as signal transduction initiated by phosphorylation of a conserved motif (ITAM) contained within the cytoplasmic domain or transmembrane cell surface receptor (e.g., TCR receptor, CAR receptor, and NKR receptor).

[0122] In some embodiments, the inducible promoter is a human promoter.

[0123] In a preferred embodiment according to the present invention, the inducible promoter is selected from the group consisting of: NFAT promoter, NF-kB 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 or IL-17 promoter. For example, in a preferred embodiment according to the present invention, the inducible promoter is selected from the group consisting of: (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), (synthetic) NF-kB promoter (Jutz et al., J Immunol Methods. 2016 Mar; 430: 10-20), (synthetic) AP-1 promoter (Jutz et al., J Immunol Methods. 2016 Mar; 430: 10-20). As understood by those skilled in the art, such NFAT promoter, NF-kB promoter and AP-1 promoter are also referred to as synthetic promoters because they (may) contain multiple copies of NFAT, NF-kB or AP-1 binding motifs.Similarly, in a preferred embodiment according to the present invention, the inducible promoter is selected from the group consisting of: the (native) CD69 promoter (Redondo-Antón 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), the (native) IL-2 promoter (Skerka et al., J Biol Chem. 1995 Sep 22; 270(38):22500-6), the (native) IL-4 promoter (Davydov et al., J Immunol. 1995 Dec 1; 155(11):5273-9, Macián et al., EMBO J. 2000 Sep 1; 19(17):4783-95), the (native) IL-6 promoter (Faggioli et al., Biochim Biophys Acta. May 28, 2004; 1692(1):17-24), the (native) IL-8 promoter (Okamoto et al., J Biol Chem. Mar 18, 1994; 269(11):8582-9), the (native) IL-13 promoter (Dolganov et al., Blood. Apr 15, 1996; 87(8):3316-26), the (native) IL-17 promoter (Liu et al., J Biol Chem. Dec 10, 2004; 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.

[0124] Specifically, for example, an NFAT promoter refers to one or more NFAT response elements linked to a minimal promoter of any gene (such as a gene expressed by T cells). In some embodiments, the minimal promoter of a gene expressed by T cells is the minimal human IL-2 promoter (see, for example, Zhang et al., Mol Ther. 2011 Apr; 19(4): 751–759). The NFAT response element contains a binding motif to which one or more NFAT proteins (such as NFAT1, NFAT2, NFAT3, and / or NFAT4) bind. The NFAT promoter can contain any number of binding motifs, such as at least one, at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs. In a preferred embodiment, the sequence of the NFAT binding motif is ggaggaaaaactgtttcatacagaaggcgt (SEQ ID NO: 202). In an embodiment, the NFAT promoter contains the NFAT binding motif. In a particularly preferred embodiment, the NFAT promoter nucleotide sequence contains ggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtcgcgaattcgcggagactctagagggtatataatggaagctcgatttccag (SEQ ID NO: 203) or consists of the same. It includes 4 copies of the NFAT binding site and a minimal promoter (see also Jutz et al., J Immunol Methods. 2016 Mar; 430: 10-20).

[0125] According to the present invention, the cells of the present invention are configured with a nucleic acid comprising an inducible promoter, such as a promoter induced after ITAM-mediated signal transduction, such as a promoter induced after activation of TCR, CAR or NKR by an activating signal, such as a promoter induced after binding of a receptor to a ligand (such as an antigen). The inducible promoter contained in the nucleic acid provided to the cells according to the present invention is operably linked to a nucleic acid encoding a protein of interest. In a preferred embodiment according to the present invention, the nucleic acid encoding the protein of interest is additionally operably linked to a nucleic acid encoding an RNA degradation element (RDE), and wherein the RDE is preferably an AU-rich element (ARE). Preferably, the 3' untranslated region (UTR) used in the present invention is 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 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 derived from the IL2 or IFNγ 3' untranslated region (UTR). Preferably, the RDE, preferably the ARE, is located in the 3' untranslated region (UTR) relative to the nucleic acid encoding the protein of interest. Preferably, more than one RDE, preferably ARE, is used in the nucleic acid according to the present invention, such as 1, 2, 3, 4 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) (such as two or more), which may be referred to as a tandem 3' UTR).

[0126] RNA destabilizing elements (RDEs) are known to those skilled in the art and are nucleic acids that affect or maintain the stability of RNA molecules or the translation kinetics of RNA molecules. Some RDEs bind to polypeptides. Preferably, the RDE is an RDE that binds to an RDE-binding polypeptide, and the RDE-binding polypeptide can stabilize the RNA, thereby increasing the half-life of the RNA. Such RDEs can be used to control the expression of a protein of interest operably linked to an inducible promoter. In other words, in such preferred embodiments, the RDE is an RDE that causes RNA stabilization by interacting with an RNA-stabilizing protein.

[0127] In the context of the present invention, RDEs can contribute to the control of the expression of a target protein. 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 Kovarik 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 WO2018045177.

[0128] Preferably, the RDE is an AU-rich element (ARE). Such adenosine-uridine-rich elements are found in the 3' untranslated regions (UTRs) of many messenger RNAs (mRNAs), including those encoding proto-oncogenes, nuclear transcription factors, and cytokines. AREs are one of the most common determinants of RNA stability in mammalian cells. AREs are defined as regions in mRNAs where adenine and uridine bases occur frequently. AREs are generally classified into three classes according to sequence differences (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 that are dispersed over the 3’UTR in a generally U-rich context. Class II AREs contain multiple copies of the canonical AUUUA pentamer, which occasionally overlap to form an AUUUAUUUA nonamer sequence. In contrast, class III AREs are negatively defined as lacking the AUUUA motif but being generally U-rich. The best-characterized adenosine-uridine (AU)-rich elements have a core sequence of AUUUA in a U-rich sequence.

[0129] In a preferred embodiment, more than one RDE is used, preferably ARE. Repeats of the base sequence, the core AUUUA element, are generally required for function.

[0130] In a preferred embodiment, the RDE can be a class I AU-rich element, preferably derived from the 3'UTR of a gene encoding, for example, c-myc, c-fos, β1-AR, PTH, interferon γ, MyoD, p21, cyclin A, cyclin B1, cyclin D1, PAI-2 or NOS HANOS.

[0131] In a preferred embodiment, the RDE can also be a class II AU-rich element and is preferably derived 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 can be a class III AU-rich element, which is preferably derived from the 3′UTR of a gene encoding, for example, c-jun, GLUT1, p53, hsp 70, myogenin, NF-M or GAP-43.

[0132] In highly preferred embodiments of the present invention, the RDE, preferably the ARE, preferably the ARE, binds to RNA binding proteins (RBPs) such as NF90 and HuR. For example, the 3' untranslated regions (UTRs) 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), TNFa (NM_000594.4), CSF2 (NM_000758.4), FasL (NM_000639.3), c-fos (NM_005252.4) and other 3' untranslated regions (UTRs) 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., 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. May 2019;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, the present invention uses the AU-rich elements (AREs) from these 3' untranslated regions (UTRs).

[0133] 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 the mRNA destabilizing factor increases mRNA stability and protein expression levels. Importantly, according to the present invention, the rheostat-mediated control of payload production surprisingly applies to regulatory gene elements from different classes (e.g., promoters and / or 3'UTRs) that share the property of responding to ITAM signals. ) mediated control of payload production is surprisingly applicable to regulatory gene elements from different classes (e.g., promoters and / or 3'UTRs) that share the property of responding to ITAM signals.

[0134] Specifically, the RDE (preferably ARE) is an RDE (preferably ARE) that binds to an RNA binding protein (RBP), thereby increasing the stability of the mRNA. In a preferred embodiment, 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, the 3’UTR containing the ARE decreases the stability of the mRNA, while in the presence of ITAM signaling, RNA binding proteins (RBPs) such as NF90 and HuR are exported from the nucleus, bind to the 3’UTR containing the ARE and increase the stability of the mRNA and the export of the mRNA from the nucleus, thereby producing the POI. In some preferred embodiments, the ARE is or from the 3’untranslated region (UTR) of IL2 or IFNγ.

[0135] Those skilled in the art know how to provide a nucleic acid comprising an inducible promoter according to the present invention and comprising a nucleic acid encoding a protein of interest, wherein the nucleic acid encoding the protein of interest is operably linked to a nucleic acid encoding an RDE, preferably an ARE. If the nucleic acid is transcribed (using the inducible promoter), a transcript (mRNA) encoding a protein linked to the RDE is obtained.

[0136] The present invention is not particularly limited to the type of cell, such as the type of cell that has been or will be modified to provide a modified cell according to 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 or human cell, such as 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, such as a pluripotent stem cell, such as an induced pluripotent stem cell. In a preferred embodiment, the cell is a human immune cell. In a preferred embodiment where the cell is an immune cell, the immune cell is preferably selected from the group consisting of: T cells, T cells expressing a TCR, TCR-modified T cells, CAR-T cells, NK cells, CAR-NK cells, tumor-infiltrating lymphocytes (TIL), and macrophages. 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 cell is a CAR NK cell. In some embodiments, the cell is a macrophage, including CAR macrophages (see, for example, 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 cell is a tumor-infiltrating lymphocyte (TIL). In some embodiments, the cell is a human cell. Preferably, the immune cell is preferably selected from the group consisting of: human T cells, human CAR-T cells, human TCR-modified T cells (sometimes also referred to as engineered T cell receptor T cells), human NK cells, human CAR-NK cells, human tumor-infiltrating lymphocytes (TIL), and human macrophages.

[0137] Those skilled in the art are well aware of the existence of different cells (including different immune cells) and know how to identify these cells. For example, T cells, or T lymphocytes, play a central role in cell-mediated immunity. They 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 cell surfaces. CAR-T cells are T cells that express the CAR complex. There are multiple types of T cells, including but not limited to T helper cells (TH cells), cytotoxic T cells, and regulatory T cells. TH cells express CD4 on their surfaces and are activated when peptide antigens are presented to them on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, which secrete different cytokines to promote different types of immune responses. Cytotoxic T cells (TC cells or CTLs) can destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surfaces. These cells recognize their targets by binding to antigen-associated MHC class I, which is present on the surface of all nucleated cells. Regulatory T cells (Tregs) inhibit the immune response, for example, by secreting molecules such as IL-10, and these cells are characterized by the expression of the transcription factor FOXP3.

[0138] Another example is memory T cells, which are a subset of antigen-specific T cells that can persist for a long time after an infection has resolved. When re-exposed to their cognate antigen, they rapidly expand into large numbers of effector T cells, thus providing the immune system with a "memory" against past infections. Memory cells can be CD4+ or CD8+. Preferably, the T cells are CD4-positive T cells. Preferably, the T cells are CD8-positive T cells.

[0139] Natural killer cells (or NK cells) are a type of cytotoxic 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 type of cell that differentiates from the common lymphoid progenitor that gives rise to B lymphocytes 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, for example, Zhang et al., Biomarker Research, Volume 10, Article number: 12 (2022)).

[0140] Macrophages are a type of white blood cell in the immune system that can phagocytose and digest pathogens, such as cancer cells, microorganisms, cell debris, and foreign substances. Macrophages do not have proteins on their surface that are specific for healthy somatic cells. Another type of immune cell that can be suitably used according to 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.

[0141] In combination with a drug-regulated protein stability domain that allows for dose-dependent expression of a chimeric protein in engineered cells (such as T cells) according to the present invention, the system provides an efficient and reliable way to precisely regulate T cell activation in response to an activation signal (such as an antigen) of an activating receptor (such as a TCR), and thereby regulate the induction of an inducible promoter and the expression (production) of a protein (the protein of interest). In addition, the production and secretion of cytokines can be regulated in this way. In this way, T cell activity can be regulated, which can lead not only to the regulated production of the POI, but also to the maintained, restored, safe, and controllable functions of T cells in vitro and in vivo (i.e., in the treatment of cancer or other conditions, such as autoimmune diseases that rely on the use of T cells, including T cells modified to express new or additional TCRs and / or CARs). Similarly, in addition to regulating the production of the protein of interest (biological product or payload), the function of NK cells can also be regulated.

[0142] Immune cells, such as T cells (including CAR T cells) or NK cells (including CAR NK cells), which contain receptors, chimeric proteins, and an inducible promoter operably linked to a protein of interest and express or contain molecules according to the present invention, can be obtained using the patient's own peripheral blood or from the peripheral blood of a donor. Alternatively, cells can be obtained using conventional methods known to those skilled in the art, by ex vivo proliferation and / or differentiation of stem cells of immune cell progenitors.

[0143] According to the present invention, the receptor can be any suitable receptor. The receptor is a receptor capable of receiving an activation signal that causes the receptor to induce, for example 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 a receptor that can transduce a signal into the cell (i.e., via cell transmission) upon receiving an activation signal (such as a binding event), such that the inducible promoter is transduced and induced, resulting in 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 (such as a complex comprising 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. For example, this allows for the reception of extracellular signals (here meaning signals not from the cell containing the receptor). In a preferred embodiment according to the present invention, the receptor is selected from the group consisting of: T cell receptor (TCR), chimeric antigen receptor (CAR), and NK cell receptor (NKR) and / or wherein the receptor is an antibody against an antigen, such as a tumor antigen.

[0144] According to the present invention, the docking domain can be any suitable docking domain. The docking domain is a domain that is capable of binding (preferably reversibly binding) to the receptor (such as binding to the intracellular or cytoplasmic domain of the receptor), and by binding to the receptor, inhibits, reduces or blocks the signal transduction induced when the receptor receives an activation signal (for example, blocks the signal transduction when the CAR binds to an antigen). By inhibiting, reducing or blocking the signal transduction from the receptor to the cell, the docking domain thus inhibits, reduces or blocks the activation of the cell, although the receptor has been activated by an activation signal (such as an antigen, such as a tumor antigen, or any other suitable receptor activation molecule).

[0145] In a preferred embodiment according to the present invention, the chimeric protein according to the present invention and contained in the engineered cell according to the present invention is characterized by the presence of a docking domain that comprises (as a first part) an SH2 domain from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM). In other preferred embodiments according to the present invention, the docking domain comprises an SH2 domain that is capable of binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) contained in the receptor, preferably after receiving an activation signal, preferably wherein the SH2 domain is from a protein selected from the group consisting of Zap70, Syk, and Lck.

[0146] WO2021080427 provides such docking domains (and pharmaceutically-modulated protein stability domains) in detail, including examples of suitable SH2 domains, and the present invention specifically contemplates and preferably such docking domains or SH2 domains disclosed therein. Preferably, the SH2 domain according to the present invention can be any SH2 domain from a protein capable of binding to (phosphorylated) ITAM. Those skilled in the art are well aware of the SH2 domains suitable for the chimeric proteins of the present invention and / or can easily identify such suitable SH2 domains or proteins containing such SH2 domains capable of binding phosphorylated (ITAM).

[0147] Those skilled in the art should understand that a suitable SH2 domain can be selected according to the ITAM contained in the receptor according to the present invention capable of receiving an activation signal. For example, the receptor is the TCR / CD3 complex and / or CAR and / or NK cell receptor (NKR) complex targeted by the designed chimeric protein or polypeptide according to the present invention. In other words, the chimeric protein according to the present invention contains an SH2 domain from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), and wherein the ITAM is contained in, for example, the TCR or CAR complex to be targeted in the context of the present invention. In other words, the SH2 domain in the docking domain of the chimeric protein can interact / bind with the phosphorylated ITAM contained in the receptor, for example, after the receptor receives an activation signal. In other words, according to the present invention, in these preferred embodiments, when the receptor (e.g., TCR) receives an activation signal (e.g., after binding to an antigen), the ITAM contained in the receptor will be phosphorylated, thereby allowing the SH2 domain contained in the docking domain to bind to the phosphorylated ITAM contained in the receptor.

[0148] In fact, SH2 domains typically bind to phosphorylated tyrosine residues in the context of longer peptide motifs in target proteins. SH2 domains themselves lack any intrinsic catalytic activity, but they can localize coupled functional domains in polypeptides near appropriate substrates, activators, or inhibitors (Ngoenkam et al., Immunology. 2018 Jan;153(1):42–50). For example, some SH2 domains may interact with proteins having phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs), while other SH2 domains interact with proteins having phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). In the present invention, SH2 domains from proteins that bind to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) are preferred. The surprising finding is that while SH2 domains from proteins that bind to phosphorylated ITAMs are important for modulating T cell activity 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 according to the present invention.

[0149] In a preferred embodiment, the SH2 domain is from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) present in a TCR complex, an NKR complex, and / or a CAR.

[0150] 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). The SH2 domain can be found in various databases well-known to those skilled in the art, for example (see, e.g., 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 sequences according to SEQ ID NOs 164-168, or 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 sequences according to SEQ ID NOs 169-171. Those skilled in the art will understand that sequences as defined above are also included, and in which 1, 2, 3, 4, 5, 6, 10 amino acids are deleted, substituted or inserted.

[0151] Cells according to the invention are also provided, wherein the SH2 domain is from a protein selected from the group consisting of Zap70, Syk and Lck. It has been found according to the invention that chimeric proteins according to the invention, in particular those comprising a first part, are very suitable, said first part comprising the SH2 domain from Zap70, Syk and Lck.

[0152] ZAP70 is a protein that is normally expressed near the cell membrane of T cells and natural killer cells. It plays a key role in T cell signaling. It has a molecular weight of 70 kDa and consists of 2 N-terminal SH2 domains and a C-terminal kinase domain. It is a member of the protein tyrosine kinase family. The UniProtKB accession number for human ZAP70 protein is P43403. The sequence length is 619 amino acids 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 has the same domain structure, namely 2 N-terminal SH2 domains and a C-terminal kinase domain. In B cells, the deletion of Syk can be rescued by Zap70 (Kong et al., Immunity. 1995 May;2(5):485-92). Similarly, in T cells, the deletion of ZAP70 can be rescued by Syk (Williams et al., Mol Cell Biol. 1998 Mar;18(3):1388-99). The UniProtKB accession number for human Syk protein is P43405. The sequence length is 635 amino acids and is shown as SEQ ID NO 154.

[0153] Lck (also known as p56-LCK) is expressed in lymphocytes. Lck plays a crucial role in the TCR signal transduction pathway. In T cells, it is constitutively associated with the cytoplasmic domains of CD4 and CD8 co-receptors. Activation of the TCR by the peptide-MHC complex brings Lck close to the TCR complex, and thus the ITAM residues in the CD3 subunits are phosphorylated by Lck. The phosphorylated ITAM can serve 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 required for interaction with the phosphorylated ITAM, and SH4 is required for membrane association (Ngoenkam et al., Immunology. 2018 Jan;153(1):42–50). The UniProtKB accession number for human Lck protein is P06239. The sequence length is 509 amino acids and is shown as SEQ ID NO 155. Preferably, Zap70, Syk, and Lck are human Zap70, Syk, and Lck.

[0154] In another preferred embodiment according to the present invention, the ITAM present in the receptor is present in a TCR, CAR or NKR, and more preferably, the ITAM is derived from or located in a CD3ζ chain, CD3ε chain, CD3δ chain, CD3γ chain, FceRIγ chain or DAP12.

[0155] In a preferred embodiment, the engineered cell is a T cell expressing a TCR complex and / or a CAR complex. Preferably, the TCR complex and / or the CAR complex comprises a CD3ζ chain domain containing an ITAM, or any other domain with an ITAM disclosed herein.

[0156] In other preferred embodiments, the engineered cell is an NK cell expressing an NKR complex and / or a CAR complex. Preferably, the NKR complex or the CAR complex comprises a CD3ζ chain domain containing an ITAM, or any other domain with an ITAM disclosed herein.

[0157] In some embodiments according to the present invention, the ITAM is the ITAM contained in a T cell receptor (TCR) complex and / or a chimeric antigen receptor (CAR) and / or an NKR complex, preferably the ITAM contained in a CD3ζ chain, CD3ε chain, CD3δ chain, CD3γ chain, γ chain of the immunoglobulin receptor FceRI and DAP12.

[0158] As discussed, the SH2 domain can be from a protein that binds to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs are present in cell signaling molecules such as the CD3ζ, CD3ε, CD3γ and CD3δ chains of the T cell receptor complex, as well as in the intracellular domains of Fc receptors such as FceRI (Love et al., Cold Spring Harb Perspect Biol. 2010 Jun;2(6):a002485).

[0159] The chimeric protein is designed to interact with phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) in a TCR / CD3 complex and / or a CAR and / or an NK cell receptor (NKR) and / or a macrophage CAR complex, which complexes contain signaling molecules with ITAMs, such as DAP12, the γ chain of the immunoglobulin receptor FceRI or the CD3ζ chain (Lanier et al., Nat Immunol. 2008 May;9(5):495–502).

[0160] The tyrosine residues within the ITAM motif become phosphorylated upon interaction of the receptor molecule with its ligand (activation signal) and form a docking domain for other proteins involved in the cellular signaling pathway. Through the interaction of the chimeric protein of the present invention with the TCR and / or CAR, T cell activation (and subsequent cytotoxic effects and / or cytokine secretion) is inhibited. Similarly, through the interaction of the chimeric protein according to the present invention with the NKR and / or CAR in NK cells, NK cell activation (and subsequent cytotoxic effects and / or cytokine secretion) of NK cells is inhibited.

[0161] In NK cells, certain activating NK cell receptors (NKRs) form complexes with signaling molecules bearing ITAMs (such as the CD3ζ chain, the γ chain of the immunoglobulin receptor FceRI, and DAP12). For example, the NK cell receptors (NKRs) NKp46 and NKp30 associate with the γ chain of the immunoglobulin receptor FceRI and the CD3ζ chain, while NKp44 associates with the signal transduction adapter DAP12 (Barrow et al., Front Immunol. 2019; 10:909). Thus, in an embodiment of the present invention, the cell according to the present invention is an NK cell.

[0162] The domain bearing ITAM is also used in chimeric antigen receptor (CAR) design. The CD3ζ chain contains three ITAMs, while the CD3ε chain, the γ chain of the immunoglobulin receptor FceRI, and the DAP12 signaling domain contain one ITAM, and they are 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, et al., J Immunol. 2015 Apr 1; 194(7):3201 - 12).

[0163] The hemITAM tag can be easily recognized as a tyrosine that is separated from a leucine or isoleucine by any two other amino acids, resulting in the tag YxxL / l. Two of these tags are spaced 6 to 8 amino acids apart, constituting the consensus ITAM sequence of YxxL / lx(6 - 8)YxxL / l.

[0164] In a preferred embodiment, the ITAM-containing domain can be or comprise the CD3ζ chain domain. In another preferred embodiment, the ITAM-containing domain can be or comprise the CD3ε chain domain. However, in another preferred embodiment, the ITAM-containing domain can be or comprise the gamma chain of the immunoglobulin receptor FceRI. However, in another preferred embodiment, the ITAM-containing domain can be or comprise the DAP12 domain.

[0165] In preferred embodiments, the chimeric protein is designed to interact with phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) in, for example, the TCR / CD3 complex and / or CAR and / or NK cell receptor (NKR) complexes, which complexes contain signaling molecules bearing ITAMs, such as DAP12, the gamma chain of the immunoglobulin receptor FceRI or the CD3ζ chain (Lanier et al., Nat Immunol. May 2008;9(5):495–502).

[0166] The tyrosine residues within these ITAM motifs become phosphorylated upon interaction of the receptor molecule with its ligand (activation signal) and form docking sites for other proteins involved in cellular signaling pathways. By interaction of the chimeric protein according to the invention with the TCR and / or CAR, T cell activation (and subsequent cytotoxic effects and / or cytokine secretion) is inhibited. Similarly, by interaction of the chimeric protein according to the invention with the NKR and / or CAR in NK cells, NK cell activation (and subsequent cytotoxic effects and / or cytokine secretion) of NK cells is inhibited.

[0167] In some embodiments, the ITAM is the 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 according to SEQ ID NOs 172 - 177.

[0168] According to a preferred embodiment of the present invention, the docking domain comprised in the chimeric protein comprises, in addition to an SH2 domain capable of binding a phosphorylated ITAM motif (e.g., present in a receptor that has received an activation signal), an immunoreceptor tyrosine-based switch motif (ITSM) and / or an ITIM, preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM). Such motifs are present, for example, in the inhibitory tail of PD1 and are thought to be involved in the immunosuppressive action of PD1 (Boussiotis et al., Cancer J. 2014 Jul-Aug;20(4):265–271).

[0169] The presence of these ITSM, ITIM or ITSM and ITIM motifs in the docking domain also improves the blocking of receptor signaling (in this case ITAM signaling) by the chimeric protein according to the present invention. Thus, although the presence of the SH2 domain already provides blocking of ITAM signaling, the presence of these ITSM, ITIM or ITSM and ITIM motifs in the docking domain further enhances this blocking, thereby enabling strict regulation of the signaling through the cell and subsequently strict regulation of the production of the biological product (the protein of interest), since the inducible promoter used in this case is dependent on said signaling (here ITAM signaling) / induced by said signaling (here ITAM signaling).

[0170] Surprisingly, it has been found that the presence of such an ITSM, preferably such an ITSM and such an ITIM, in the docking domain of the chimeric protein allows the chimeric protein to effectively inhibit signal transduction through a receptor, e.g., through a TCR, NKR or CAR (after ligand binding to the receptor). The inventors have demonstrated that these ITSM, preferably the ITSM and ITIM present in the inhibitory tail of an inhibitory immunoreceptor protein such as PD1, can be utilized to inhibit TCR and / or CAR signaling in T cells and NKR signaling in NK cells without the presence of the extracellular domain of an inhibitory protein or interaction with its ligand (e.g., PD-L1 of PD1). As described herein, the system according to the present invention in combination with a pharmaceutically regulated protein stability domain allows for dose-dependent expression of the chimeric protein in a cell (e.g., a T cell), thereby providing an effective and reliable way to precisely regulate T cell activation and thus regulate the induction of the inducible promoter according to the present invention and the production of the protein of interest.

[0171] According to a preferred embodiment, the docking domain comprises a portion containing an immunoreceptor tyrosine-based switch motif (ITSM), an immunoreceptor tyrosine-based inhibitory motif (ITIM) or preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0172] 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 part), the protein stability domain related to the drug (discussed elsewhere herein; hereinafter referred to as the second part), or ITSM, ITIM, or ITSM and ITIM (hereinafter referred to as the third part) are fused (e.g., gene-linked) at the N-terminus or C-terminus of the chimeric protein, or are present inside the chimeric protein. In other words, in the chimeric protein, the first, second, or third part can be located at the C-terminus, at the N-terminus, or can be flanked by other parts at the C-terminus and / or N-terminus. Examples of suitable orders of the first part (P1), the second part (P2), and the third part (P3) can generally be xP1xP2xP3x, xP1xP3xP2x, xP2xP1xP3x, xP2xP3xP1x, xP3xP1xP2x, or xP3xP2xP1x, where x at any position can independently refer to the absence of additional amino acid residues or the presence of one or more additional amino acid residues that do not form part of P1, P2, and / or P3. For the order of the first part and the second part, for example, in embodiments where the third part is absent, similar to the above examples, examples of suitable orders of the first part (P1) and the second part (P2) can generally be xP1xP2x or xP2xP1x, where x at any position can independently refer to the absence of additional amino acid residues or the presence of one or more additional amino acid residues that do not form part of P1 and / or P2.

[0173] Meanwhile, those skilled in the art should understand that in addition to the SH2 domain from a protein that binds to the immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated, the first part can also contain other domains or amino acids, such as one or several amino acids that are usually flanked (on one or both sides) by the SH2 domain of a protein that binds to the immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated.

[0174] Meanwhile, those skilled in the art should understand that in addition to the drug-regulated protein stability domain, the second part can also contain other domains or amino acids on one or both sides.

[0175] Meanwhile, those skilled in the art should understand that in addition to the immunoreceptor tyrosine-based switch motif (ITSM), preferably the ITSM and the immunoreceptor tyrosine-based inhibitory motif (ITIM), the third part can also contain other domains or amino acids, such as one or several amino acids that are usually flanked (on one or both sides) by these motifs.

[0176] Those skilled in the art should understand that as long as the position of the drug-regulated protein stability domain in the chimeric protein is such that the chimeric protein degrades upon contact with a drug that interacts with the drug-regulated protein stability domain, and inhibition of signal transduction, such as through activation of receptors (e.g., TCR, NKR, or CAR), is released due to the breakdown of the chimeric protein, resulting in signal transduction, T cell activation, induction of an inducible promoter, and subsequent production of the protein of interest, the first, second, and third portions of the chimeric protein according to the present invention can be present in the chimeric protein in any order.

[0177] Thus, due to the presence of the drug-regulated protein stability domain, the present disclosure can tightly regulate, for example, T cell and NK cell activation through the chimeric protein according to the present invention, and the drug-regulated protein stability domain is used to regulate (e.g., reduce or increase) the expression of the chimeric protein according to the present invention in a time- and / or dose-dependent manner.

[0178] Preferably, the ITIM and / or ITSM are from an inhibitory receptor protein, preferably from an inhibitory immune receptor protein, preferably from a protein selected from the group consisting of: PD1, BTLA, SIRPα, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, and LY9. Preferably, the inhibitory receptor protein, the inhibitory immune receptor protein, or the protein selected from the group consisting of PD1, BTLA, SIRPα, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, or LY9 is from a human.

[0179] PD1 (also known as PD-1) is encoded by the PDCD1 gene. PD1 is a type I transmembrane protein. Interaction with its ligands PD-L1 / PD-L2 leads to downregulation of cytotoxic T effector functions. The UniProtKB accession number for human PD1 is Q15116. The sequence is 288 amino acids in length. The cytoplasmic domain of PD1 contains ITIM and ITSM motifs. Phosphorylated ITSM in the cytoplasmic domain of PD1 recruits the SHP-2 phosphatase, which dephosphorylates key signaling molecules in the TCR signaling pathway (such as ZAP70, PKCθ, and CD3ζ (CD247)), and leads to 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 for use in a chimeric protein / protein according to the present invention is characterized by SEQ ID NO 156 (representing the cytoplasmic domain of PD1), said suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0180] B and T lymphocyte attenuator (BTLA) is mainly expressed in T cells, B cells, and mature lymphocytes (Yue et al., Front Immunol. 2019; 10:617). It is an immune regulatory receptor that plays a key role in immune tolerance. Similar to 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 the human BTLA protein is Q7Z6A9. The sequence is 289 amino acids in length. The cytoplasmic domain of BTLA contains ITIM and ITSM motifs.

[0181] For example, a suitable portion for use in a chimeric protein according to the present invention is characterized by SEQ ID NO 157 (representing the cytoplasmic domain of BTLA), said suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0182] SIRPA (also known as SIRPalpha, SIRPα, BIT, MFR, MYD1, PTPNS1, SHPS1, SIRP) is expressed in myeloid cells. Upon engagement with 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 is mainly associated with SHP-1 (Veillette et al., J Biol Chem. 1998 Aug 28;273(35):22719-28). SIRPA is a type I transmembrane protein. The UniProtKB accession number for the human SIRPA protein is P78324. The sequence is 504 amino acids in length. The cytoplasmic domain of SIRPA contains two ITIMs and one ITSM motif.

[0183] For example, a suitable portion for use in a chimeric protein according to the invention is characterized by SEQ ID NO 158 (representing the cytoplasmic domain of SIRPa), said suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0184] PECAM1 (also known as PECAM-1, CD31) is expressed in 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 cell and B cell signaling by recruiting 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 results in the recruitment of SHP-1 and SHP2, the production of TNF-a, IL-6, and IFN-β, and the downregulation of TLR4 signaling (Rui et al., J Immunol. 2007 Dec 1; 179(11):7344-51). PECAM1 negatively regulates the platelet signaling pathway (Jones et al., FEBS Lett. 2009 Nov 19; 583(22):3618-24). PECAM1 is a type I transmembrane protein. The UniProtKB accession number for the human PECAM1 protein is P16284. The sequence is 738 amino acids in length. The cytoplasmic domain of PECAM1 contains ITIM and ITSM motifs. For example, a suitable portion for use in a chimeric protein according to the invention is characterized by SEQ ID NO 159 (representing the cytoplasmic domain of PECAM1), said suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0185] Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a group of immunomodulatory receptors that are mainly expressed on hematopoietic system cells ( et al., Dev Comp Immunol. 2018 Sep; 86: 219 - 231). SIGLEC5 (also known as CD33L2, OBBP2) is expressed in monocytes, neutrophils, and B cells, SIGLEC9 is expressed in neutrophils, monocytes, dendritic cells, and NK cells, while SIGLEC11 is expressed in macrophages (Macauley et al., Nat Rev Immunol. 2014 Oct; 14(10): 653–666). Most Siglecs have inhibitory ITIM / ITSM motifs that can recruit SHP1 and SHP2 and act 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. May 20, 2005; 280(20): 19843 - 51, Haas et al., Cancer Immunol Res. May 2019; 7(5): 707 - 718, Angata et al., J Biol Chem. Jul 5, 2002; 277(27): 24466 - 74). SIGLEC5, SIGLEC9, and SIGLEC11 are type I transmembrane proteins. They contain ITIM and ITSM motifs in their cytoplasmic domains. The UniProtKB accession number for the human SIGLEC5 protein is O15389. The sequence length is 551 amino acids. The UniProtKB accession number for the human SIGLEC9 protein is Q9Y336. The sequence length is 463 amino acids. The UniProtKB accession number for the human SIGLEC11 protein is Q96RL6. The sequence length is 698 amino acids. For example, suitable portions for use in the chimeric proteins according to the 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), said suitable portions comprising immunoreceptor tyrosine - based switch motifs (ITSM), preferably ITSM and immunoreceptor tyrosine - based inhibitory motifs (ITIM).

[0186] T lymphocyte surface antigen Ly - 9 (also known as LY9, SLAMF3, CD229) is expressed in thymocytes and mature T and B lymphocytes (de la Fuente et al., Blood. 2001 Jun 1; 97(11): 3513 - 20). It has been reported to interact with SHIP - 1 and SHP - 2( -Ortiz et al., Front Immunol. 2018 Nov 16; 9:2661), and promotes peripheral cellular tolerance by acting as a negative regulator of the immune response (de Salort et al., Front Immunol. 2013; 4:225). LY9 is a type I transmembrane protein. The UniProtKB accession number for the human LY9 protein is Q9HBG7. The sequence is 655 amino acids in length. The cytoplasmic domain of LY9 contains two ITSM motifs. For example, a suitable portion for use in the chimeric proteins according to the invention is characterized by SEQ ID NO 163 (representing the cytoplasmic tail of LY9), said suitable portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0187] Thus, the ITSM and / or ITIM contained in the third part of the chimeric polypeptide can be the ITSM and / or ITIM contained in SEQ ID NOs 156 - 163, or a 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 with the amino acid sequences according to SEQ ID NOs 156 - 163. Those skilled in the art will understand that sequences as defined above are also included, and in which 1, 2, 3, 4, 5, 6, 10 amino acids are deleted, substituted or inserted. The third part may also contain additional (multiple) amino acid sequences adjacent to the amino acids defined above, provided that the third part of the chimeric polypeptide remains functional within the scope of the present invention.

[0188] In some embodiments, the ITSM and ITIM are obtained from or derived from the same inhibitory protein, and in other embodiments, the ITSM and ITIM are each derived from different inhibitory proteins.

[0189] Cells according to the invention are also provided, wherein the chimeric protein comprises ITSM and ITIM.

[0190] Surprisingly, it has been found that the simultaneous presence of ITSM and ITIM in the third part contained in the chimeric proteins according to the invention is particularly advantageous (see Examples).

[0191] In some embodiments according to the invention, the drug-regulated protein stability domain is a CRBN polypeptide substrate domain which is capable of binding to the CRBN protein in response to a drug, preferably thereby promoting ubiquitin pathway-mediated degradation of the chimeric protein.

[0192] In this embodiment, the chimeric protein according to the invention comprises a drug-regulated protein stability domain which is capable of interacting with and binding to the CRBN protein in the presence of a drug. For example, various IMiDs (including those described herein) have been shown to bind to the CRBN protein, 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.

[0193] 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 many other proteins. Studies have shown that thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide), and CC-885 can all bind to CRBN (see, for example, Lopez-Girona et al., Leukemia 26:2326-2335).

[0194] Those skilled in the art know that the CRBN polypeptide substrate domain can bind to the CRBN protein in response to a drug, thereby promoting the degradation of the chimeric protein mediated by the ubiquitin pathway and is suitable for use in the present invention.

[0195] In a preferred embodiment, the drug-regulated protein stability domain comprises a Cys2-His2 zinc finger domain capable of drug-induced binding to a CRBN polypeptide, preferably wherein the Cys2-His2 zinc finger domain is a heterologous zinc finger domain. Such a drug-regulated protein stability domain comprising a Cys2-His2 zinc finger domain is also referred to as a "zinc finger degron".

[0196] The Cys2His2-like fold group (C2H2) is a well-characterized class of zinc fingers that are extremely 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 having the amino acid sequence motif (Pabo et al., Annual Review of Biochemistry (2001). 70:313-40) X2-Cys-X2,4-Cys-X12-His-X3,4,5-His.

[0197] In some embodiments, the chimeric protein comprises a CRBN polypeptide substrate domain containing one or more zinc fingers (i.e., zinc finger degrons).

[0198] Although not particularly limited to a specific 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, or a fragment capable of small molecule-induced binding to the 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).

[0199] In another preferred embodiment, the CRBN polypeptide substrate domain comprises a heterologous fusion polypeptide, wherein the heterologous fusion polypeptide comprises at least a first fragment of a first C2H2 zinc finger protein and a second fragment of a second C2H2 zinc finger protein, and the combination of the first fragment and the second fragment in the heterologous fusion polypeptide is capable of drug-induced binding to the CRBN polypeptide. For example, in some embodiments, the beta-turn (formed by two short beta-strands) from the first C2H2 zinc finger protein can be fused to the alpha-helix of the second C2H2 zinc finger protein. Also provided are cells according to the invention, which comprise a chimeric protein according to the invention containing such a small molecule-regulated protein stability domain.

[0200] Although the present invention is not particularly limited to specific chimeric fusion polypeptides that can be formed or can be included in the CRBN polypeptide substrate domain, in a preferred embodiment, the chimeric fusion polypeptide comprises a first fragment of a beta-turn selected from ZFP91 ZF4 (LQCEICGFTCR; SEQ ID NO: 142), ZFN653 ZF4 (LQCEICGYQCR; SEQ ID NO: 143), ZNF276 ZF4 (LQCEVCGFQCR; SEQ ID NO: 144), ZNF827 ZF1 (FQCPICGLVIK; SEQ ID NO: 145), and a second fragment of an alpha-helix selected from IKZF1 ZF2 (QKGNLLRHIKLH; SEQ ID NO: 146), and any possible combinations. Preferably, the chimeric fusion polypeptide comprises the beta-turn of ZFP91 ZF4 and the alpha-helix of IKZF1 ZF2, and preferably, wherein the chimeric fusion polypeptide comprises one selected from SEQ ID NOs 147–151. Also provided are cells according to the present invention that comprise a chimeric protein according to the present invention with such a small molecule-regulated protein stability domain.

[0201] According to another embodiment, the CRBN polypeptide substrate binding domain used in the method of the present invention comprises or further comprises IKZF1 ZF3 (FKCHLCNYACRRRDALTGHLRTH; SEQ ID NO: 152), and preferably wherein the CRBN polypeptide substrate binding domain comprises the beta-turn of ZFP91 ZF4, the alpha-helix of IKZF1 ZF2, and IKZF1 ZF3. Preferably, IKZF1 ZF3 is located at the C-terminus of the second part of the chimeric protein according to the present invention. Also provided are cells according to the present invention that comprise a chimeric protein according to the present invention with such a small molecule-regulated protein stability domain.

[0202] Those skilled in the art should understand that in some embodiments, the chimeric protein according to the present invention comprises one or more CRBN polypeptide substrate domains that are capable of binding to CRBN in response to a drug, thereby promoting ubiquitin pathway-mediated degradation of the chimeric protein according to the present invention. The zinc finger degron polypeptide domain (one or more CRBN polypeptide substrate domains that are capable of binding to CRBN in response to a drug, thereby promoting ubiquitin pathway-mediated degradation of the chimeric protein) can be included as a single degron polypeptide domain or multiple degron polypeptide domains, optionally wherein the multiple degron polypeptide domains are linked in tandem or in an array, optionally using a polypeptide linker, such as those known in the art.

[0203] Those skilled in the art should also understand that within the CRBN polypeptide substrate-binding domain used in the method according to the present invention, different portions (e.g., β-turns and α-helices) can be directly adjacent to each other, or a polypeptide linker (including short amino acid sequences), such as those known in the art, can be used to connect them. Zinc finger degrader suitable for the present invention is well known to those skilled in the art.

[0204] Drugs (e.g., small molecules) suitable for modulating the degradation of chimeric proteins according to the present invention that comprise one or more such C2H2 zinc finger proteins, fragments or domains include so-called immunomodulatory imide drugs (IMiDs), which include but are not limited to thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide) and CC-885 (see, for example, 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 skilled in the art know how to select a suitable drug, e.g., an IMiD suitable for use in the method according to the present invention.

[0205] Accordingly, there is provided a cell according to any one of the preceding claims, wherein the drug that allows the CRBN polypeptide substrate domain to bind to the CRBN protein, thereby promoting ubiquitin pathway-mediated chimeric protein degradation, is an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide) and CC-885.

[0206] The immunomodulatory imide drug (IMiD)-induced zinc finger degradation system (i.e., an example of a drug-regulated stability domain according to the present invention, e.g., comprising a Cys2-His2 zinc finger domain, e.g., the above-mentioned hybrid Cys2-His2 zinc finger domain (or hybrid zinc finger domain)) is known per se in the prior art and has been described, for example, by Sievers et al., Science. 2018 Nov 2; 362(6414): eaat0572. As described above, in the context of the present invention, the degradation stator system employs a fusion of at least a docking domain of a chimeric protein according to the present invention with a short zinc finger degrader (sometimes also referred to as (hybrid) zinc finger domain, Cys2-His2 zinc finger domain, and / or zinc finger polypeptide), which helps to recruit the chimeric protein according to the present invention to the IMiD / CRBN E3 ligase complex, e.g., in the presence of a small molecule called IMiD. It is generally believed that IMiD binds to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, and CRBN can recruit a (fusion) protein containing a zinc finger degrader (as a drug-regulated protein stability domain) through interaction with the zinc finger degrader, and this interaction is mediated by IMiDs such as thalidomide and its derivatives. This zinc finger degradation system is based on human protein sequences, thereby limiting the risk of immune-mediated rejection. In addition, protein stability is regulated by clinically approved small molecules, such as thalidomide, pomalidomide, and lenalidomide (as examples of IMiDs), thereby facilitating the clinical development of these systems.

[0207] In preclinical work, this protein stability control system has demonstrated its value in clinically relevant applications, such as regulating CAR-T cell activity when such zinc finger degraders are fused to CARs (Jan et al., Sci Transl Med. 2021 Jan 6; 13(575): eabb6295). In addition, the inventors reported the use of the IMiD / zinc finger system in a chemically regulated and SH2-delivered inhibitory tail (CRASH-IT) switch platform, which, in a preferred embodiment, is also used in the present invention (and is referred to as a rheostat switch and which comprises, for example and which allows control of the cell activity levels of various cell therapy platforms, such as CAR-T cells, TCR-T cells, and NK cells (Sahillioglu et al., WO2021080427).

[0208] Although the fact that the zinc finger degrader is composed of human sequences and can be controlled using approved molecules favors the clinical application of the system, at therapeutically effective concentrations, the clinical use of these IMiD molecules in the treatment of patients with hematological malignancies is associated with severe side effects. For example, a study evaluating maintenance therapy with high-dose (25 mg / day) and low-dose (5 mg / day) lenalidomide in patients with multiple myeloma showed that the lenalidomide dose was associated with both toxicity and efficacy, and dose reduction was common in the high-dose lenalidomide cohort due to toxicity, particularly neutropenia. (Fenk et al., Clin Cancer Res. 2020 Nov 15;26(22):5879-5886).

[0209] Thus, preferably, in the context of the present invention, a drug-related protein stability domain responsive to lower drug doses is used, preferably a drug-related protein stability domain comprising a Cys2-His2 zinc finger domain capable of drug-induced binding to a CRBN polypeptide.

[0210] Thus, in a preferred embodiment, the Cys2-His2 zinc finger domain is a chimeric zinc finger domain (or chimeric zinc finger degrader) 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, preferably, wherein the chimeric zinc finger domain comprises one, two, or more amino acid substitutions relative to 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 chimeric zinc finger domain comprises two amino acid substitutions relative to 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 chimeric zinc finger domain comprises three amino acid substitutions relative to 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 chimeric zinc finger domain comprises four amino acid substitutions relative to 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 chimeric zinc finger domain is the chimeric zinc finger domain disclosed herein.

[0211] Such zinc finger degraders (as drug-regulated stability domains) allow the use of reduced concentrations of small molecules or drugs to regulate the drug-regulated stability domain, particularly IMiD, to control the protein degradation, expression, and / or stability of chimeric proteins (and, for example, cellular activity (total activity in cells).

[0212] Thus, such zinc finger degron (degron tag) according to the present invention can better control the protein degradation, expression, and / or stability of chimeric proteins at similar or reduced concentrations of small molecules (especially IMiDs). At the same time, it can allow for a more sensitive regulation of the degradation, expression, level, and / or stability of chimeric proteins.

[0213] The drug-regulated stability domain preferably comprises a Cys2-His2 zinc finger domain, preferably wherein the Cys2-His2 zinc finger domain is preferably a hybrid zinc finger domain comprising a first part and a second part. The first part and the second part are each independently composed of an amino acid sequence. The first part and the second part comprised in the hybrid zinc finger domain may be directly adjacent to each other or may not be directly adjacent. In some embodiments, the first part and the second part are connected by a linker peptide of, for example, one, two, three, four, five, or more amino acids. Thus, in some embodiments, the first part and the second part may be connected to each other by an additional linker peptide, such as a linker peptide comprising one, two, three, four, five, six, seven, or more amino acids. However, in some embodiments, there is no additional linker peptide between the first part of the hybrid zinc finger domain and the second part of the hybrid zinc finger domain. In such embodiments, the first part and the second part are directly adjacent to each other. Preferably, the first part and the second part are directly adjacent to each other.

[0214] The first part of the hybrid zinc finger domain can be the N-terminus or the C-terminus of the second part of the hybrid zinc finger domain. In a preferred embodiment, the first part of the hybrid zinc finger domain is the N-terminus of the second part of the hybrid zinc finger domain.

[0215] In a preferred embodiment, the first part consists of 8–30 amino acids, with a sequential increase preferably of 11-30, 10–20, 11–20, 10–14, 11-14, 10–11 amino acids, and most preferably 11 amino acids.

[0216] In a preferred embodiment, the second part consists of 8–30 amino acids, with a sequential increase preferably of 12-30, 11–20, 12–20, 11–14, 12-14 amino acids, and most preferably 12 amino acids.

[0217] In some embodiments, preferably, the non-natural hybrid zinc finger domain consists of about 16-60 amino acids, preferably about 20-36 amino acids, about 20-30 amino acids, such as 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 wherein the hybrid zinc finger domain comprises at least two subdomains (referred to herein as parts), and wherein each subdomain is from a different wild-type zinc finger. For example, wherein the first part of the hybrid zinc finger domain is from a first (wild-type) zinc finger, and wherein the second part of the hybrid zinc finger domain is from a second different wild-type zinc finger domain. Based on the disclosure herein, one of ordinary skill in the art can select the first part of the hybrid zinc finger domain from the first wild-type zinc finger and / or can select the second part of the hybrid zinc finger domain from the second wild-type zinc finger.

[0218] For example, he can appropriately select such parts from wild-type zinc finger domains (especially wild-type Cys2-His2 (C2H2) zinc finger domains), which are available from various scientific publications and public and well-known gene and protein databases, such as from (Sievers et al., Science. 2018 Nov 2; 362(6414): eaat0572), and for example are known or expected to destabilize proteins (e.g., wild-type proteins containing such wild-type zinc finger domains) in the presence of an IMiD (e.g., thalidomide), and thereby can control the degradation of such proteins.

[0219] In certain embodiments, the first part of the hybrid zinc finger domain comprises the amino acid sequence represented by sequence X1X2C3X4X5C6X7X8X9X 10 X 11 wherein each X (X1, X2,... etc.) independently represents any amino acid, more specifically wherein each X represents any natural or proteogenic 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 of representing the amino acid sequence contained in the first part of the hybrid zinc finger domain is (X)2C(X)2C(X)5. However, since amino acid substitutions at specific positions in the first part of the hybrid zinc finger domain and parts such as this are part of the present invention, it is preferred to use X1X2C3X4X5C6X7X8X9X 10 X 11 , indicating each amino acid and its position.

[0220] Those skilled in the art will understand that, for example, X1 can represent another amino acid different from, for example, X2, or it can represent the same amino acid. Those skilled in the art understand that C3 indicates the presence of cysteine at the third position of the amino acid sequence contained in the first part of the hybrid zinc finger domain. Similarly, C6 indicates the presence of cysteine at the sixth position. In a preferred embodiment, the first part of the hybrid zinc finger domain has the amino acid sequence (X1X2C3X4X5C6X7X8X9X 10 X 11 ) as indicated above, and wherein the amino acid sequence is the first Cys2-His2 zinc finger domain (e.g., wild-type Cys2-His2 zinc finger domain). In some embodiments, the indicated amino acid sequence of the first part of the hybrid zinc finger domain may include an additional short segment of amino acid sequence at its N-terminus, such as, for example, 1-10, preferably 1-5, such as 1, 2, 3, 4, or 5 amino acids of the first or second Cys2-His2 zinc finger domain. For example, Figure 11 An example of a zinc finger degron (i.e., a drug-regulated protein stability domain) according to the present invention is provided (where the positions of amino acid substitutions are indicated), wherein there is a short segment of amino acid sequence GERP at the N-terminus of the hybrid zinc finger domain (derived from IKZF1 in this case; in some other embodiments, this fragment may come from another wild-type zinc finger domain, such as it may be GEKP). Experiments have confirmed that although such short segments of sequence may be included, it does not significantly affect the results obtained using the degron (i.e., a drug-regulated protein stability domain).

[0221] As detailed elsewhere herein, in a preferred embodiment, the hybrid zinc finger domain contained in the drug-regulated protein stability domain according to the present invention is a hybrid zinc finger containing a Cys2 His2 (C2H2) zinc finger domain, and wherein the hybrid zinc finger domain contains at least two subdomains, namely a first part and a second part, and wherein the amino acid sequence of the first part is from a wild-type zinc finger, more specifically from a wild-type Cys2 His2 (C2H2) zinc finger.

[0222] In a specific embodiment, the second part of the hybrid zinc finger domain contains the amino acid sequence represented by X 12 X 13 X 14 X 15 X 16 X 17 X18H 19 X 20 X 21 X 22 H 23 wherein each X (X 12 、X13 and the like) independently represent any amino acid, and more specifically each X represents any natural or proteogenic amino acid (such as 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 part of the hybrid zinc finger domain is (X)7H(X)3H. However, since amino acid substitutions at specific positions in the second part of the hybrid zinc finger domain and parts such as this are part of the present invention, it is preferred to use X 12 X 13 X14X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 , indicating each amino acid and its position. In some embodiments, it is also contemplated that the second part of the hybrid zinc finger domain is (X)6H(X)3H.

[0223] Those skilled in the art should understand that, for example, X 12 can represent an amino acid different from, for example, X 13 , or it can represent the same amino acid. Those skilled in the art understand that H 19 indicates the presence of histidine at the nineteenth position of the amino acid sequence contained in the second part of the hybrid zinc finger domain. Similarly, H 23 indicates the presence of histidine cysteine at the 23rd position in this sequence. In a preferred embodiment, the second part of the hybrid zinc finger domain has 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 ), and wherein the amino acid sequence is the second Cys 13 -His2 zinc finger domain.

[0224] In some embodiments, the shown amino acid sequence of the second part of the hybrid zinc finger domain may include an additional short stretch of amino acid sequence at its C-terminus, such as, for example, 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4, or 5 amino acids of the second Cys2-His2 zinc finger domain. In a preferred embodiment, the first part of the hybrid zinc finger domain is the N-terminus of the second part of the hybrid zinc finger domain. In such embodiments, the second part is C-terminal relative to the first part.

[0225] In other words, in a preferred embodiment, the hybrid zinc finger domain contained in the zinc finger degron (i.e., the drug-regulated protein stability domain) according to the present invention is a hybrid zinc finger domain containing a Cys2His2 (C2H2) domain, and wherein the hybrid zinc finger domain contains at least two sub-domains, namely a first part and a second part, and wherein the amino acid sequence of the first part is from a wild-type zinc finger, more specifically from a wild-type Cys2His2 (C2H2) zinc finger (i.e., the first Cys2-His2 zinc finger domain), and wherein the amino acid sequence of the second part is from a wild-type zinc finger, more specifically from a wild-type Cys2His2 (C2H2) zinc finger (i.e., the second Cys2-His2 zinc finger domain), and wherein preferably, the second Cys2-His2 zinc finger domain is different from the first Cys2-His2 zinc finger domain (e.g., wherein the first Cys2-His2 zinc finger domain is a wild-type zinc finger different from the second Cys2-His2-zinc finger region). Preferably, the amino acid sequence of the first part comprises or is X1X2C3X4X5C6X7X8X9X 10 X 11 and / or the amino acid sequence of the second part comprises or is X 12 X 13 X 14 X 15 X 16 X17X 18 H 19 X 20 X 21 X 22 H 23 .

[0226] As described above, the first and second parts together form a hybrid zinc finger domain, and wherein the first part is preferably a first Cys2-His2 zinc finger domain, such as a wild-type, naturally occurring Cys2-His2 zinc finger domain, and the second part is preferably a second different Cys2-His2 zinc finger domain, such as a different wild-type, naturally occurring Cys2-His2 zinc finger domain. In such embodiments, the hybrid zinc finger domain formed by the first and second parts may be referred to as a hybrid Cys2-His2 (C2H2) zinc finger domain, such as a Cys2-His2 (C2H2) zinc finger domain that does not exist in wild-type zinc finger proteins and / or in nature.

[0227] Generally, the C2H2 zinc finger domain (including, for example, the hybrid zinc finger domain according to the present invention) comprises a β-hairpin and an α-helix subdomain. Generally, the C2H2 zinc finger domain consists of about 20-36 amino acids, about 20-30 amino acids, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. The C2H2 zinc finger domain generally comprises an N-terminal β-hairpin containing two conserved cysteine residues, followed by an α-helix containing two conserved histidine residues at its C-terminus (see, for example, Fedotova et al., Acta Naturae, 2017 Apr-Jun; 9(2):47-58). This motif is also present in the zinc finger domains according to the present invention.

[0228] In a preferred embodiment, the zinc finger degron (i.e., the drug-regulated protein stability domain) according to the present invention is further characterized by the presence of two amino acid substitutions in the hybrid zinc finger domain, and wherein the amino acid substitution in the first part of the hybrid zinc finger domain is relative to the first part of the first Cys2-His2 zinc finger domain, and wherein the amino acid substitution in the second part of the hybrid zinc finger domain is relative to the second part of the second Cys2-His2 zinc finger domain, and wherein the substitutions do not occur at any position in C3, C6, X7, H 19 or H 23 In some embodiments, X7 is glycine.

[0229] As discussed herein, in an embodiment, the hybrid zinc finger domain consists of a first part from a first Cys2-His2 zinc finger domain, particularly from a first wild-type Cys2-His2 zinc finger domain, and wherein the first part comprises the amino acid sequence X1X2C3X4X5C6X7X8X9X 10 X 11(i.e., as present in a first wild-type Cys2-His2 zinc finger domain). As discussed herein, in an embodiment, the heterozygous zinc finger domain consists of a second portion from a second Cys2-His2 zinc finger domain, particularly from a second wild-type Cys2-His2 zinc finger domain, and 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., as present in a second wild-type Cys2-His2 zinc finger domain).

[0230] In a preferred embodiment, the Cys2-His2 zinc finger domain according to the invention is further characterized in that two amino acid substitutions are present in the heterozygous zinc finger domain, and wherein the amino acid substitutions are located in the first portion of the heterozygous zinc finger domain and are amino acid substitutions relative to the first portion of the first Cys2-His2 zinc finger domain from which the sequence is obtained. For example, if X2 is valine in the first (wild-type) Cys2-His2 zinc finger domain from which the sequence is obtained and the valine is replaced, for example, with lysine in a zinc finger degron according to the invention (i.e., a drug-regulated protein stability domain), then in the zinc finger degron according to the invention, X2 will be lysine and the amino acid substitution to lysine is relative to the valine contained in the wild-type Cys2-His2 zinc finger domain from which the amino acid substitution to lysine is obtained. In a similar manner, amino acid substitutions in the second portion of the heterozygous zinc finger domain are defined herein. For example, if X 20 is proline in the second (wild-type) Cys2-His2 zinc finger domain from which the second portion sequence is obtained and the proline is replaced, for example, with leucine in a zinc finger degron according to the invention, then in the zinc finger degron according to the invention, X 20 will be leucine and the amino acid substitution to leucine is relative to the proline contained in the wild-type Cys2-His2 zinc finger domain from which the amino acid substitution to leucine is obtained.

[0231] As disclosed above, relative to portions of the wild-type first Cys2-His2 zinc finger domain and the second Cys2-His2 zinc finger domain, the hybrid zinc finger domain contains two amino acid substitutions. Those skilled in the art are fully capable of providing, preparing, and identifying such amino acid substitutions relative to the wild-type first Cys2-His2 zinc finger domain and the second Cys2-His2 zinc finger domain, for example, by comparing the amino acid sequences of the first portion and / or the second portion of the hybrid zinc finger domain.

[0232] In some embodiments, both of the amino acid substitutions (i.e., replacing an amino acid in the wild-type sequence with a different amino acid) are in the first portion. In some embodiments, both of the amino acid substitutions are located in the second portion. In some other embodiments, one amino acid substitution is in the first portion and one amino acid substitution is in the second portion. Preferably, the hybrid zinc finger domain contains no more than two amino acid substitutions.

[0233] In a preferred embodiment, the substitutions do not occur at any of positions C3, C6, X7, H 19 、or H 23 . In some embodiments, X7 is G7.

[0234] Surprisingly, it has been found that by introducing two amino acid substitutions in the first portion and / or the second portion of the hybrid zinc finger domain and 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, a Cys2-His2 zinc finger domain can be provided that has a greatly enhanced or increased sensitivity to IMiD molecules (such as thalidomide or analogs) relative to the wild-type zinc finger domain and relative to a hybrid zinc finger domain composed of wild-type subdomains (as described herein) and that does not contain two amino acid substitutions. Interestingly, while it has been found that introducing one amino acid substitution may also enhance or increase sensitivity to IMiD molecules, unexpectedly introducing additional amino acid substitutions may even further enhance or increase sensitivity to IMiD molecules, as shown in the examples and described herein.

[0235] According to this aspect of the invention, there is provided a hybrid Cys2-His2 zinc finger domain that contains two amino acid substitutions relative to the wild-type zinc finger domain (portions thereof) that make up the hybrid zinc finger domain and that has an enhanced or increased sensitivity to IMiD molecules.

[0236] In some embodiments, two amino acid substitutions are located directly 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), such as shown in the attached tables, figures, and examples.

[0237] Although any amino acid substitution is in principle allowed, preferably, the amino acid substitutions at specific positions in the hybrid zinc finger domain are the substitutions as described herein (either with respect to the amino acid being substituted or with respect to the substituting amino acid). A person skilled in the art can provide such amino acid substitutions, as well as the Cys2-His2 zinc finger domains according to the invention, without undue burden based on the information disclosed herein.

[0238] Also provided are hybrid Cys2-His2 zinc finger domains, wherein the first part is the β-hairpin loop of a first Cys2-His2 zinc finger domain and the second part is the α-helical region of a second different Cys2-His2 zinc finger domain. Such hybrid Cys2-His2 zinc finger domains do not occur naturally in the cells according to the invention or, preferably, do not occur in nature.

[0239] The zinc finger domain includes a β-hairpin loop and an α-helical region, and a person skilled in the art knows how to provide the β-hairpin loop portion and / or the α-helical region portion of a (wild-type) Cys2-His2 zinc finger domain. Representative examples are well known to a person 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, ZNF276, 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).

[0240] As discussed above, a zinc finger degrader and / or hybrid zinc finger polypeptide according to the present invention may include one or more amino acid residues relative to the N-terminus of the β-hairpin portion, one or more amino acid residues located between the β-hairpin portion and the α-helix portion, and one or more amino acid residues relative to the C-terminus of the α-helix portion, provided that the zinc finger degrader and / or hybrid zinc finger polypeptide according to the present invention is a substrate of the CRBN-IMiD complex, and / or exhibits enhanced or increased sensitivity to an IMiD molecule (e.g., relative to the wild-type situation). These additional amino acids may correspond to residues in the native Cys2-His2 zinc finger domain or may be different, provided that the zinc finger degrader maintains a zinc finger-like fold and exhibits the properties disclosed herein.

[0241] In an embodiment of the present invention, there is provided a zinc finger degrader and / or hybrid zinc finger polypeptide according to the present invention, wherein the first substitution is located in the second portion and the second substitution is located in the first portion or the second portion. Although both amino acid substitutions may be included in the first portion, or may both be included in the second portion, or one amino acid substitution is included in the first portion and the second amino acid substitution is included in the second portion, in a preferred embodiment, at least one amino acid substitution is present in the second portion. It has been found that particularly suitable zinc finger degraders according to the present invention can be provided, and wherein at least one amino acid substitution is located in the second portion, i.e., in the α-helix portion of the hybrid zinc finger domain. The second amino acid substitution may be present in the second portion or may be present in the first portion (i.e., in the β-hairpin loop).

[0242] There is also provided a zinc finger degrader and / or hybrid zinc finger polypeptide according to the present invention, wherein at least one substitution is located at a position selected from the group consisting of: X1, X4, X 12 、X 13 、X 14 、X 15 、X 17 、X 21 and X 22 . It has been found that an amino acid substitution at one of these positions, particularly in the hybrid zinc finger domain, can provide increased or enhanced sensitivity to an IMiD molecule for the zinc finger degrader and / or hybrid zinc finger polypeptide according to the present invention. Without being bound by theory, in particular, as shown in the examples, it is believed that these positions are important positions in the hybrid zinc finger domain that allow for an observed improvement or increase in sensitivity to IMiD. Preferably, the substituted amino acids are as disclosed herein.

[0243] In a preferred embodiment, there is provided a zinc finger degrader and / or a chimeric zinc finger polypeptide according to the present invention, and wherein at least one substitution present in the zinc finger degrader and / or the chimeric zinc finger polypeptide according to the present invention (or present in the chimeric zinc finger domain) is selected from those listed in Table 1. Table 1 lists the respective positions of X1–X 22 and, in a preferred embodiment, substitutions may be present in the zinc finger degrader and / or the chimeric zinc finger polypeptide according to the present invention, as well as substituted amino acids (i.e., including the amino acid at that position in the zinc finger degrader and / or the chimeric zinc finger polypeptide according to the present invention; single-letter code). Preferably, both amino acid substitutions (both the position and the substituted 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 any other table or list provided herein), each amino acid substitution is at a different position. At the same time, those skilled in the art will understand that the substituted amino acid is intended to represent an amino acid different from the natural amino acid (i.e., different from the amino acid present in the wild-type Cys2-His2 zinc finger domain used for the first or second part of the chimeric zinc finger domain). In this regard, it is noted that the present invention is not particularly limited to a specific wild-type Cys2-His2 zinc finger domain for providing the first part, the second part (and / or the third and fourth parts as discussed herein).

[0244] Similarly, the present invention also encompasses the use of the positions and substitutions listed in Table 1 (or any other table or list provided herein) in any suitable first or second part of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a chimeric zinc finger domain according to the present invention. In other words, the positions and substituted amino acids listed in Table 1 (or any other table or list provided herein) are applicable to any suitable chimeric zinc finger domain, provided that the zinc finger degrader and / or the chimeric zinc finger polypeptide according to the present invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., sensitivity to IMiD in the context of protein degradation).

[0245] However, in a preferred embodiment, the positions and substitutions are relative to the chimeric zinc finger domain used in the examples herein, i.e., wherein the first part of the chimeric zinc finger domain is the β-hairpin region of ZFP91 ZF4, and the second part of the chimeric zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11 ; wherein the chimeric zinc finger domain of the 'parental zinc finger degrader' used in the examples is schematically represented).

[0246] Table 1 - Preferred positions and substitutions (substituted amino acids)

[0247]

[0248]

[0249] In a preferred embodiment, there is provided a zinc finger degrader and / or a chimeric zinc finger polypeptide according to the present invention, and wherein 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 chimeric zinc finger domains described herein according to the present invention (each row shows a preferred combination of two positions within the chimeric zinc finger domain and the corresponding substituted amino acids; for example, 4R and 12L indicate that in this combination, the amino acid at position 4 in the chimeric zinc finger domain is replaced by arginine (R), and the amino acid at position 12 in the chimeric zinc finger domain is replaced by leucine (L) - in other words, the original amino acids in the portion obtained from the wild-type Cys2-His2 zinc finger domain are replaced by arginine at position 4 and by leucine at position 12). In a preferred embodiment of the present invention, the two substitutions are selected from any combination shown in Table 2.

[0250] Table 2 - Preferred Combinations of Positions and Substitutions (Substituted Amino Acids)

[0251]

[0252]

[0253] Those skilled in the art should understand that the substituted amino acids are intended to represent amino acids different from the natural amino acids (i.e., different from the amino acids present in the wild-type Cys2-His2 zinc finger domain used for the first or second part of the chimeric zinc finger domain). In this regard, it is noted that the present invention is not particularly limited to a specific wild-type Cys2-His2 zinc finger domain used to provide the first part, the second part (and / or the third and fourth parts as discussed herein).

[0254] Likewise, the present invention also encompasses the use of the positions and substitutions listed in Table 2 (or any other table or list provided herein) in any suitable first or second part of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a chimeric zinc finger domain according to the present invention. In other words, the positions and substituted amino acids listed in Table 2 (or any other table or list provided herein) are applicable to any suitable chimeric zinc finger domain, provided that the zinc finger degrader and / or the chimeric zinc finger polypeptide according to the present invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., sensitivity to IMiD in the context of protein degradation).

[0255] However, in the preferred embodiments, the positions and substitutions are relative to the chimeric zinc finger domains used in the examples herein, i.e., where the first part of the chimeric zinc finger domain is the β-hairpin region of ZFP91 ZF4, and the second part of the chimeric zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11 ; where the chimeric zinc finger domain of the 'parental zinc finger degrader' used in the examples is shown schematically).

[0256] In addition, the major amino acid substitutions Q12R, Q12K, N15R, and L22R, which are frequently observed in the examples, when combined with the minor amino acid substitutions listed in Table 8 (see below), further increase the sensitivity to IMiDs in a synergistic manner (i.e., EIQ12R / K13V = 0.006877267, while EIQ12R = 0.167217557). Accordingly, in a particularly preferred embodiment of the present invention, a combination of the amino acid substitutions mentioned in Table 8 is used / provided, such as 12R and 13T or 22R and 14L, etc.

[0257] In a preferred embodiment, there is provided a zinc finger degrader and / or a chimeric zinc finger polypeptide according to the present invention, wherein the amino acid substituted in the first part and / or the amino acid substituted in the second part is selected from those listed in Table 3.

[0258] Table 3 lists the amino acids preferably substituted in the chimeric zinc finger domain of the zinc finger degrader and / or the chimeric zinc finger polypeptide according to the present invention. In other words, in the preferred embodiment of the present invention, the positions and / or the positions and amino acids substituted are selected from those listed in Table 3. Accordingly, in the preferred embodiment of the present invention, the two amino acid substitutions are selected from the combinations at positions 1, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, and 22, and wherein the amino acids substituted are a combination of two amino acids listed in Table 3.

[0259] Table 3 - Preferred positions and amino acids to be substituted

[0260] L1 E4 I5 F8 T9 C10 R11 Q12 K13 G14 N15 L16 L17 R18 I20 K21 L22

[0261] Preferably, both of the two amino acids substituted (both the position and the substituted amino acid) are selected from those listed in Table 3. Those skilled in the art will appreciate that an amino acid (such as the preferred amino acids shown in Table 3) can be substituted with any suitable amino acid as long as it provides a zinc finger degrader and / or a chimeric zinc finger polypeptide according to the present invention. However, in the preferred embodiment, the amino acids listed in Table 3, preferably the amino acid combinations, are replaced with the corresponding amino acids listed in Table 1 or the corresponding amino acid combinations shown in Table 2 (where corresponding means, for example, at the corresponding positions in the chimeric zinc finger domain disclosed herein).

[0262] Those skilled in the art will understand that when both of the substituted amino acids are selected from those listed in Table 3 (or any other table or list provided herein), each amino acid substitution is at a different position. At the same time, those skilled in the art will understand that the substituted amino acid is intended to represent an amino acid that is substituted by another amino acid and is different from the native amino acid (i.e., different from the amino acids present in the wild-type Cys2-His2 zinc finger domain used for the first or second part of the hybrid zinc finger domain). In this regard, it is noted that the present invention is not particularly limited to a specific wild-type Cys2-His2 zinc finger domain for providing the first part, the second part (and / or the third and fourth parts as discussed herein).

[0263] Similarly, the present invention also encompasses the use of the positions and substitutions listed in Table 3 (or any other table or list provided herein) in any suitable first or second part of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a hybrid zinc finger domain according to the present invention. In other words, the substituted positions and amino acids listed in Table 3 (or any other table or list provided herein) are applicable to any suitable hybrid zinc finger domain, provided that the zinc finger degrader and / or hybrid zinc finger polypeptide according to the present invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., sensitivity to IMiD in the context of protein degradation).

[0264] However, in a preferred embodiment, the positions and substitutions are relative to the hybrid zinc finger domain used in the examples herein, i.e., where the first part of the hybrid zinc finger domain is the β-hairpin region of ZFP91 ZF4, and the second part of the hybrid zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11 ; where the hybrid zinc finger domain of the 'parental zinc finger degrader' used in the examples is schematically represented).

[0265] In a preferred embodiment, there is provided a zinc finger degrader and / or hybrid zinc finger polypeptide according to the present invention, wherein the two amino acids in the substituted first part and / or second part are selected from those listed in Table 4.

[0266] Table 4 lists preferred combinations of two amino acids that are substituted at two different positions in the hybrid zinc finger domains described herein according to the invention (each row shows a preferred combination of two positions within the hybrid zinc finger domain and the corresponding substituted amino acids; for example, E4 and Q12 indicate that in this combination, the amino acid at position 4 in the hybrid zinc finger domain is replaced by glutamate (E), and the amino acid at position 12 is replaced by glutamine (Q) - in other words, the original amino acids in the portion obtained from the wild-type Cys2-His2 zinc finger domain are glutamate at position 4 and glutamine at position 12. In a preferred embodiment of the invention, the two amino acids used as substitutions are selected from any combination shown in Table 4. Those skilled in the art will understand that the amino acids (such as the preferred amino acids shown in Table 4) can be replaced by any suitable amino acid as long as it provides a zinc finger degron and / or hybrid zinc finger polypeptide according to the invention. However, in a preferred embodiment, the amino acids, preferably the amino acid combinations listed in Table 4, are replaced by the corresponding amino acids listed in Table 1 or the corresponding amino acid combinations shown in Table 2 (where corresponding means, for example, at the corresponding positions in the hybrid zinc finger domains disclosed herein).

[0267] Table 4 - Preferred Combinations of Positions and Amino Acids to be Substituted

[0268]

[0269]

[0270]

[0271] Those skilled in the art will understand that the substituted amino acids are intended to represent amino acids that are different from the native amino acids being substituted (i.e., different from the amino acids present in the wild-type Cys2-His2 zinc finger domain used for the first or second part of the hybrid zinc finger domain). In this regard, it is noted that the invention is not particularly limited to a specific wild-type Cys2-His2 zinc finger domain used to provide the first part, second part (and / or third and fourth parts as discussed herein).

[0272] Similarly, the invention also encompasses the use of the positions and substitutions listed in Table 4 (or any other table or list provided herein) in any suitable first or second part of a (wild-type) Cys2-His2 zinc finger domain that can be used to provide a hybrid zinc finger domain according to the invention. In other words, the positions and amino acids substituted and listed in Table 4 (or any other table or list provided herein) are applicable to any suitable hybrid zinc finger domain as long as the zinc finger degron and / or hybrid zinc finger polypeptide according to the invention maintains a zinc finger-like fold and exhibits the properties disclosed herein (e.g., sensitivity to IMiDs in the context of protein degradation).

[0273] However, in a preferred embodiment, the positions and amino acids being replaced are relative to the chimeric zinc finger domain used in the examples herein, i.e., where the first portion of the chimeric zinc finger domain is the β-hairpin region of ZFP91 ZF4, and the second portion of the chimeric zinc finger domain is the α-helical region of IKZF1 ZF2 (see Figure 11 ; where the chimeric zinc finger domain of the 'parental zinc finger degrader' used in the examples is schematically represented).

[0274] In other embodiments, there is provided a zinc finger degrader and / or chimeric 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, ZNF827 ZF1, preferably IKZF1 ZF2.

[0275] In other words, in some embodiments, the amino acid sequences of the Cys2-His2 zinc finger domains for the selection of the first portion and / or the second portion (see below) are selected from the group consisting 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–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).

[0276] In highly preferred embodiments, the β-hairpin loop (or the first portion) of the hybrid zinc finger domain is from ZFP91 ZF4, and the α-helix (or the second portion) of the hybrid zinc finger is from IKZF1 ZF2. In other highly preferred embodiments, the β-hairpin loop (or the third portion) of other hybrid zinc finger domains is from IKZF1 ZF3 and the α-helix (or the fourth portion) of other hybrid zinc fingers is from ZFP91 ZF5.

[0277] As briefly discussed above, in embodiments of the present invention, a zinc finger degrader according to the present invention is also provided, wherein two amino acid substitutions are introduced into a heterozygous zinc finger domain, the first part of which has an amino acid sequence according to SEQ ID NO: 101 (LQCEICGFTCR-ZFP91 ZF4 (first part)), and / or the second part of the heterozygous zinc finger domain has an amino acid sequence according to SEQ ID NO: 102 (QKGNLLRHIKLH-IKZF1 ZF2 (second part)), and / or the heterozygous zinc finger domain has an amino acid sequence according to SEQ ID NO: 103 (LQCEICGFTCRQKGNLLRHIKLH-(ZFP91 ZF4 / IKZF1 ZF2 heterozygous zinc finger domain)). In a preferred embodiment, two amino acid substitutions according to the present invention can be introduced into these sequences.

[0278] In other preferred embodiments, a zinc finger degrader according to the present invention is provided, wherein two amino acid substitutions are introduced, and wherein the zinc finger degrader has an amino acid sequence according to SEQ ID NO: 107 (LQCEICGFTCRQKGNLLRHIKLHSGEKPFKCHLCNYACRRKDSVVAHKAKSH). In a preferred embodiment, two amino acid substitutions according to the present invention can be introduced into these sequences.

[0279] In a preferred embodiment, a zinc finger degrader and / or a heterozygous zinc finger polypeptide according to the present invention is provided, and wherein the heterozygous zinc finger domain containing two substitutions is selected from those listed in Table 5. Table 5 lists preferred heterozygous zinc finger domains (or heterozygous zinc finger polypeptides) according to the present invention. These preferred zinc finger domains are those that show the most beneficial EI index (enrichment index) calculated according to the Examples section. The results are summarized in Table 6.

[0280] Table 5 - Preferred heterozygous zinc finger domains

[0281]

[0282]

[0283]

[0284] Table 6 - EI index (enrichment index) of the preferred heterozygous zinc finger domains listed in Table 5.

[0285]

[0286]

[0287]

[0288]

[0289]

[0290] In fact, it has surprisingly been found that, using two substitutions in the hybrid zinc finger domain described herein, the zinc finger degrader provided according to the invention has enhanced or increased IMiD sensitivity, particularly compared to those in which, for example, the hybrid zinc finger domain contains only one substitution. For example, when the hybrid zinc finger domain used in the examples contains only one mutation (substitution L1Y, providing the sequence YQCEICGFTCRQKGNLLRHIKLH (SEQ ID NO: 108)), the corresponding EI value is at least 10 - 15 times higher compared to the EI value determined for SEQ ID.NO 36 ( = indicates a zinc finger degrader and / or hybrid zinc finger polypeptide that is less sensitive to IMiD) (and at least 5 times higher compared to SEQ ID.NO 100). This shows that, despite the introduction of a second substitution in the hybrid zinc finger domain, the sensitivity of the resulting hybrid zinc finger domain (with two substitutions) is unexpectedly significantly increased. Table 7 shows exemplary EI values obtained for various single mutations in the hybrid single domain used in the examples herein.

[0291] Table 7 - EI index (enrichment index) of selected single mutations in the hybrid single domain.

[0292] Position Mutation EI Index L1 H 0.636477685 L1 W 0.637400065 Q2 D 0.917930978 Q2 T 0.946831062 E4 A 0.365806836 E4 W 0.392513782 15 V 0.418917893 F8 Y 0.452021407 T9 K 0.424730805 T9 R 0.533275349 C10 S 0.35751446 R11 K 0.608245997 R11 G 0.645443413 Q12 F 0.307819119 Q12 Y 0.34576953 K13 Q 0.278922437 K13 P 0.280186349 G14 K 0.306447148 N15 K 0.205033159 N15 T 0.298069121 L16 F 0.516568047 L16 M 0.552891676 L17 Y 0.35840278 L17 Q 0.457417453 R18 K 0.285725983 R18 F 0.361286698 20I Y 0.3727609 20I R 0.453876376 K21 S 0.294182544 K21 Q 0.300387833 L22 R 0.372469467 L22 S 0.401515932

[0293] Although in a preferred embodiment, the zinc finger degrader according to the invention comprises a hybrid zinc finger domain containing any of the sequences listed in Table 5, the invention also contemplates that the hybrid zinc finger domain of the zinc finger degrader according to the invention comprises both the β - hairpin of the hybrid zinc finger domain listed in Table 5 and the α - helix of the hybrid zinc finger domain listed in Table 5, and further comprises different α - helix and β - hairpin structural parts respectively. For example, in a preferred embodiment, it is contemplated that the hybrid zinc finger domain is composed of a combination of the β - hairpin part of any one of the sequences listed in Table 5 and the α - helix part of any one of the sequences listed in Table 5. In some embodiments, the hybrid zinc finger domain is composed of a combination of the β - hairpin part of any one of the sequences listed in Table 5 and another α - helix part not necessarily listed in Table 5. In some embodiments, the hybrid zinc finger domain is composed of a β - hairpin part not necessarily listed in Table 5 and the α - helix part of any one of the sequences listed in Table 5.

[0294] In some embodiments, the hybrid zinc finger domain of the zinc finger degrader and / or zinc finger polypeptide according to the present invention is any one listed in Table 5, preferably any one of SEQ ID 1–90, SEQ ID 1–80, SEQ ID 1–70, SEQ ID 1–60, SEQ ID 1–50, SEQ ID 1–40, SEQ ID 1–30, SEQ ID 1–20, SEQ ID 1–10. In another embodiment, the hybrid zinc finger domain of the zinc finger degrader and / or zinc finger polypeptide according to the present invention is any one of SEQ ID1–10, SEQ ID 11–20, SEQ ID 21–30, SEQ ID 31–40, SEQ ID 41–50, SEQ ID 51–60, SEQ ID61–70, SEQ ID 71–80, SEQ ID 81–90, SEQ ID 91–100. In other embodiments, as Figure 11 shown, the hybrid zinc finger domain (ZFP91 ZF4–IKZF1 ZF2) is replaced by any of the sequences listed in Table 5.

[0295] According to another aspect of the present invention, there is provided a method of providing a zinc finger degrader according to the present invention, a non-natural hybrid zinc finger polypeptide according to the present invention, a fusion protein according to the present invention, or a nucleic acid encoding these, the method comprising:

[0296] (A) providing a hybrid zinc finger domain comprising a first part and a second part, wherein

[0297] (1) the first part comprises the amino acid sequence X1X2C3X4X5C6X7X8X9X of a first Cys2-His2 zinc finger domain 10 X 11 , wherein X represents any amino acid;

[0298] (2) the second part comprises the amino acid sequence X 12 X 13 X 14 X 15 X 16 X 17 X 18 H19X 20 X 21 X 22 H 23 , wherein X represents any amino acid, and wherein the second Cys2-His2 zinc finger domain is different from the first Cys2-His2 zinc finger domain;

[0299] (3) the second part is C-terminal relative to the first part;

[0300] (B) introducing two different amino acid substitutions in the heterozygous zinc finger domain, and wherein said substitutions are not located at any of positions C3, C6, X7, H 19 , or H 23 ; and

[0301] (C) using the heterozygous zinc finger domain obtained in step (B), or a nucleic acid sequence encoding the heterozygous zinc finger domain obtained in step (B), to prepare a zinc finger degrader according to the present invention, a non-natural heterozygous zinc finger polypeptide according to the present invention, a fusion protein according to any one of the present invention, or a nucleic acid encoding these.

[0302] Methods for preparing a zinc finger degrader according to the present invention, a non-natural heterozygous zinc finger polypeptide according to the present invention, a fusion protein according to the present invention, or a nucleic acid encoding these are well known to those skilled in the art. Such methods are readily available in the prior art and include, for example, those described in the examples.

[0303] Those skilled in the art will also understand that the same considerations, features, and preferences as described elsewhere herein apply to the first part, the second part, and with respect to the first and second Cys2-His2 zinc finger domains.

[0304] Those skilled in the art should also understand that for the two different amino acid substitutions introduced in the method of the present invention, these can be made at any position in the heterozygous zinc finger domain comprising the first part and the second part, and can be replaced with any amino acid (as long as the amino acid being replaced is different from the substituting amino acid). Those skilled in the art should also understand that in a preferred embodiment, the position, the amino acid being replaced, and / or the substituting amino acid are those as described in any one of Tables 1-6 and 8, including combinations of the two substitutions, and the indicated preferences.

[0305] The method may include the step of testing the sensitivity of a heterozygous zinc finger domain comprising two substitutions obtained by the method of the present invention and / or a zinc finger degrader according to the present invention, a non-natural heterozygous zinc finger polypeptide according to the present invention, a fusion protein according to the present invention, or a nucleic acid encoding these to IMiD, for example as described in the examples, for example by determining the EI value or index as described herein.

[0306] Based on the EI value or index, it can be determined whether the obtained hybrid zinc finger domain containing two substitutions, and / or the zinc finger degrader according to the present invention, the unnatural hybrid zinc finger polypeptide according to the present invention, the fusion protein according to the present invention, or the nucleic acid encoding these should be discarded. Thus, in a preferred embodiment, the method includes the step of analyzing the sensitivity of the obtained hybrid zinc finger domain containing two substitutions, and / or the zinc finger degrader according to the present invention, the unnatural hybrid zinc finger polypeptide according to the present invention, the fusion protein according to the present invention, or the nucleic acid encoding these to IMiD, such as described in the Examples section, in order to determine its usefulness as a zinc finger degrader, etc.

[0307] Accordingly, embodiments are provided for establishing the sensitivity of the zinc finger degrader according to the present invention, the unnatural hybrid zinc finger polypeptide according to the present invention, the fusion protein according to the present invention (e.g., obtained by the method according to the present invention) to immunomodulatory imide drugs (IMiDs)-induced degradation, preferably wherein the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, ibedimod, their salts and analogs.

[0308] The description of the above specific embodiments will fully disclose the general nature of the present invention. Others can easily modify and / or adapt such specific embodiments for various applications by applying the technical knowledge in the art (including the content of the references cited herein), without undue experimentation and without departing from the general concept of the present invention. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to fall within the meaning and scope of the equivalents of the disclosed embodiments.

[0309] According to some embodiments, a drug-regulated protein stability domain is also provided, preferably wherein the drug-regulated protein stability domain comprises a Cys2-His2 zinc finger domain, preferably wherein the Cys2-His2 zinc finger domain is a hybrid zinc finger domain, such as the Cys2-His2 zinc finger domain and / or hybrid zinc finger domain disclosed herein, and comprises another second Cys2-His2 zinc finger domain, preferably another hybrid zinc finger domain.

[0310] Regarding the other or second zinc finger domain (Cys2-His2 zinc finger domain), it has surprisingly been found that preferably, the second zinc finger domain contained in the zinc finger degrader (drug-regulated protein stability domain) according to the present invention is a hybrid zinc finger domain, as described above for the (first) hybrid zinc finger domain (Cys2-His2 zinc finger domain). The second zinc finger domain can 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.

[0311] For the second zinc finger domain (second Cys2-His2 zinc finger domain), second hybrid zinc finger domain, and / or third or fourth part described herein, in some embodiments, they may be sensitive or insensitive to IMiD per se (i.e., in the absence of the first zinc finger degron (Cys2-His2 zinc finger domain)), i.e., they may or may not cause protein instability in the presence of IMiD (such as thalidomide), and thereby control the degradation of such proteins. Preferably, it is insensitive to IMiD per se.

[0312] The drug-regulated stability domain may further comprise a second native or non-native zinc finger domain, such as a hybrid zinc finger domain (Cys2-His2 zinc finger domain), in addition to the Cys2-His2 zinc finger domain. In the zinc finger degron according to the invention, the second zinc finger domain is located C-terminal relative to the first (hybrid or non-hybrid) zinc finger domain. In such embodiments, the first (hybrid or non-hybrid) zinc finger domain in the zinc finger degron according to the invention is located N-terminal relative to the second (hybrid or non-hybrid) zinc finger domain.

[0313] In some embodiments, the first zinc finger domain and the second zinc finger domain are directly adjacent to each other without a peptide linker. 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 linked to each other by a short peptide linker. In embodiments where the first (hybrid or non-hybrid) zinc finger domain and the second (hybrid or non-hybrid) zinc finger domain are linked by a linker peptide, such linker peptide may consist of, for example, one, two, three, four, five, six, seven or more amino acids.

[0314] For example, in some embodiments, the linker may be a short segment of amino acid sequence derived from the wild-type zinc finger domain, such as from the N-terminal portion or the portion adjacent to the N-terminal of the wild-type zinc finger domain. Such amino acid sequence may be, for example, the SGEKP sequence (see Figure 11 )(in this case derived from the IKZF1 protein - the SGEKP sequence is the endogenous linker between IKZF1ZF2 and IKZF1 ZF3 in the IKZF1 protein; in the same way, other endogenous linkers may be used as linkers in the context of the present invention, such as similar endogenous linkers from other C2H2 zinc finger proteins; in some other embodiments, this segment of sequence may be from another wild-type zinc finger domain, such as GERP). Experiments have confirmed that although such a short segment of sequence may be included, it does not significantly affect the results obtained with the zinc finger degron, and those skilled in the art are fully capable of providing additional linkers suitable within the scope of the present invention.

[0315] For a second (heterozygous or non-heterozygous) zinc finger domain, it has been found that including such a second (heterozygous or non-heterozygous) zinc finger domain can further improve the sensitivity of the zinc finger degron of the present invention in controlling protein degradation, protein levels, and / or cellular activity. Thus, according to this aspect of the present invention, there is provided a zinc finger degron, wherein the zinc finger degron comprises the first and second (heterozygous or non-heterozygous) zinc finger domains as detailed herein. According to a preferred embodiment, the present invention provides a zinc finger degron, wherein the zinc finger degron comprises the first and second zinc finger domains (Cys2-His2 zinc finger domains) as detailed herein.

[0316] The second zinc finger domain comprises a third portion and a fourth portion. The third portion and the fourth portion are each independently composed of an amino acid sequence. The third portion and the fourth portion contained in the second zinc finger domain may be directly adjacent to each other or may not be directly adjacent to each other. In some embodiments, the third portion and the fourth portion are connected by a linker peptide of, for example, one, two, three, four, five or more amino acids. Thus, in some embodiments, the third portion and the fourth portion may be connected to each other by an additional linker peptide, such as a linker peptide comprising one, two, three, four, five, six, seven or more amino acids. However, in some embodiments, there is no additional linker peptide 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.

[0317] The third portion of the second zinc finger domain may be the N-terminus or the C-terminus of the fourth portion of the second zinc finger domain. In a preferred embodiment, the third portion is the N-terminus of the fourth portion.

[0318] In a preferred embodiment, the third portion is composed of 8 - 30 amino acids, preferably increasing in sequence by 11 - 30, 10 - 20, 11 - 20, 10 - 14, 11 - 14, 10 - 11 amino acids, most preferably 11 amino acids.

[0319] In a preferred embodiment, the fourth portion is composed of 8 - 30 amino acids, preferably increasing in sequence by 12 - 30, 11 - 20, 12 - 20, 11 - 14, 12 - 14 amino acids, most preferably 12 amino acids.

[0320] In some embodiments, preferably, the non-natural second zinc finger domain consists of about 16-60 amino acids, preferably about 20-36 amino acids, about 20-30 amino acids, such as 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 wherein the heterozygous zinc finger domain comprises at least two subdomains (referred to herein as parts), and wherein each subdomain is from a different wild-type zinc finger. For example, wherein the third part of the second zinc finger domain is from a third wild-type zinc finger, and wherein the fourth part of the second zinc finger domain is from a fourth different wild-type zinc finger domain. Based on the disclosure herein, one of ordinary skill in the art is able to select the third part of the second zinc finger domain from the third wild-type zinc finger and / or is able to select the fourth part of the second zinc finger domain from the fourth wild-type zinc finger. For example, he can appropriately select such parts from wild-type zinc finger domains (especially wild-type Cys2-His2 (C2H2) zinc finger domains), which can be obtained from various scientific publications and public and well-known gene and protein databases, such as from (Sievers et al., Science. 2018 Nov 2; 362(6414): eaat0572), and which are known or expected to destabilize proteins (e.g., wild-type proteins containing such wild-type zinc finger domains) in the presence of an IMiD (e.g., thalidomide), and thereby can control the degradation of such proteins.

[0321] In a specific embodiment, the third part of the second zinc finger domain comprises the amino acid sequence represented by (Z)2C(Z)2C(Z) 5-6 wherein Z represents any amino acid. Thus (Z)2 represents two amino acids, while (Z) 5-6 represents 5 or 6 amino acids. By way of example, the third part of the second zinc finger domain can, for example, comprise the amino acid sequence Z1Z2C3Z4Z5C6Z7Z8Z9Z 10 Z 11 or Z1Z2C3Z4Z5C6Z7Z8Z9Z 10 Z 11 Z 12, and each Z (Z1, Z2, etc.) independently represents any amino acid, more specifically, each Z represents any natural or proteogenic 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 understand that, for example, Z1 can represent a different amino acid than, for example, Z2, or it can represent the same amino acid. Those skilled in the art understand that C3 indicates the presence of cysteine at the third position of the amino acid sequence contained in the third part of the second zinc finger domain. Similarly, C6 indicates the presence of cysteine at the sixth position. In a preferred embodiment, the third part of the second zinc finger domain has the amino acid sequence (Z)2C(Z)2C(Z) 5-6 , even more preferably (Z)2C(Z)2C(Z)6, and wherein the amino acid sequence is the amino acid sequence of the third Cys2-His2 zinc finger domain (e.g., the amino acid sequence of the wild-type Cys2-His2 zinc finger domain). In some embodiments, the indicated amino acid sequence of the third part of the second zinc finger domain may include an additional short segment of amino acid sequence at its N-terminus, such as 1-10, preferably 1-5, e.g., 1, 2, 3, 4, or 5 amino acids of the third or fourth (wild-type) Cys2-His2 zinc finger domain. For example, Figure 11 Examples of zinc finger degrons according to the present invention are provided (indicating the positions of amino acid substitutions), and as discussed above, there is a short segment of amino acid sequence SGEKP at the N-terminus of the second zinc finger domain. Experiments have confirmed that although such a short segment sequence may be included, it does not significantly affect the results obtained using the degron.

[0322] As detailed herein, and compared to the first hybrid zinc finger domain contained in the zinc finger degron according to the present invention, in a preferred embodiment, the second zinc finger domain is also a hybrid zinc finger containing a Cys2 His2 (C2H2) zinc finger domain, and wherein the hybrid zinc finger domain contains at least two subdomains (i.e., the third part and the fourth part), and wherein the amino acid sequence of the third part is from a wild-type zinc finger, more specifically from a wild-type Cys2 His2 (C2H2) zinc finger.

[0323] In a particular embodiment, the fourth part of the second zinc finger domain contains the amino acid sequence represented by (Z)6H(Z) 3-4 H represents the amino acid sequence. Thus, (Z)6 represents 6 amino acids, while (Z) 3-4 represents 3 or 4 amino acids. By way of example, the fourth part of the second zinc finger domain may, for example, contain the amino acid sequence Z 13Z 14 Z 15 Z 16 Z 17 Z 18 H 19 Z 20 Z 21 Z 22 H 23 or Z 13 Z 14 Z 15 Z16Z 17 Z 18 H 19 Z 20 Z 21 Z 22 Z 23 H 24 , wherein each Z (Z 13 , Z 14 …, etc.) independently represents any amino acid, and more specifically, wherein each Z represents any natural or proteogenic 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 understand that, for example, Z 13 can represent a different amino acid from, for example, Z 14 , or it can represent the same amino acid. Those skilled in the art understand that H 19 indicates the presence of histidine at the 19th position of the amino acid sequence contained in the second part of the first hybrid zinc finger domain. Similarly, H 23 or H 24 indicates the presence of histidine-cysteine at the 23rd position or at the 24th position of the sequence (depending on whether (Z) 3-4 is associated with 3 or 4 amino acids). It should be noted that, for example, Z13 does not indicate the position relative to the first hybrid zinc finger domain, because as explained elsewhere herein, in some embodiments, there may be a linker 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 part of the second (hybrid) zinc finger can vary and can be, for example, 11 amino acids in length. In a preferred embodiment, the fourth part of the second zinc finger domain has the amino acid sequence (Z)6H(Z) 3-4 H, and wherein the amino acid sequence is that of the second Cys2-His2 zinc finger domain, i.e., more particularly the amino acid sequence of the wild-type Cys2His2 (C2H2) zinc finger.

[0324] In other words, in a preferred embodiment, the second zinc finger domain contained in the zinc finger degradation resolver according to the present invention is a hybrid zinc finger domain containing a Cys2His2 (C2H2) domain, and wherein the hybrid zinc finger domain contains at least two subdomains (i.e., the third part and the fourth part), and wherein the amino acid sequence of the third part is from a wild-type zinc finger, more particularly from a wild-type Cys2 His2 (C2H2) zinc finger (i.e., the third Cys2-His2 zinc finger domain), and wherein the amino acid sequence of the fourth part is from a wild-type zinc finger, more particularly from a wild-type Cys2 His2 (C2H2) zinc finger (i.e., the fourth Cys2-His2 zinc finger domain), and wherein preferably, the third Cys2-His2 zinc finger domain is different from the fourth Cys2-His2 zinc finger domain (e.g., wherein the third Cys2-His2 zinc finger domain is a wild-type zinc finger different from the fourth Cys2-His2 zinc finger domain). Preferably, the amino acid sequence of the third part comprises or is (Z)2C(Z)2C(Z) 5-6 , and / or the amino acid sequence of the fourth part comprises or is (Z)6H(Z) 3-4 H.

[0325] In a preferred embodiment, the fourth part is C-terminal relative to the third part.

[0326] Those skilled in the art should understand that in addition to the above domains, parts and linkers, the zinc finger degradation resolver according to the present invention may also contain other amino acids, such as the N-terminus from the first hybrid zinc finger domain or the C-terminus from the second zinc finger domain.

[0327] In other embodiments, there is provided a zinc finger degradation resolver and / or a hybrid zinc finger polypeptide according to the present invention, and 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, ZNF692 ZF5, preferably ZFP91 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, ZNF692 ZF5, preferably ZFP91 ZF5.

[0328] The present invention is not particularly limited to a specific (wild-type) Cys2-His2 zinc finger domain that provides the first, second, third, and / or fourth portions in the first heterozygous zinc finger domain and the second zinc finger domain, respectively. A variety of wild-type or naturally occurring Cys2-His2 zinc finger domains have been described in the art and are suitable for use in a degron system that includes the use of IMiDs (see, for example, those described by (Sievers et al., Science. 2018 Nov 2; 362(6414): eaat0572)). Those skilled in the art will understand that such Cys2-His2 zinc finger domains, which contain a typical β-hairpin loop and α-helix, can thus be suitably used in the present invention and provide, for example, the first, second, third, and / or fourth portions in the first heterozygous zinc finger domain and the second zinc finger domain (particularly the second zinc finger domain), and wherein two amino acids in the first and / or second portions can be substituted and are detailed herein. Thus, those skilled in the art can readily provide such zinc finger degrons according to the present invention that retain the zinc finger-like fold (β-hairpin loop and α-helix) and exhibit the properties as disclosed herein (e.g., sensitivity to IMiDs in the context of protein degradation).

[0329] In other words, in some embodiments, the amino acid sequence of the Cys2-His2 zinc finger domain for selection of the third portion and / or fourth portion (see below) is 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).

[0330] As discussed herein, in certain embodiments, the second zinc finger in a zinc finger degrader and / or a heterozygous zinc finger degrader according to the present invention can be a heterozygous zinc finger domain, or alternatively, can be a non-heterozygous zinc finger domain. In the latter case, the second zinc finger can thus also be selected from any one of the third or fourth Cys2-His2 zinc finger domains listed above (providing both a first and a second part from the same Cys2-His2 zinc finger domain).

[0331] 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., the ZF4 domain from ZFP91 and the 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., both the first Cys2-His2 zinc finger domain and the fourth Cys2-His2 zinc finger domain are from ZFP91 (zinc finger domains ZF4 and ZF5 respectively) and both the second Cys2-His2 zinc finger domain and the third Cys2-His2 zinc finger domain are from a different zinc finger protein, such as IKZF1 (zinc finger domains ZF2 and ZF3 respectively). When providing two different Cys2-His2 zinc finger domains from the same zinc finger protein, the Cys2-His2 zinc finger domain that is N-terminal relative to the other in the wild-type zinc finger protein is preferably also N-terminal to the other in the zinc finger degrader according to the present invention.

[0332] The listed Cys2-His2 zinc finger domains, their sequences and structures are well known to those skilled in the art and are available from various scientific publications and databases.

[0333] Although it is not necessary for the present invention that the zinc finger depletor and / or zinc finger polypeptide according to the present invention includes a second zinc finger domain, or includes a second zinc finger domain (as described below), it has surprisingly been found that the sensitivity of the zinc finger depletor and / or zinc finger polypeptide according to the present invention can be further enhanced or improved by combining a first hybrid zinc finger domain (e.g., which has two substitutions) with an additional second zinc finger domain (preferably a second zinc finger domain as described herein). Although various second zinc finger domains and / or second zinc finger domains can be used (discussed elsewhere herein; e.g., those obtained from IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, ZNF692 ZF5 (see also Uniprot accession numbers: Q13422, Q9UKT9, Q96JP5, Q96CK0, Q8N554, Q17R98, Q9BU19 respectively)), in a preferred embodiment, there is provided a zinc finger depletor and / or hybrid zinc finger depletor according to the present invention, and wherein the third part of the second zinc finger domain comprises the amino acid sequence according to SEQ ID NO: 104 (FKCHLCNYACRR), the fourth part of the second zinc finger domain comprises the amino acid sequence according to SEQ ID NO: 105 (KDSVVAHKAKSH), and / or wherein the second zinc finger comprises the amino acid sequence according to SEQ ID NO: 106 (FKCHLCNYACRRKDSVVAHKAKSH). In a preferred embodiment regarding the second zinc finger, the second zinc finger is composed of the β-hairpin part of IKZF1 ZF3 and the α-helix part of ZFP91 ZF5.

[0334] Thus, in an embodiment, there is provided a non-natural hybrid zinc finger polypeptide according to the present invention, and wherein the non-natural hybrid zinc finger comprises a first hybrid zinc finger domain as defined herein.

[0335] Also provided are unnatural hybrid zinc finger polypeptides according to the invention that further comprise a second zinc finger domain, wherein the second zinc finger domain is a non-hybrid zinc finger domain. As explained herein, although including a second zinc finger domain in the hybrid zinc finger polypeptides according to the invention may be beneficial, it has been determined that such a second zinc finger domain is not necessary to provide the observed enhanced or increased sensitivity to IMiDs. However, in some embodiments, the unnatural hybrid zinc finger polypeptides according to the invention 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 can be a wild-type Cys2-His2 zinc finger domain, such as those disclosed herein, such as IKZF1 ZF3, IKZF3 ZF3, as well as ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, and / or ZNF827 ZF2, ZNF692 ZF5.

[0336] The second zinc finger domain is preferably oriented C-terminally relative to the first hybrid zinc finger domain and, as described elsewhere herein, can be directly adjacent to the first hybrid zinc finger domain or can be linked to the first hybrid zinc finger domain as such.

[0337] In some embodiments, the second zinc finger domain is a second zinc finger domain such as those described elsewhere herein. In those embodiments where the second zinc finger domain in the hybrid zinc finger domain according to the invention is non-hybrid (e.g., a wild-type or naturally occurring zinc finger domain or a variant thereof), the second zinc finger domain can preferably be selected from, for example, IKZF1 ZF3 (SEQ ID NO: 109 (FKCHLCNYACRRRDALTGHLRTH)) or IKZF3 ZF3 (SEQ ID NO: 110 (FKCHLCNYACQRRDALTGHLRTH)) or comprise the amino acid sequence according to SEQ ID NO: 109 or SEQ ID NO: 110. Other 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 NO: 109–115, etc.

[0338] In some embodiments according to the present invention, the drug is an immunomodulatory imide drug (IMiD), preferably where the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, ibedilomide, CC-885, their salts, and analogs. In some embodiments, the concentration used is from 0.01 nM to 1000 nM, and the concentration of the IMiD (such as lenalidomide) is sequentially increased to be from 0.05 to 1000 nM, 0.1 - 750 nM, and 0.5 nM to 500 nM. In some embodiments, the daily dose of the IMiD provided to the patient can be from 0.005–50 mg per day, such as 0.005–50 mg of lenalidomide per day.

[0339] Thus, in some embodiments, there are provided cells according to the present invention, where the drug is an immunomodulatory imide drug (IMiD), preferably where the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, ibedilomide, CC-885, their salts, and analogs. Alternatively, there are also provided cells according to the present invention, where the cells further comprise an immunomodulatory imide drug (IMiD), preferably where the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, ibedilomide, CC-885, their salts, and analogs.

[0340] The protein of interest can be any protein of interest. For example, the protein of interest can be a protein that helps treat a condition or disease, such as treating cancer, for example, aiming to treat tumors. Thus, in some embodiments, there are provided nucleic acids encoding a protein of interest that encode cytokines, interleukins, interferons, chemokines, receptors, ligands, antibodies or antibody fragments, bispecific antibodies, T cell engagers, bispecific T cell engagers, checkpoint inhibitor antagonists, agonists, enzymes, regulatory elements, transcription factors, or DNA-binding domains of transcription factors. Those skilled in the art will understand how to provide nucleic acids encoding such proteins of interest, and where the nucleic acid encoding the protein of interest is operably linked to an inducible promoter according to the present invention.

[0341] In some embodiments, the cytokine is a natural 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.

[0342] In some embodiments, the antibody or antibody fragment or bispecific antibody is an anti-IL-6, anti-IL-6R, anti-IL-6Ra, anti-TNFa, anti-IL-1, anti-PD1, anti-CD25, anti-CD3, anti-CD20, anti-CD40 agonistic antibody, anti-IL-8, anti-MCP1, anti-MIP-1, anti-TGFβ, anti-CD47, anti-CSF1R, anti-CD28, anti-TIGIT, anti-VEGFR or anti-FAP.

[0343] In some embodiments, the checkpoint inhibitor antagonist is an anti-PD-L1, anti-PD-1, anti-CTLA4, anti-LAG3, anti-TIM3, anti-2B4 or anti-CD160, anti-CD5.

[0344] In some embodiments, the bispecific antibody comprises, but is not limited to, a functional domain of any one 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 agonistic antibody, anti-IL-8, anti-MCP1, anti-MIP-1, anti-TGFβ, anti-CD47, anti-CSF1R, anti-CD28, anti-TIGIT, anti-VEGFR or anti-FAP.

[0345] In some embodiments, the chemokine is CCL5, XCL-1, XCL-2, CCR-7, CCL-19 or CCL-21.

[0346] In some embodiments, the transcription factor or regulatory element is T-bet, TCF7, EOMES, a Runx family member, BLIMP1, Bcl2, Bcl6, FoxP3, FoxO1, FoxO1-3A.

[0347] In an embodiment according to the invention, the nucleic acid encoding the protein of interest encodes a chemokine. In an embodiment according to the invention, the chemokine is CCL5, CCL19 or CCL21. In an embodiment according to the invention, the nucleic acid encoding the protein of interest encodes a cytokine receptor. In an embodiment according to the invention, the cytokine receptor is TGFBR (TGF-β receptor) or TGFBR2.

[0348] In some embodiments, cells according to the invention are also provided, and nucleic acids encoding a receptor, nucleic acids encoding a chimeric protein, and / or (preferably) nucleic acids encoding a protein of interest are provided to the cells (and wherein the nucleic acids encoding the protein of interest are operably linked to an inducible promoter, e.g., wherein the nucleic acids provided to the cells comprise an inducible promoter and a gene encoding the protein of interest). In such embodiments, by transcription and translation, the cells according to the invention will be provided with the receptor and chimeric protein according to the invention, and, in the case where the inducible promoter according to the invention is induced, will also be provided with the protein of interest (since the protein of interest is then expressed).

[0349] In some embodiments, the cells according to the invention have expressed a receptor, such as a TCR, NKR, or CAR, and the cell is provided with nucleic acids encoding a chimeric protein and nucleic acids encoding a protein of interest (and wherein the nucleic acids encoding the protein of interest are operably linked to an inducible promoter, e.g., wherein the nucleic acids provided to the cell comprise an inducible promoter and a gene encoding the protein of interest).

[0350] In preferred embodiments, the nucleic acids encoding a receptor as disclosed herein, and / or nucleic acids encoding a chimeric protein, and / or nucleic acids encoding a protein of interest are provided to the cells using a vector to introduce the nucleic acids into the cells from the outside. In other preferred embodiments, the nucleic acids encoding a chimeric protein and / or nucleic acids encoding a protein of interest as disclosed herein are provided to the cells using a vector to introduce the nucleic acids into the cells from the outside. In preferred embodiments, the nucleic acids encoding a receptor as disclosed herein, and / or nucleic acids encoding a chimeric protein, and / or nucleic acids encoding a protein of interest are or have been prepared using recombinant DNA techniques.

[0351] In some embodiments, the nucleic acids encoding a receptor as disclosed herein, and / or nucleic acids encoding a chimeric protein, and / or nucleic acids encoding a protein of interest are not integrated into the genomic DNA of the cells, and / or wherein the nucleic acids are extrachromosomal. In some embodiments, the nucleic acids encoding a receptor as disclosed herein, and / or nucleic acids encoding a chimeric protein, and / or nucleic acids encoding a protein of interest are integrated into the genomic DNA of the cells, and / or wherein the nucleic acids are not extrachromosomal.

[0352] In some embodiments, the nucleic acids encoding a receptor, nucleic acids encoding a chimeric protein, and / or nucleic acids encoding a protein of interest (wherein the nucleic acids encoding the protein of interest are operably linked to an inducible promoter, e.g., wherein the nucleic acids provided to the cells comprise an inducible promoter and a gene encoding the protein of interest) are located on one and the same vector. In these embodiments, the cells are provided with the vector, which comprises the nucleic acids encoding a receptor, nucleic acids encoding a chimeric protein, and / or nucleic acids encoding a protein of interest (under the control of an inducible promoter).

[0353] In other embodiments according to the present invention, more than one type of vector may be used. For example, a first vector contains a nucleic acid encoding a receptor, and a second vector encodes a chimeric protein according to the present invention. In such embodiments, the nucleic acid encoding the protein of interest may be contained in the first vector, the second vector, or both. Those skilled in the art should understand that any type of combination of a nucleic acid encoding a receptor, a nucleic acid encoding a chimeric protein, and a nucleic acid encoding a protein of interest may be contained in the vector, including combinations in which one nucleic acid (e.g., a nucleic acid encoding a receptor or a nucleic acid encoding a chimeric protein or a nucleic acid encoding a protein of interest) is not contained in the vector. In the latter embodiments, other vectors may be used, which contain nucleic acids not contained in the first vector, or in which the cells already express a protein (e.g., a receptor). 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 more than one vector. In some embodiments, the nucleic acid encoding the chimeric protein is contained in more than one vector. In some embodiments, the nucleic acid encoding the protein of interest is present in more than one 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 may be in opposite, reverse orientations.

[0354] Accordingly, in a preferred embodiment of the present invention, there is provided a (modified) cell according to the present invention, which comprises:

[0355] a receptor that is capable of receiving an activation signal;

[0356] a nucleic acid that is operably linked to a promoter and encodes a chimeric protein, the chimeric protein comprising:

[0357] a docking domain that is capable of binding to the receptor and inhibiting signal transduction induced when the receptor receives an activation signal;

[0358] a drug-regulated protein stability domain; and

[0359] an inducible promoter that is operably linked to a nucleic acid encoding a protein of interest, wherein the inducible promoter is induced when the receptor receives an activation signal in the absence of the chimeric protein.

[0360] Those skilled in the art should understand that the preferences, features, and embodiments discussed herein with respect to the present invention equally apply to this aspect of the present invention.

[0361] Those skilled in the art will understand that in the preferred embodiments, cells according to the invention are provided which comprise a nucleic acid operably linked to a promoter and encoding a (cytoplasmic) chimeric protein and a nucleic acid encoding a protein of interest (operably linked to an inducible promoter), wherein the cell expresses the chimeric protein. In other embodiments, cells according to the invention are provided which comprise a nucleic acid operably linked to a promoter and encoding a (cytoplasmic) chimeric protein 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 (e.g., as described herein, in the presence of a drug (e.g., an IMiD such as lenalidomide) that modulates a drug-regulated stability domain in the chimeric protein, modulating a zinc finger degron (as the drug-related stability domain)); and under conditions in which a receptor in the cell according to the invention receives an activating signal (e.g., by ligand binding). Those skilled in the art will understand that in such embodiments, the cell also expresses a receptor capable of receiving the activating signal.

[0362] Thus, in these embodiments of the invention, such cells according to the invention are provided and wherein the chimeric protein is expressed and / or the protein of interest is expressed.

[0363] In view of the disclosure herein, those skilled in the art will understand that by using the cells according to the invention and by using a drug capable of modulating a drug-regulated stability domain comprised in the chimeric protein, the expression of the protein of interest can be regulated by providing an activating signal to the cell or to a receptor expressed by the cell or once the cell or a receptor expressed by the cell has received an activating signal. Regulation or modulation of the expression of the protein of interest can be achieved by providing to the cell an effective amount of a drug capable of modulating the drug-regulated protein stability domain. For example, in some embodiments, the amount of the drug provided is sufficient to allow the protein of interest to be expressed from a nucleic acid encoding the protein of interest and operably linked to an inducible promoter at a level of at least or not more than 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the maximum expression of the protein of interest in the cells according to the invention. For example, maximum expression can be established in the absence of (expression of) the chimeric protein according to the invention and under conditions in which the receptor according to the invention has been activated by an activating signal.

[0364] Thus, in such embodiments according to the present invention, a method for regulating the expression of a target protein is provided, the method comprising providing an activation signal to a cell according to the present invention in vitro or in vivo in the absence of a drug that is present in an effective amount and that can modulate the protein stability domain regulated by the drug. Those skilled in the art should understand that, in the context of such embodiments, the effective amount of the drug refers to the drug concentration that allows the expression of the target protein (e.g., at the expression level as shown above). Those skilled in the art should also understand that the preferences, features, and embodiments discussed herein with respect to the present invention are equally applicable to this aspect of the present invention.

[0365] Those skilled in the art should understand that the present invention also allows the regulation of the expression (level) of a target protein in a subject in need (e.g., a patient). At the same time, the present invention allows the regulation of the activity of various cells in response to an activation signal. For example, once the TCR recognizes an antigen on a tumor cell and is activated, the activity of T cells against the tumor cell can be regulated, leading to the intracellular transduction of ITAM-mediated signals. By being able to regulate the expression of a target protein in response to an activation signal and regulate the activity of cells, the present invention allows for a powerful treatment of a condition to be treated (e.g., cancer / tumor), while allowing the precise regulation of the cell activity level and the target protein expression level to a desired level in a subject to be treated (e.g., a patient).

[0366] Therefore, a method for regulating the expression of a target protein in a subject is also provided, the method comprising administering to the subject an effective amount of a drug that can modulate the protein stability domain regulated by the drug, wherein the subject comprises a cell according to the present invention.

[0367] In such embodiments, a patient is provided with a cell according to the invention or the patient comprises a cell according to the invention, and a patient is provided with an effective amount of a drug (e.g., an IMiD such as lenalidomide), e.g., when using a zinc finger degron as the drug-regulated protein stability domain. As discussed above, an effective amount of a drug refers to the drug concentration that allows the expression of the protein of interest to the desired level (e.g., the expression level as shown above). Thus, the use of cells according to the invention in a subject allows for a response to an activation signal and for the regulation of the expression of the protein of interest by providing to the subject a drug that regulates the drug-regulated protein stability domain, and, e.g., in the case where the cell is an immune cell (e.g., a T cell, NK cell, tumor infiltrating lymphocyte or macrophage), for the regulation of the activity of the cell in response to an activation signal received (e.g., binding to an antigen present on cancer / tumor cells). In some embodiments, a first effective amount of a drug is provided to a subject and then a second or other amount of the drug is provided to the patient at a later time point, and wherein the first effective amount and the second or other effective amount of the drug can be the same or different. In other embodiments, there is a period of time between the first effective amount and the second or other effective amount during which the subject is not treated with an effective amount of the drug, e.g., such that during this period between the two effective amounts of the drug, signal transduction from the receptor receiving the activation signal and / or induction of the inducible promoter and / or expression of the protein of interest is inhibited or suppressed to a certain extent (e.g., to the greatest possible extent in cells according to the invention). Those skilled in the art should also understand that more than one drug capable of regulating the drug-regulated protein stability domain can be used simultaneously or sequentially. For example, it is contemplated that a first drug may be more effective than a second drug in regulating the drug-regulated protein stability domain, and that by achieving a certain ratio between the two drugs, the expression of the protein of interest (and / or the activity of the cell, e.g., the activity of an immune cell such as a T cell, tumor infiltrating lymphocyte or NK cell) can be regulated more precisely.

[0368] Those skilled in the art should also understand that the preferences, features, and embodiments discussed herein with respect to the invention apply equally to this aspect of the invention.

[0369] In another embodiment of the invention, a method of regulating the expression of a protein of interest in a subject is provided, wherein the method further comprises administering to the subject a cell according to the invention.

[0370] Those skilled in the art should understand that the cell can be any type of cell suitable for administration to a subject (e.g., a human patient). The cell can be an autologous cell (i.e., obtained from the subject) that has been modified (engineered) and subsequently engineered to obtain the cell according to the present invention. The cell can also be an allogeneic cell. The cell is preferably a T cell (e.g., a T cell expressing a TCR and / or a CAR), an NK cell (including a CAR NK cell), a tumor-infiltrating lymphocyte, or a macrophage (including a CAR macrophage).

[0371] In some embodiments, the cell according to the present invention is administered to the subject only once. In other embodiments, the cell according to the present invention is administered to the subject at multiple time intervals. Determining an effective amount of the cell according to the present invention to be administered to the patient is within the knowledge of those skilled in the art. In one example, for example, using a T cell or a CAR T cell, the amount of the cell according to the present invention administered to the subject can be, for example, an amount equivalent to the amount or number of cells typically administered to the subject during a T cell therapy and / or a CAR T cell therapy.

[0372] In some embodiments, the cell according to the present invention can be administered to the subject in combination with other agents, provided that the additional agent does not adversely affect the ability of the cell according to the present invention to deliver the desired therapy.

[0373] As shown herein, the present invention can be used to treat various conditions or diseases, for example, in a human patient, where regulation of the production of a protein of interest may be required in response to an activation signal. For example, it is contemplated that the cell according to the present invention can be used to treat various immune diseases, which include autoimmune diseases. Also, as exemplified herein, the cell according to the present invention (particularly an immune cell according to the present invention, such as a T cell, an NK cell, a tumor-infiltrating lymphocyte, or a macrophage) can be suitably used to treat a subject suffering from cancer. That is, in the treatment of a tumor, by allowing the production of a protein of interest at a desired level in time and space (which level can be altered by using different effective amounts of a drug and / or different drug combinations that can modulate the protein stability domain of the drug). Accordingly, a method of regulating the expression of a protein of interest in a subject is also provided, wherein the method is for treating cancer in the subject and / or for treating a tumor in the subject.

[0374] In view of the disclosure herein, there is provided a cell according to the present invention for use as a medicament, preferably for treating cancer in a subject and / or for treating a tumor in a subject, preferably wherein the treatment comprises administering the cell according to the present invention to the subject and optionally administering a drug capable of modulating the protein stability domain of the drug.

[0375] Those skilled in the art should also understand that the preferences, features, and embodiments of the present invention discussed herein are equally applicable to this aspect of the present invention.

[0376] Accordingly, there is provided a cell according to the present invention, which is used as a medicament, preferably for treating cancer in a subject and / or for treating a tumor in a subject, wherein administering a drug that can regulate a drug-regulated protein stability domain includes changing the dose of the drug that can regulate a drug-regulated protein stability domain. By changing the dose, the expression level of the target protein can be regulated. For example, such a level can be monitored from a blood sample using techniques such as ELISA.

[0377] In other embodiments according to the present invention, the present invention provides a drug, which is used as a medicament, preferably for treating cancer in a subject and / or for treating a tumor in a subject, wherein the treatment includes administering to the subject a cell according to the present invention, and administering a drug, wherein the drug can regulate a drug-regulated protein stability domain.

[0378] Those skilled in the art should also understand that the preferences, features, and embodiments of the present invention discussed herein are equally applicable to this aspect of the present invention.

[0379] Preferably, the drug is at least one IMiD, preferably selected from the groups disclosed herein, preferably lenalidomide.

[0380] Finally, in some embodiments, there is provided a vector, namely comprising as Figure 6A DNA vector of any design as shown and understood by those skilled in the art, wherein, in the context of these embodiments, a specifically indicated polyadenylation signal (such as SV40 pA), a constitutive transport element (CTE) (such as CTE), a specific gene encoding a receptor capable of receiving an activation signal (such as CD19 CAR), a specific promoter (such as pCMV or pMSCV), a specific gene encoding a chimeric protein according to the present invention (such as RheoBrick), a specific post-transcriptional regulatory element (such as WPRE), a specific 3' UTR (such as IL2 3' UTR), and / or a specific inducible promoter (in the context of the present invention; such as pNFAT) can 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 according to the present invention, post-transcriptional regulatory element, 3' UTR, and / or a specific inducible promoter (in the context of the present invention). In some embodiments, a vector or a combination of vectors is provided, which comprises at least one or more genes encoding a receptor capable of receiving an activation signal according to the present invention, and / or one or more genes encoding a chimeric protein according to the present invention, and / or one or more inducible promoters according to the present invention and a gene encoding a protein of interest according to the present invention and operably linked to the inducible promoter; in the case of a combination of vectors, a first vector and a second vector, wherein the first vector and the second vector each independently comprise at least one of the following: a gene encoding a receptor capable of receiving an activation signal according to the present invention, a gene encoding a chimeric protein according to the present invention, or an inducible promoter according to the present invention and a gene encoding a protein of interest according to the present invention and operably linked to the inducible promoter; and, preferably, wherein the vector or the combination of vectors comprises one or more RNA degradation elements according to the present invention, and even more preferably, wherein the vector or the combination of vectors has a configuration (and has or does not have other elements as shown therein) with respect to the following: one or more genes encoding a receptor capable of receiving an activation signal according to the present invention, one or more genes encoding a chimeric protein according to the present invention, one or more inducible promoters according to the present invention and a gene encoding a protein of interest according to the present invention and operably linked to the inducible promoter, and optionally one or more RNA degradation elements according to the present invention. Figure 6 as shown in

[0381] In certain embodiments, one of the multiple vectors according to the present invention comprises (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 according to the present invention, and at least one inducible promoter and a nucleic acid encoding a protein of interest operably linked to the inducible promoter.

[0382] In certain embodiments, one of the plurality of vectors according to the invention comprises (alone or in combination) at least one gene encoding a chimeric protein according to the invention, as well as at least one inducible promoter and a nucleic acid encoding a protein of interest operably linked to the inducible promoter. In certain embodiments, one of the plurality of vectors according to the invention comprises (alone or in combination) at least one gene encoding a chimeric protein according to the invention (e.g., in embodiments where the cell containing the vector endogenously expresses a receptor capable of receiving an activation signal and contains an endogenous locus into which the gene encoding the protein of interest has been integrated, and the locus contains a promoter controlled by the inducible promoter according to the invention). In some embodiments, the vector may comprise additional elements as disclosed herein, particularly an RNA degradation element (RDE) as disclosed and described herein.

[0383] In some embodiments, a nucleic acid (or vector) is provided and used to form a modified cell according to the invention, the nucleic acid (or vector) comprising, alone or in combination, a nucleic acid encoding a chimeric protein according to the invention operably linked to a promoter, and / or a nucleic acid encoding a protein of interest operably linked to an inducible promoter according to the invention. In some embodiments, a vector is additionally provided that comprises or further comprises a nucleic acid encoding a receptor according to the invention, the nucleic acid being operably linked to a promoter.

[0384] Particularly preferred vectors provided to a cell to provide a cell according to the invention are vectors that combine a nucleic acid encoding a chimeric protein of the invention, a nucleic acid encoding a receptor (e.g., an antigen receptor), and a nucleic acid encoding a protein of interest, the nucleic acid encoding the protein of interest being 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 thereto). Figure 6 An example CC30 of such a design is shown. As described above, the different nucleic acids can be in the same orientation or in opposite, reverse orientations. Preferably, the orientation is as shown in CC30.

[0385] In another embodiment, a combination of at least two vectors is provided, the at least two vectors being provided to a cell to provide a cell according to the invention, and wherein both vectors comprise a nucleic acid encoding a chimeric protein according to the invention. One of the vectors may further comprise a nucleic acid encoding a receptor, while the other vector may further comprise a nucleic acid encoding a protein of interest, the nucleic acid encoding the protein of interest being 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 thereto). An example of such a combination of at least two vectors is as Figure 6The combination of the vectors CC39 and CC1 as shown. As described above, different nucleic acids can be in the same orientation or in opposite, reverse orientations. Preferably, the orientation is as shown for CC39 and CC1.

[0386] In addition, the above-described vector or vector combination preferably comprises at least one 3'UTR, said 3'UTR comprising at least one ARE, preferably more than one ARE.

[0387] For the various embodiments disclosed herein, the protein of interest is preferably a protein secreted by a cell according to the invention and / or modified such that the protein is secreted by a cell according to the invention, or is a cell surface protein, such as a transmembrane cell surface protein. In some embodiments, the protein of interest itself is another receptor that can receive other activating signals.

[0388] Those skilled in the art should understand that due to the sharing mechanism shown herein, the present invention is not particularly limited to a specific combination of cell types, receptors capable of receiving activation signals, chimeric proteins, docking domains, drug-regulated protein stability domains, inducible promoters, and nucleic acids encoding the protein of interest. Those skilled in the art should understand that the teachings of the present application can use any suitable combination thereof. Therefore, for those skilled in the art, based on the disclosure of different embodiments herein, any combination thereof can be disclosed. 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 the CRBN polypeptide, especially any of the hybrid zinc finger domains 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 imide drug. For those skilled in the art, in the embodiments of the present invention, any Cys2-His2 zinc finger domain capable of drug-induced binding to the CRBN polypeptide disclosed herein, especially any hybrid zinc finger domain, can be replaced by any other Cys2-His2 zinc finger domain capable of drug-induced binding to the CRBN polypeptide, especially any of the hybrid zinc finger domains disclosed herein. For those skilled in the art, in the embodiments of the present invention, any protein of interest disclosed herein can be replaced by any other protein of interest disclosed herein. For those skilled in the art, in the embodiments of the present invention, any cell type disclosed herein can be replaced by any other cell type disclosed herein. For those skilled in the art, in the embodiments of the present invention, any immunomodulatory imide drug disclosed herein can be replaced by any other immunomodulatory imide drug disclosed herein. For those skilled in the art, such substitutions and the combinations resulting therefrom are also directly and clearly disclosed.

[0389] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are hereby incorporated by reference in their entirety, including all data, tables, charts, and text provided in the cited references. In addition, the entire content of the references cited in the references cited herein is also incorporated by reference in its entirety.

[0390] It should be understood that the language or terms herein are for descriptive purposes and not for limitation, such that those skilled in the art can interpret the terms or language in this specification according to the teachings and guidance presented herein and in combination with the knowledge of those of ordinary skill in the art.

[0391] It should be understood that all the details, examples, and preferences discussed with respect to one aspect of the embodiments of the present invention are equally applicable to any other aspect or embodiment of the present invention. Therefore, it is not necessary to separately elaborate on all these details, embodiments, and preferences for all aspects.

[0392] The present invention has been generally described above. The present invention will be more readily understood by reference to the following examples, which are provided by way of illustration only and are not intended to limit the present invention. Other aspects and embodiments will be apparent to those skilled in the art. Examples

[0393] Example 1

[0394] Introduction, Results and Discussion

[0395] As discussed elsewhere herein, there is a need to improve the therapeutic window of cell therapies, particularly immunotherapies such as T cell-based therapies, for example in the treatment of tumors (such as solid tumors) in various cancers. The inventors of the present application have found that the invention disclosed herein can improve the therapeutic window of such therapies (such as cancer therapies), thereby providing new methods that can effectively deliver biologics (such as cytokines) and other biological agents (such as antibodies, T cell engagers, and any protein of interest (such as a therapeutically active protein)) to tumor cells, particularly solid tumors.

[0396] Local production of such proteins of interest, such as biologic agent compounds with anti-tumor activity, within a limited in vivo environment (such as within the tumor microenvironment) can be achieved by generating these molecules from adoptively transferred cells. This method is referred to 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), where the biologic agent payload with anti-tumor properties can be released from the adoptively transferred cells after antigen receptor activation. Although this method ensures local delivery of the payload of interest in the tumor microenvironment, the timing and dose of biologic production by CAR-T cells are uncontrolled, which can lead to the production of excessive payload and fatal toxicity (Zhang et al., Clin Cancer Res. May 15, 2015; 21(10):2278–2288).

[0397] Here, we disclose a new method that allows the generation of biologics from genetically modified cells, particularly immune cells (CAR-T, TCR-T, TIL, NK cells, and other suitable cells known to those skilled in the art), in a manner that strictly depends on activation signals (such as antigens), activates the receptors that receive such signals, and can be regulated using drugs (such as small molecules).

[0398] As extensively illustrated in the examples, the present invention enables precise control of the timing and dose of biologics (target proteins) production using genetically modified cells (particularly immune cells), thereby enabling the production of an appropriate amount of biologics at the disease site (i.e., when such cells are provided to a patient in need) to optimize the therapeutic window and prevent overproduction of such biologics to avoid side effects.

[0399] To achieve this goal, the inventors created a transgenic design in which the production of the biologic payload (biologic product, target protein) is operably associated with the level of ITAM signaling in immune cells, and in which the level of ITAM signaling is regulated by a rheostat switch. Examples of such immune cell rheostat switches (also known as CRASH-IT switches) are described in WO2021 / 080427, and in some embodiments, such rheostat switches are preferred. In the current examples, the rheostat switch can also be referred to by its registered trademark for reference.

[0400] The rheostat switch used in the invention disclosed herein, particularly strictly controls immune cell activation using an orally administered, clinically approved drug or small molecule (such as lenalidomide) (WO2021 / 080427). In the absence of the drug, immune cell function (such as immune cell activation) is strictly inhibited, while when the drug is provided, immune cell function is restored, thereby providing remote control ( Figure 1 ).

[0401] Structurally, the rheostat switch (such as ) platform consists of three functional domains: a docking domain that forms a reversible interaction with the antigen receptor, an inhibitory domain that inhibits the TCR / CAR / NKR (NK cell receptor) signaling pathway, and a degron domain that controls the stability of the rheostat switch (such as switch) in a drug-dependent manner (such as a small molecule-dependent manner).

[0402] In some embodiments, and as used in the examples, the rheostat switch (for example, The switch) comprises a docking domain derived from the Zap70 (2xSH2) domain, an inhibitory signaling domain of the ITIM / ITSM-containing signaling domain derived from the Siglec11 protein, and a synthetic zinc finger (SynFinger) degron sequence, and we have optimized the synthetic zinc finger (SynFinger) degron sequence to show several-fold increased sensitivity to lenalidomide (as described herein, see also Dutch patent application NL2031325). However, those skilled in the art should understand that the present invention is not limited to such optimized rheostat switches. In other embodiments of the rheostat switch, for example the switch, the docking domain is derived from the SH2 domain of the Syk or Lck protein, the inhibitory signaling domain is derived from the ITIM / ITSM-containing signaling domain of PD1, BTLA, SIRPa, SIGLEC5, SIGLEC9, PECAM1, Ly9, and the drug-regulated protein stability domain comprises a self-cleaving degron (such as the SMASh tag), a PROTAC-binding domain (such as FKBP12F36V (SEQ ID NO:135)) or other zinc finger degrons. Examples of preferred rheostat switches used in the present invention (for example, the switch) are described in WO2021 / 080427 and NL2031325 and herein.

[0403] In addition to developing a technology capable of strictly controlling T cell activity in an antigen-dependent and small molecule-regulated manner, the inventors have surprisingly found that this technology is particularly useful and beneficial for controlling the production of potent payloads (the protein of interest, such as biologics, such as cytokines, including IL-12) in the immunosuppressive tumor microenvironment (TME) and T cell adapters.

[0404] Surprisingly, this is achieved by creating a transgenic design in which the production of such payloads (the protein of interest, biologics) is directly controlled by the level of T cell activation. According to the present invention, in a rheostat switch (such as the switch), the activation of T cells, the modified immune cells (such as T cells) are strictly controlled by signals from activation receptors (such as antigens, such as antigens expressed by tumor cells) and drugs (such as small molecules) ( Figure 1 - 2 ).

[0405] Taking advantage of this, the present invention provides that such transgenes are produced specifically at the site of the activation signal (such as an antigen) (such as at the tumor site), and the T cell activity (or signaling) is controlled to the desired extent by using a drug (small molecule).

[0406] Importantly, in the absence of a rheostat switch (such as In the case of a switch), the use of a transgene controlled by a promoter that is induced after activation of a cell (by a receptor capable of receiving an activation signal) surprisingly shows that the production of a payload (biological product or protein of interest) can be tightly regulated. This is illustrated herein by using an NFAT-controlled EGFP transgene, establishing evidence for the feasibility of controlling payload production, and when lenalidomide is provided in CD4 and CD8 cells, the total EGFP produced increased by ~20-fold ( Figure 2 ). As Figure 7 shown, this is also demonstrated by the IL-12 data discussed below. Note that in this example, although T cells modified with 2 lentiviral vectors (CC3 + CC1) - the vectors encode a rheostat switch, such as switch, CD19CAR, and NFAT-EGFP - only moderately express EGFP in the absence of lenalidomide, but they strongly upregulate EGFP expression in the presence of lenalidomide (drug) and CD19+ Nalm6 cells (activation signal). On the other hand, T cells modified only with vectors encoding CD19CAR and NFAT-EGFP (CC23 + CC22) showed the same EGFP expression in Nalm6 co-culture in the absence or presence of lenalidomide. Note that the rheostat switch (such as switch) may be included in one or more vectors used to modify immune cells. In this example, compared to the vector combinations present in one vector (CC23 + CC1 or CC3 + CC22), the presence of the rheostat switch (such as switch) in both lentiviral vectors (CC3 + CC1) has proven to improve the fold induction. Thus, in some embodiments involving vectors, the rheostat switch (such as switch) is included in at least one vector, preferably included in more than one vector, such as included in two vectors, for modifying cells (such as immune cells) according to the present invention.

[0407] Synthetic promoters that respond to ITAM signaling (e.g., NFAT, NF-kB, AP-1, Jutz et al., J Immunol Methods. 2016 Mar;430:10-20) and native promoters that respond to ITAM signaling (including IL-2 (Gene ID: 3558), IL-4, (3565), IL-6 (Gene ID: 3569), IL-8 (3576), IL-13 (3596), IL-17 (3605), IFNγ (Gene ID: 3458), TNFα (Gene ID: 7124), CD69 (Gene ID: 969), CD137 (Gene ID: 3604), Sousa et al BMC Genomics. 2019 Jul 19;20(1):593, Glinos et al Genes Immun. 2020;21(6):390–408, Redondo-Antón et al Front Genet. 2020 Oct 27;11:552949, Skerka et al J Biol Chem. 1995 Sep 22;270(38):22500-6, Goldfeld et al J Exp Med. 1993 Oct 1;178(4):1365–1379, Cockerill et al Mol Cell Biol. 1995 Apr;15(4):2071–2079, Davydov et al J Immunol. 1995 Dec 1;155(11):5273-9, Macián et al EMBO J. 2000 Sep 1;19(17):4783-95, Dolganov et al Blood. 1996 Apr 15;87(8):3316-26, Faggioli et al Biochim Biophys Acta. May 28, 2004;1692(1):17-24, Kim et al FEBS Lett. 2003 Apr 24;541(1-3):163-70, Gonsky et al J Immunol. 2000 Feb 1;164(3):1399-407, Liu et al J Biol 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.Meanwhile, for example, the 3' untranslated regions (UTRs) 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), IFNγ (NM_000619.3), TNFα (NM_000594.4), CSF2 (NM_000758.4), FasL (NM_000639.3), c-fos (NM_005252.4) and other 3' untranslated regions (UTRs) 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., 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. May 2019; 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).

[0408] Specifically, induction of ITAM signaling increases the nuclear export of mRNA stabilizing factors (such as HuR and NF90), which bind to the AREs in the 3' UTR and replace 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). Replacing the mRNA destabilizing factors increases mRNA stability and protein expression levels. Importantly, according to the present invention, the rheostat (such as )The control of payload production mediated by is surprisingly applicable to regulatory gene elements from different classes (e.g., promoters and / or 3'UTRs), all of which have the property of responding to ITAM signals.

[0409] Activation of the ITAM receptor complex in lymphocytes 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(Suppl2):44, Fric et al., Blood. 2012 Aug 16;120(7):1380-9). In addition, RNA-binding proteins (RBPs) such as HuR control mRNA stability in T cells, NK cells, and macrophages (Kim et al., ImmuneNetw. 2009 Aug;9(4):115–121, et al., Front Immunol. May 23, 2018;9:1094). Thus, modulating ITAM signaling and / or subsequent production of the protein of interest by the methods of the present invention is not limited to T cells, but also applicable to other cells (including immune cell subsets, including NK cells and macrophages).

[0410] Although some (protein of interest) leaky expression may be allowed, such leaky expression is preferably restricted. By combining multiple control mechanisms responsive to the activation state of immune cells, leaky expression can be reduced while increasing the fold induction. We hypothesize that if an antigen-dependent cargo production increase of ~10-fold can be achieved for the NFAT promoter or 3’UTR sequence element when used alone, an expression cassette combining the two elements in the same expression cassette can reach an induction level of up to ~100-fold. In addition, we hypothesize that through the regulation of a small molecule-based rheostat (e.g., ) the precise cargo (payload, protein of interest, biologic) expression level may be surprisingly further enhanced within this increased dynamic range ( Figure 3 ).

[0411] The inventors surprisingly confirmed the above by replacing the constitutive mRNA transport element (CTE) and SV40 polyA downstream of the cargo expression cassette with 3’UTR elements derived from the IL2 or IFNg genes ( Figure 4 A). In Nalm6 co-culture experiments, using a rheostat (e.g., ) T cells modified with CD19 CAR and NFAT-scIL-12 cargo cassettes (the cargo cassettes containing downstream CTE SV40pA, IL2 3’UTR or IFNg 3’UTR) showed 24-fold, 112-fold and 78-fold induction of IL-12 production in the presence of lenalidomide compared to the vector control. Figure 4 B).

[0412] By combining ITAM-responsive regulatory gene elements from different classes (e.g., NFAT promoter and regulatory 3’UTR), the fold induction was significantly improved, which enabled the inventors to test whether even greater fold induction improvements might be achieved by combining multiple regulatory elements from the same class (IL2 and IFNg 3’UTR). In fact, a direct comparison found that CAR-T cells modified with a rheostat (e.g., ) and an IL-12-encoding vector with a combined IL2 3’UTR and IFNg 3’UTR showed higher fold induction compared to vectors encoding a single type of 3’UTR. Figure 5 )

[0413] The generality 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 Figure 16 shown in A, primary human T cells were transduced with lentiviral vectors encoding CDK4 TCR, NY-ESO-1 TCR, CD19 CAR, PSMA CAR or fibronectin EDB CAR or no antigen receptor together with the RheoBrick switch and the ITAM signaling-responsive scIL12 expression cassette. Analysis of IL12 cargo production after co-culture with antigen-positive Nalm6, PC3-PSMA, NKIRTIL006, A549, A375 or Mel624 cells in the absence or presence of 500 nM lenalidomide showed that IL12 cargo production was upregulated 42-fold to 684-fold in the presence of lenalidomide compared to the vector control, while minimal IL12 cargo production was detected in cultures without target cells. Figure 16 B).

[0414] The inventors also created a large number of vectors to determine the optimal vector design for small molecule- and antigen-dependent cargo production applications. Thus, although all the different vectors described herein and, for example, the vectors provided for the engineered cells according to the invention are suitable for the present invention, it was surprisingly found that some vectors are more suitable than others. Single vector designs (e.g., single lentiviral designs) that combine all 3 components (rheostat, e.g., Antigen receptor and cargo expression cassettes), as well as 2-vector designs (e.g., dual-vector lentiviral expression systems)( Figure 6 A). Induction of EGFP cargo production was observed in all designs( Figure 6 B), although the 2-vector expression system (where T cells were modified with 2 lentiviral vectors (e.g., CC39 and CC1)) showed the highest fold induction( Figure 6 C), where the first vector encodes the cargo expression cassette and the second vector encodes the antigen receptor, and both vectors encode one copy of a rheostat, e.g., Higher fold inductions were observed in vector designs containing the IL23’UTR or IFNg 3’UTR (CC39+CC1 and CC40+CC1, respectively) compared to vectors containing CTE SV40 pA, both in 2-vector and single-vector configurations( Figure 6 C).

[0415] The ability to precisely control the expression levels of potent payloads (e.g., cytokines, chemokines, costimulatory or opsonizing antibodies, T cell engagers, NK cell engagers, etc.) in adoptively transferred cells in a small molecule concentration-sensitive and antigen-dependent manner opens up new possibilities. To demonstrate that the modified CAR-T cells according to the invention can regulate the secretion level of IL-12 (POI) as a function of drug concentration, we titrated lenalidomide in CAR-T cell / Nalm6 co-cultures. Potent small molecule-dependent IL-12 secretion was observed even at low nanomolar drug concentrations, and IL-12 secretion levels were induced by 0.5 nM to 500 nM lenalidomide. At the high 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 thus allows for the strict regulation of the expression level of the protein of interest according to the invention by modulating the drug-regulated protein stability domain by adjusting the level or concentration of a drug (e.g., a small molecule).

[0416] Notably, while there have been previous reports of the concept of small molecule-regulated IL-12 cargo secretion (e.g., using a doxycycline-inducible promoter to control IL-12 expression, Alsaieedi et al., Oncoimmunology. 2019;8(3):1542917), such studies did not disclose a mechanism for combining such small molecule-regulated cargo secretion with signaling through activating receptors to control the site of cargo production. Thus, in contrast to the present invention, inducing the expression of the cargo by providing or removing a small molecule in such systems results in the expression of the cargo in both antigen-positive and antigen-negative body sites, thereby affecting the therapeutic window.

[0417] To demonstrate that the technology of the present invention can serve as a building block for designing an AND-gate-based logic circuit with external (small molecule) control, we constructed a proof-of-concept logic circuit that can accept three conditional inputs and produce a tumor-killing output only when all conditional inputs are present ( Figure 8 ).

[0418] The first AND-gate accepts the conditional inputs CD19 antigen and the small molecule lenalidomide, which are detected by the second-generation CD19 CAR and the (i.e., an example of the rheostat system described herein), respectively, and creates a PSMA and CD3 bispecific T cell engager (BiTE) as an output, which is conditionally expressed by the NFAT-PSMA BiTE-IL2 3’UTR expression cassette ( Figure 8 A). The second AND-gate takes the output of the first AND-gate (produced by the PSMA BiTE) as an input, detects the third conditional input (PSMA antigen), and produces anti-PSMA+ tumor killing as the final output ( Figure 8 A).

[0419] To demonstrate the conditional secretion of the PSMA BiTE as a function of two conditional inputs, the AND-gate CAR-T cells (i.e., an example of the engineered cells described herein) were co-cultured with or without CD19-positive Nalm6 cells in the presence or absence of lenalidomide ( Figure 8 B-C). Subsequently, the supernatants were harvested, filtered, and added to a second co-culture containing unmodified PBMCs together with HCT116 PSMA cells or PSMA-negative HCT116 WT cells ( Figure 8 C).

[0420] At the end of the second co-culture, cytokine production and T cell degranulation were assayed, and positive results were found only in the HCT116 PSMA co-cultures prepared with the conditioned medium of the Nalm6 / lenalidomide / AND-gate CAR-T co-culture ( Figure 8 D-I).

[0421] To demonstrate that the RheoBrick-mediated, antigen-dependent, and small molecule-regulated BiTE secretion concept is not limited to one example of BiTE but is general, we designed another AND-gate-based logic circuit where PSMA CAR-T cells expressing RheoBrick secrete the FDA-approved CD19xCD3 bispecific T cell engager blinatumomab after exposure to PSMA antigen-positive target cells in the presence of lenalidomide. Briefly, lentiviral vectors encoding the PSMA CAR, RheoBrick, and an ITAM signaling-responsive expression cassette encoding blinatumomab or a mock cargo (luciferase) were transduced into primary human T cells ( Figure 17 A). The transduced cells were co-cultured with antigen-positive PC3-PSMA cells or antigen-negative PC3 cells in the presence or absence of lenalidomide ( Figure 17 B). Subsequently, the supernatants were harvested, filtered, serially diluted, and added to a second co-culture containing naive T cells together with a 1:1 mixture of CTFR-labeled CD19 antigen-positive Nalm6 cells and CTV-labeled CD19 antigen-negative K562 cells. As described in the methods, after 24 hours, the Nalm6 / K562 cell ratio was measured by FACS analysis to determine specific killing. Specific killing assays showed that conditioned media from the first co-culture with PC3-PSMA cells and lenalidomide induced the highest degree of Nalm6 cytotoxicity in the second co-culture, with a cytotoxic potency increase of approximately two orders of magnitude compared to conditioned media from PC3-PSMA / DMSO, PC3 / lenalidomide, and PC3 / DMSO conditions ( Figure 17 C). As a positive control, the Nalm6 / K562 cell mixture was incubated with serially diluted purified blinatumomab ( Figure 17 D).

[0422] Using the same method, And-gate T cells (i.e., an example of the engineered cells described herein) can be used to selectively secrete T cell adaptors specific for on-target / off-tumor toxicity risk-associated antigens (e.g., 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) at the tumor site, thereby limiting their exposure to healthy tissues.

[0423] To demonstrate that the RheoBrick-mediated, antigen-dependent, and small molecule-regulated cargo production technology can be translated in vivo, primary human T cells were modified with a vector encoding a PSMA CAR, RheoBrick, and an ITAM signaling-responsive expression cassette encoding a luciferase cargo Figure 18 A), where the luciferase cargo can serve as a tool to precisely measure cargo expression within solid tumors in live animals. As a control, T cells were modified with a PSMA CAR, an irrelevant protein (huEGFRt), and an ITAM signaling-responsive expression cassette encoding a luciferase cargo, representing conventional TRUCK T cells. A subcutaneous adenocarcinoma model was established by injecting 5 million PC3-PSMA cells into the flanks of immunodeficient NSG mice Figure 18 B). Two weeks after tumor injection, mice were intravenously injected with 2.5 million RheoBrick PSMA CAR-T cells or PSMA CAR-T cells without RheoBrick, both T cell populations encoding an ITAM signaling-induced luciferase cassette. Immediately after injection of the CAR-T cells, mice were treated daily by oral gavage with 1 mg / kg lenalidomide or vehicle control. On day 13 after injection of the CAR-T cells, luciferase cargo production reached a peak, and surprisingly, in vivo, when both groups of animals were treated daily with lenalidomide, the RheoBrick PSMA CAR-T cells produced 3.2-fold more cargo than the PSMA CAR-T cells without RheoBrick Figure 18C). By normalizing the luciferase signal with tumor size, the luciferase cargo production density was calculated, and surprisingly, it was found that in vivo, when both groups of animals were treated with lenalidomide daily, the cargo production density in RheoBrick PSMA CAR-T cells also increased compared to PSMA CAR-T cells without RheoBrick (the difference was 3.7-fold on day 13, Figure 18 D). Additionally, it was surprisingly found that in vivo, when both groups of animals were treated with lenalidomide daily, the cargo production of lenalidomide-treated RheoBrick PSMA CAR-T cells lasted longer than that of PSMA CAR-T cells without RheoBrick ( Figure 18 D). In the absence of lenalidomide administration, RheoBrick effectively blocked cargo production (on day 13, the cargo production in the group injected with RheoBrick PSMA CAR-T cells with lenalidomide differed by 109-fold compared to the group injected with RheoBrick PSMA CAR-T cells with vehicle). Additionally, RheoBrick PSMA CAR-T cells effectively controlled tumor growth in animals treated with lenalidomide but had no effect in the vehicle control group ( Figure 18 E), indicating that the production of luciferase cargo is related to T cell activity. Animals in the group injected with RheoBrick PSMA CAR-T cells and treated with lenalidomide had better survival compared to the vehicle control group, further confirming this finding ( Figure 18 F). Figure 18 G schematically illustrates the main findings of the animal study, which demonstrated that the RheoBrick-mediated, antigen-dependent, and small molecule-regulated cargo expression platform played a robust role in the animal model.

[0424] To our knowledge, there are no other examples of antigen- and small molecule-dependent cargo production platforms in the literature. We believe that the lower stringency of CAR-T cell switch systems in the literature has hindered the development of antigen- and small molecule-dependent cargo expression platforms. For example, in other switch platforms, such as the CAR-degron fusion concept, incomplete degradation of the CAR can lead to substantial residual T cell activation (~40% of the maximum IFNγ production remaining in the absence of drug, see reference Weber et al., Science. 2021 Apr 2; 372(6537): eaba1786) Figure 1B and 1D). This low stringency of the competitive switch platform has hindered the creation of a stringent antigen- and small molecule-dependent cargo expression platform, which would not have been obvious to a person skilled in the art. The unique T cell inhibitory mechanism of the CRASH-IT / RheoBrick switch (competing with endogenous Zap70 and simultaneously recruiting the inhibitory phosphatases SHP-1 / 2 near the TCR signaling complex) provides near-complete inhibition of T cell signaling (remaining ~1% of the maximum IFNg production in the absence of drug, Figure 19 ), and when the RheoBrick is combined with an ITAM signaling-induced promoter and a POI, it is capable of creating a stringent antigen- and small molecule-dependent cargo expression platform. Since the different embodiments of the RheoBrick switch described herein (e.g., in the dependent claims) share the same mechanism of action and can modulate the activity of the ITAM signaling-responsive promoter, any RheoBrick / ITAM signaling-induced promoter-cargo combination disclosed herein will be regarded by a person skilled in the art as a creative contribution to the prior art. The shared mechanism of action is independent of the cargo (i.e., not dependent on the nucleic acid encoding the protein of interest controlled by the inducible promoter, and wherein the inducible promoter is induced when the receptor receives an activation signal in the absence of a chimeric protein comprising the docking domain and the drug-related stability domain described herein, e.g., as defined in claim 1 and the dependent claims); preferably, the nucleic acid encoding the protein of interest (also referred to as the cargo), the inducible promoter operably linked to the nucleic acid encoding the protein of interest, or both are independently introduced into the cell according to the invention, e.g., on a vector present in the cell. In another embodiment, the nucleic acid encoding the protein of interest (which can also be referred to as the 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 the cargo), the inducible promoter operably linked to the nucleic acid encoding the protein of interest, or both are independently introduced or present in the original cell, either on a vector, incorporated into the genome (e.g., a chromosome), or both. Furthermore, it was not obvious whether RheoBrick / ITAM signaling-induced promoter-cargo / CAR-T cells would result in higher cargo expression levels in vivo compared to ITAM signaling-induced promoter-cargo / CAR-T cells without the RheoBrick switch. We surprisingly found that in vivo, PSMA CAR-T cells containing the RheoBrick switch and NFAT-luciferase cargo resulted in higher peak luciferase production and increased luciferase production persistence compared to PSMA CAR-T cells containing NFAT-luciferase cargo without the RheoBrick switch (Figure 18 )。

[0425] 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 ovarian cancer patients were modified with a lentiviral vector containing RheoBrick and an ITAM-signaling-responsive IL-12 expression cassette ( Figure 20 A). After co-culturing the modified TILs with ovarian tumor cells derived from the same patient, the production of the IL-12 cargo was strongly induced in the presence of lenalidomide compared to the vehicle control (induction rate of 10.7×, Figure 20 B). Co-culturing the modified TILs with the antigen-negative unrelated tumor cell line PC3 resulted in only minimal production of the IL-12 cargo in the presence of lenalidomide (a 3.9× reduction in cargo production in the PC3 co-culture compared to the co-culture with ovarian tumor cells).

[0426] The flexibility of the AND-gate technology (i.e., the inventions of cells, systems, methods, and features according to the present invention) allows for placing many therapeutically relevant biologic modalities downstream of the inducible promoters (such as the NFAT promoter) described herein, said biologic modalities including cytokines, interleukins, interferons, chemokines, immunocytokines, receptors, ligands, antibodies or antibody fragments, bispecific antibodies, T cell engagers, bispecific T cell engagers, checkpoint inhibitors, antagonists, agonists, enzymes, regulatory elements, transcription factors or DNA-binding domains of transcription factors, and combinations of two or more biologic cargo molecules, and for example, producing these molecules at a target site in accordance with the presence of a ligand for an activating receptor and, for example, being controlled by the amount of the provided small molecule to be produced at a desired level.

[0427] Using the present invention, the production of cargo molecules can be restricted to the cancer site by inputting antigens (including antigens that exhibit heterogeneous expression in tumor tissues) under conditions that are largely or completely restricted to the tumor microenvironment and / or tumor cells.

[0428] Examples of antigens restricted to the tumor microenvironment are 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 also highly specific for tumor cells and can tolerate high cell doses (1011 ((a certain number of cells), without dose-limiting toxicity (Nagarsheth et al., Nat Med. 2021 Mar; 27(3):419 - 425)). In addition, cancer-testis antigen NY-ESO-1-specific TCR-T cells have been tested in clinical trials, and no toxicity caused by the transferred T cells has been detected (Robbins et al., Clin Cancer Res. 2015 Mar 1; 21(5):1019 - 27). Other notable CAR-T cell therapy target antigens with high tumor-specific expression are 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).

[0429] The controlled cargo release method according to the present invention can also be used to treat non-oncological clinical indications. For example, in autoimmune diseases, those skilled in the art should understand that the local production of immunomodulatory compounds (such as IL-10 (P22301) or TGFB (P36897) or anti-inflammatory antibodies) controlled at the disease site and regulated by small molecules can be used to reversibly inhibit the local immune response.

[0430] Materials and Methods

[0431] Vector DNA preparation

[0432] Self-inactivating lentiviral vectors were created using gene synthesis. Briefly,[ Figure 2 ​The expression cassettes shown in A, 4A, 5A, 6A, 7A, and 8B are flanked by upstream lentiviral vector elements (RSV promoter, 5’LTR (truncated), HIV-1 (psi), RRE, cPPT / CTS) and downstream lentiviral vector elements (3’LTRΔU3, SV40 polyA, SV40 ori) derived from the pRRLSIN vector (#12252, Addgene), arranged as gene synthesis products and cloned into the EcoRV site of the pUC-AMP-GW vector backbone (Genewiz / Azenta). The nucleotide sequences of the complete lentiviral vectors described in the examples are summarized in Table 10, and the nucleotide sequences of the open reading frames, regulatory elements, and structural elements in these vectors are summarized in Table 11. The retroviral vector encoding MP71PSMA iresPuro was created by cloning the gene-synthesized PSMA (Uniprot ref: Q04609) coding sequence, IRES, and puromycin resistance gene into the MP71 vector (Engels et al., Hum Gene Ther 2003;14(12):1155-68).

[0433] Table 10. Summary of the nucleotide sequences of the complete lentiviral vectors described in the examples.

[0434]

[0435]

[0436] Rheobrick(2mut): Two amino acid substitutions in the heterozygous zinc finger domain as disclosed herein; Rheobrick(4mut): Four amino acid substitutions in the heterozygous zinc finger domain as disclosed herein.

[0437] Table 11. Summary of the open reading frames, regulatory elements, and structural elements according to Table 10.

[0438]

[0439]

[0440]

[0441] Cell lines and cell culture

[0442] 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), and Nalm6 cells (ATCC) were cultured in RPMI / 8% FCS / penicillin-streptomycin (Gibco). HCT116 cells, HEK293T cells, and FLYRD18 cells were passaged every 3 days with trypsin-EDTA (Gibco). All cell lines were tested for mycoplasma by PCR-based screening and the results were negative.

[0443] Production of lentivirus

[0444] Lentiviral particles were produced in HEK293T packaging cells. Briefly, 4,500,000 HEK293T packaging cells were seeded in each 10 cm culture dish one day before transfection. The next day, the cell medium was refreshed with DMEM supplemented with 8% FCS and without antibiotics. 25 μl of X-tremeGENE 9 (Roche) was mixed with 800 μl of Opti-MEM (Gibco) and incubated for 5 minutes. Subsequently, the Optimem-X-tremeGENE 9 mixture was added to 10 μg of lentiviral plasmid mixture DNA dissolved in water (this plasmid mixture contained 3.5 μg of transfer vectors as shown in Figure 2 A, 4A, 5A, 6A, 7A, and 8B, and packaging plasmids (Zhang et al., Nat Protoc. 2010 Mar; 5(3): 439–456): 3.5 μg of pCMVΔR8.74 and 3 μg of pMD2.G), incubated for 15 minutes, and the resulting transfection mixture was dropped onto the packaging cells. The supernatant containing lentivirus was harvested 48 hours after transfection, filtered through a syringe, and used immediately.

[0445] Production of retrovirus

[0446] Retroviral particles are produced in FLYRD18 packaging cells. One day before transfection, 700,000 FLYRD18 packaging cells are seeded in each 10-cm dish. The next day, the cell culture medium is refreshed with IMDM supplemented with 8% FCS and without antibiotics. 25 μl of X-tremeGENE 9 is mixed with 800 μl of Opti-MEM and incubated for 5 minutes. Subsequently, the Optimem-X-tremeGENE 9 mixture is added to 10 μg of retroviral plasmid DNA dissolved in water, incubated for 15 minutes, and then the resulting transfection mixture is added dropwise to the packaging cells. The supernatant containing retrovirus is harvested 48 hours after transfection and used immediately.

[0447] T cell isolation and activation

[0448] Peripheral blood mononuclear cells (PBMCs) are isolated from the buffy coat of healthy donors (Sanquin, Amsterdam, NL) by Ficoll-Isopaque density centrifugation (Hokland et al., J Immunol Methods. 1980;32(1):31-39) and cryopreserved until further use. To generate an activated T cell population, the PBMCs are thawed in PBS containing 5% FCS, counted, and then mixed with CD3 / CD28 Dynabeads (CTS) at a cell-to-bead ratio of 1:1 at a density of 10 7 cells / ml. After incubating the mixture on a tumbler at room temperature for 30 minutes, it is placed on a magnet and the unbound cells are removed. Subsequently, the bead-bound T cells are resuspended in RPMI / 10% human serum / penicillin-streptomycin containing 5 ng / ml of IL-7 (Peprotech) and 5 ng / ml of IL-15 (Peprotech) and seeded at a density of 0.75x10 6 cells / ml.

[0449] Spin-transduction of T cells

[0450] An untreated 6-well cell culture plate is coated with 10 μg / ml of retronectin (Takara) at 4 °C overnight. The next day, the retronectin solution is removed and the wells are blocked with 2% BSA (Sigma-Aldrich) dissolved in PBS for 30 minutes. Then 2 million activated T cells (1x10 in RPMI / 10% human serum / penicillin-streptomycin / 12.5 ng / ml of IL-7 and 12.5 ng / ml of IL-15) 6cells / ml) were mixed with 3 ml of viral supernatant in a 6-well plate coated with retronectin. For lentiviral co-transduction experiments, 1.5 ml of lentiviral supernatant from 2 vectors (e.g., CC41 and CC1) was used. At room temperature, with the brake off, the culture plates were centrifuged at 2,000 RPM for 90 minutes. No selection method was used to enrich the lentiviral vector-modified T cells listed in Table 2. HCT116 and PC3 tumor cells transduced with the retroviral MP71 PSMA iresPuro vector were selected with 0.5 μg / ml and 2 μg / ml puromycin for 3 days, respectively.

[0451] co-culture

[0452] On day 6 post-transduction, in a round-bottom 96-well plate, in the presence of the indicated concentration of lenalidomide or DMSO control, 100,000 T cells were mixed with 100,000 of the indicated target cells (or T cells alone as the no-target cell control) in T cell medium and incubated at 37 °C for 24 hours. For PSMA BiTE-induced cytokine production and degranulation assays, the conditioned medium from the first co-culture was harvested (see details in Figure 8 ), filtered and frozen at -80 °C until use. A vial of fresh PBMCs was opened and cultured overnight in standard T cell medium containing 5 ng / ml of IL-7 and 5 ng / ml of IL-15 for cytokine release and degranulation assays. 100 μl of the conditioned medium was mixed with 100,000 PBMCs, 100,000 HCT116 WT cells or HCT116 PSMA cells in T cell medium supplemented with golgi-plug (1:1000 dilution, BD, #51-2301KZ) and anti-LAMP1-APC (1:100 dilution, Biolegend, #328620) to prepare 200 μl of co-culture in a round-bottom 96-well plate. The co-culture was incubated at 37 °C for 5 hours.

[0453] For blinatumomab BiTE-induced cytotoxicity assays, the conditioned medium from the first co-culture was harvested (see details in Figure 17 ), filtered and frozen at -80 °C until use. A vial of fresh PBMCs was opened and depleted of CD14+ monocytes and CD19+ B cells by Dynabeads TM Untouched TMThe human T cell kit (Thermofisher) enriches T cells and cultures them overnight in standard T cell medium containing 5 ng / ml of IL-7 and 5 ng / ml of IL-15. For the second co-culture, 100,000 naive T cells are mixed with 25,000 CTFR-labeled Nalm6 cells (CD19+) and 25,000 CTV-labeled K562 cells (CD19-) in the presence of serial diluted conditioned medium from the first co-culture. After 24 hours, the cells are stained with a live / dead marker near-infrared dye (ThermoFisher), fixed, and analyzed by FACS to determine the Nalm6 / K562 cell ratio. The specific killing rate is calculated according to the formula of Noto et al., J Vis Exp. 2013;(82):51105): 100 - (the average of three wells containing target cells and effector cells without conditioned medium) x 100 of (antigen+ / antigen-) in the presence of effector cells and conditioned medium). Antigen-specific cell killing induced by the T cell supernatant producing blinatumomab is normalized by the T cell supernatant producing an unrelated cargo (luciferase). For the positive control, target cells are co-cultured with naive T cells in the presence of purified blinatumomab (#100441-2, BPS Bioscience)...

Claims

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

2. The cell according to any one of the preceding claims, wherein the inducible promoter is selected from the group consisting of: NFAT promoter, NF-kB 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 or IL-17 promoter.

3. The cell according to any one of the preceding claims, wherein the nucleic acid encoding the protein of interest is operably linked to a nucleic acid encoding an RNA degradation element (RDE), preferably wherein the RDE is an AU-rich element (ARE), preferably wherein the ARE is or is 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.

4. The cell according to any one of the preceding claims, wherein the cell is selected from the group consisting of: T cells, T cells expressing a TCR, T cells expressing a modified TCR, CAR T cells, NK cells, CAR NK cells, tumor-infiltrating lymphocytes, macrophages and CAR macrophages.

5. The cell according to any one of the preceding claims, wherein the receptor is selected from the group consisting of: T cell receptor (TCR), chimeric antigen receptor (CAR) and NK cell receptor (NKR), and / or wherein the receptor is antigen-specific, such as for a tumor antigen.

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

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

8. The cell according to any one of the preceding claims, wherein the docking domain further comprises an immunoreceptor tyrosine-based switch motif (ITSM) and / or an immunoreceptor tyrosine-based inhibitory motif (ITIM), or ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), preferably wherein the ITIM and / or ITSM are from an inhibitory receptor protein, preferably an inhibitory immunoreceptor protein, preferably from a protein selected from the group consisting of PD1, BTLA, SIRPα, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1, and LY9.

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

10. The cell according to any one of the preceding claims, wherein the drug-regulated protein stability domain comprises a Cys2-His2 zinc finger domain capable of drug-induced binding to the CRBN polypeptide, preferably wherein the Cys2-His2 zinc finger domain is a heterologous zinc finger domain.

11. The cell according to any one of the preceding claims, wherein the Cys2-His2 zinc finger domain is a heterologous 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, preferably wherein the heterologous zinc finger domain comprises one, two, three, four, or more amino acid substitutions relative to 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.

12. The cell according to any one of the preceding claims, wherein the drug-regulated protein stability domain comprises additional, e.g., a second Cys2-His2 zinc finger domain, preferably comprises additional, e.g., a second heterologous zinc finger domain.

13. The cell according to any one of the preceding claims, wherein the drug is an immunomodulatory imide drug (IMiD), preferably wherein the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, ibalidomide, CC-885, their salts, and analogs.

14. The cell according to any one of the preceding claims, wherein the cell further comprises an immunomodulatory imide drug (IMiD), preferably wherein the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, ibalidomide, CC-885, their salts, and analogs.

15. The cell according to any one of the preceding claims, wherein 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.

16. The cell according to any one of the preceding claims, 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 or IL-21, and / or wherein the chemokine is CCL5, CCL19 or CCL21, and / or wherein the protein is a cytokine receptor, and / or wherein the cytokine receptor is TGFBR (TGF-β receptor) and / or TGFBR2.

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

18. The cell according to claim 17, wherein the chimeric protein is expressed and / or wherein the protein of interest is expressed.

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

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

21. The method according to any one of the preceding claims, wherein the method comprises administering to the subject the cell according to any one of the preceding claims.

22. The method according to any one of the preceding claims, wherein the method is for treating cancer in a subject and / or for treating a tumor in a subject.

23. The cell according to any one of the preceding claims, which is used as a medicament, preferably for treating cancer in a subject and / or for treating a tumor in a subject, preferably wherein the treatment comprises administering to the subject the cell according to any one of the preceding claims, and optionally administering a drug capable of regulating a drug-regulated protein stability domain.

24. A cell according to any one of the preceding claims, for use as an agent according to any one of the preceding claims, wherein administering a drug capable of modulating a drug-modulated protein stability domain comprises altering the dose of the drug capable of modulating the drug-modulated protein stability domain.

25. A drug for use as an agent, preferably for treating cancer and / or for treating a tumor in a subject, wherein the treatment comprises administering to the subject a cell according to any one of the preceding claims and administering the drug, wherein the drug is capable of modulating a drug-modulated protein stability domain.

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

Images