Zinc finger degron sequence

Abstract

The present invention generally relates to novel zinc finger degron sequences, which comprise at least one non-natural zinc finger domain in the form of a hybrid composed of a first portion from a first zinc finger and a second portion from a second zinc finger, preferably the first portion comprises the beta hairpin of the first zinc finger and the second portion comprises the alpha helix of the second zinc finger. In a preferred embodiment, the degron tag further comprises a second non-natural hybrid zinc finger domain.

Description

[Technical field]

[0001] The present invention generally relates to novel zinc finger degron sequences, which comprise at least one non-natural zinc finger domain in the form of a hybrid composed of a first portion from a first zinc finger and a second portion from a second zinc finger, preferably the first portion comprises the beta hairpin of the first zinc finger and the second portion comprises the alpha helix of the second zinc finger. In a preferred embodiment, the degron tag further comprises a second non-natural hybrid zinc finger domain. In a preferred embodiment, the zinc finger domain comprises at least two amino acid substitutions. Also provided are fusion proteins comprising the degron tag according to the invention, and the use of the degron tag according to the invention in methods involving controlling the levels, cellular activity, expression, etc. of a protein of interest. [Background technology]

[0002] The Background Description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the claimed invention, nor that any publications referenced specifically or implicitly are prior art.

[0003] Cellular behavior is controlled to a great extent by the expression levels of various proteins, including key regulatory proteins. To achieve such control over cellular behavior, synthetic or artificial protein stability control tools have been developed that allow reversible and titratable expression of proteins of interest (POIs), e.g., on a time scale of minutes, by exogenously provided factors, e.g., (small) compounds.

[0004] In particular, strategies have been developed to control POI proteolysis rates (i.e., the rate of protein degradation, e.g., the rate of cellular protein degradation) in response to exogenously provided factors, such as membrane-permeable small molecules. Well-known examples include the SMASh / Asunaprevir (Chung et al. Nat Chem Biol. 2015 Sep; 11(9): 713-20), FKBP12F36V / dTAG-13 (Nabet et al. Nat Chem Biol. 2018 May; 14(5): 431-441), zinc finger degron / IMiD (Sievers et al. Science. 2018 Nov 2; 362(6414): eaat0572), and DD / Shield1 (Haugwitz et al. Biotechniques. 2008 Mar; 44(3): 432-3) platforms.

[0005] These platforms and the principles on which they are based are well known to those skilled in the art and have been widely described in both the scientific and patent literature (e.g., for zinc finger degrons / IMiDs see WO2019007869, WO2020132039, WO2021188286, or WO2021080427).

[0006] Most of these platforms and the methods used therein involve the fusion of a POI with a small molecule-dependent degron domain (i.e., a domain or portion of a protein that is important in regulating the rate of protein degradation), and preferential interaction of the degron (domain) with the proteasome complex in the presence of the small molecule regulator leads to subsequent degradation of the POI-degron fusion protein.

[0007] Alternatively, the POI can be fused to a degron domain that is intrinsically unstable and therefore prone to proteolysis, and stabilization of the folded protein state by small molecule addition is used to prevent such proteolysis.

[0008] The POI can also be fused to a regulatory domain, including a constitutively active degron domain, a protease, and a corresponding protease cleavage site in the linker between the POI and the degron tag. In the absence of a protease inhibitor, the degron domain is proteolytically removed from the POI, thereby, for example, stabilizing POI expression. In contrast, in the presence of a protease inhibitor, degradation of the fusion protein is induced.

[0009] Despite the great interest in developing protein stability control systems that can be used for cell therapy applications, several challenges remain. For example, preferred protein stability regulators used in clinical cell therapy products should be composed of human sequences to minimize the risk of immune-mediated rejection, and the immunogenicity of, for example, the SMASh-tag degron system based on the Hepatitis C virus (HCV)-derived NS3 / 4A protease (Tan et al., PLoS One. 2017;12(7):e0181578) is likely to limit its clinical utility.

[0010] In addition, small molecule regulators of protein stability control domains should be safe and preferably clinically approved. The lack of clinical approval of dTAG-13 PROTACs used in the FKBP12F36V tag degron system complicates the clinical development of FKBP12F36V-based regulatory systems. In addition, the large size of PROTAC molecules is considered a challenge for administration and metabolism of this drug class (Hu et al. Chembiochem. 2021 Sep 8. doi:10.1002 / cbic.202100270. Online ahead of print).

[0011] Moreover, importantly, small molecule concentrations sufficient to induce biologically meaningful control over protein levels, e.g., meaningful control over protein degradation (e.g., degradation of a POI fused to a degron), should preferably be compatible with clinical use, especially in the context of long-term small molecule administration, where side effects of such administration may be a concern.

[0012] The immunomodulatory imide drug (IMiD)-inducible zinc finger degron system described by Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572, for example, utilizes the fusion of a POI with a short zinc finger degron tag (sometimes referred to as a zinc finger domain and / or zinc finger polypeptide) that promotes recruitment of the POI-zinc finger fusion degron protein to the IMiD / CRBN E3 ligase complex in the presence of a small molecule known as an IMiD. In general, IMiDs bind to cereblon (CRBN), a substrate receptor for the CRL4CRBN E3 ubiquitin ligase, and CRBN can recruit (fuse) proteins containing zinc finger degron tags through an interaction with the zinc finger degron, which is believed to be mediated by IMiDs such as thalidomide and its derivatives. This zinc finger degron system is based on human protein sequences, thus 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 clinical development of these systems.

[0013] In preclinical studies, this protein stability control system has demonstrated its value in clinically relevant applications such as modulating CAR-T cell activity when such zinc finger degrons are fused to CARs (Jan et al. Sci Transl Med. 2021 Jan 6;13(575):eabb6295). Furthermore, the inventors recently reported the use of the IMiD / zinc finger system in the Chemically Regulated SH2 Delivery Inhibitory Tail (CRASH-IT) switch platform, which allows control of the cellular activity levels of various cell therapy platforms such as CAR-T, TCR-T, and NK cells (Sahillioglu et al., WO2021080427).

[0014] The fact that zinc finger degrons are composed of human sequences and can be regulated using approved molecules facilitates the clinical application of this system, however, clinical use of these IMiD molecules in the treatment of patients with hematological cancers is associated with significant side effects at therapeutically effective concentrations. As an example, in a study evaluating high-dose (25 mg / day) and low-dose (5 mg / day) lenalidomide maintenance treatment in multiple myeloma patients, lenalidomide dose was shown to correlate with both toxicity and efficacy, and dose reductions due to toxicity, particularly neutropenia, were frequent in the high-dose lenalidomide cohort. (Fenk et al. Clin Cancer Res. 2020 Nov 15;26(22):5879-5886). / pct

[0015] Due to concerns associated with the use of IMiDs at higher doses, the identification of novel zinc finger degron sequences that can be regulated at lower drug doses is attractive.

[0016] In view of this, novel products, compositions, methods and uses for controlling the (expression) level of a protein of interest using improved novel zinc finger degron sequences are highly desirable, but are not yet readily available. In particular, there is a clear need in the art for reliable, efficient and reproducible products, compositions, methods and uses that allow for regulating the (expression) level of a protein of interest using novel zinc finger degron sequences (zinc finger degron tags, domains, polypeptides) in a manner compatible with clinical use, for example in the treatment or prevention of disease, and allow for the use of low concentrations of small molecules (lower drug doses), in particular IMiDs, that regulate protein degradation / stability by interacting with the degron sequence fused to the protein of interest. Thus, the technical problem underlying the present invention can be seen in the provision of such products, compositions, methods and uses to meet any of the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and in the following specification. Summary of the Invention

[0017] The scope of the invention is defined by the claims. Subject matter not encompassed by the claims does not form a part of the claimed invention. [Brief description of the drawings]

[0018] Embodiments of the invention are further described below with reference to the accompanying drawings.

[0019] [Figure 1-1]The sensitivity of zinc finger degrons to IMiDs can be improved by modification of the second zinc finger sequence. (A) Schematic of a panel of zinc finger degrons containing ZFP91 ZF4 beta hairpin and IKZF1 ZF2 alpha helix as the hybrid first zinc finger. In this panel, the degron contains a second zinc finger sequence selected from IKZF1 ZF3 (single hybrid, dual zinc finger degron), IKZF1 ZF3 beta hairpin-ZFP91 ZF5 alpha helix (double hybrid degron), or the degron does not contain a second zinc finger (single hybrid, single zinc finger degron). (B-E) Primary human T cells engineered with HLA class I restricted CDK4 TCR+Zap70-PD1-degron CRASH-IT switch, in which the degron sequence is selected from (A), were pretreated with the indicated concentrations of thalidomide. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP high, CD8+ T cells upon co-culture with NKIRTIL006 melanoma cells in the continued presence of the indicated concentrations of thalidomide. Error bars represent standard deviation (n=2). Data are representative of two independent experiments. [Figure 1-2]The sensitivity of zinc finger degrons to IMiDs can be improved by modification of the second zinc finger sequence. (A) Schematic of a panel of zinc finger degrons containing ZFP91 ZF4 beta hairpin and IKZF1 ZF2 alpha helix as the hybrid first zinc finger. In this panel, the degron contains a second zinc finger sequence selected from IKZF1 ZF3 (single hybrid, dual zinc finger degron), IKZF1 ZF3 beta hairpin-ZFP91 ZF5 alpha helix (double hybrid degron), or the degron does not contain a second zinc finger (single hybrid, single zinc finger degron). (B-E) Primary human T cells engineered with HLA class I restricted CDK4 TCR+Zap70-PD1-degron CRASH-IT switch, in which the degron sequence is selected from (A), were pretreated with the indicated concentrations of thalidomide. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP high, CD8+ T cells upon co-culture with NKIRTIL006 melanoma cells in the continued presence of the indicated concentrations of thalidomide. Error bars represent standard deviation (n=2). Data are representative of two independent experiments.

[0020] [Figure 2-1] FIG. 1 is a schematic diagram of a non-limiting embodiment according to the present invention. [Figure 2-2] FIG. 1 is a schematic diagram of a non-limiting embodiment according to the present invention.

[0021] [Diagram 3]Design of the Synthetic Zinc Finger (SynFinger) library. The SynFinger library contains Zap70-PD1-degron CRASH-IT switch variants, where the parent zinc finger degron contains single or double amino acid substitutions or no substitutions. The figure shows the sequence of the double hybrid degron used as the parent zinc finger sequence in the SynFinger library screen. The amino acids indicated by the arrows are substituted with any other genetically encoded amino acid except cysteine. The two cysteines and the conserved glycine in the ZFP91 ZF4 beta hairpin, as well as the two histidines in the IKZF1 ZF2 alpha helix, were kept constant. The substitution mutations are numbered starting from the first amino acid of the ZFP91 ZF4 beta hairpin (marked with an asterisk).

[0022] [Figure 4] Enrichment of specific amino acid substitutions in the top 100 degrons. SynFinger degrons were ranked according to enrichment index (EI) value after removing SynFingers with low sequence reads (see Examples). The graph shows the number of times each amino acid substitution was found in combination with other amino acid substitutions in the top 100 SynFinger degrons. For example, Q12R mutation was observed in 11 double amino acid substitution combinations with either K13T, L17M, K13V, E4L, L17Y, L22H, C10A, I20R, N15S, E4W and E4Q in the top 100 SynFingers.

[0023] [Figure 5-1]SynFingers fusion proteins allow sensitive control of immune cell function. A subset of SynFingers identified in the SynFinger library screen was individually validated. (A-H) Primary human T cells were engineered with HLA class I-restricted CDK4 TCR+Zap70-Siglec11-degron CRASH-IT switch, where the degron sequence includes the parent zinc finger degron (double hybrid degron shown in Figures 1 and 3) or the parent zinc finger degron with the following mutations: G14N / K21A, G14M / N15R, L17I / K21L, E4R / Q12L, Q12R / K13T, or Q12R / K13V. As a control, primary human T cells engineered with HLA class I-restricted CDK4 TCR+vector control were used. Cells were pretreated with 5 nM lenalidomide or left untreated. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP intermediate, CD8+ T cells when co-cultured with NKIRTIL006 melanoma cells in the continued absence (E-H) or presence (A-D) of 5 nM lenalidomide. Dashed horizontal lines indicate cytokine production and degranulation levels of cells engineered with the CRASH-IT switch containing the parental zinc finger degron. Error bars represent standard deviation (n=2). SynFinger and vector control groups were compared to the parental zinc finger group using a one-tailed t-test: *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, ns=not significant. Data are representative of two independent experiments. [Figure 5-2]SynFingers fusion proteins allow sensitive control of immune cell function. A subset of SynFingers identified in the SynFinger library screen was individually validated. (A-H) Primary human T cells were engineered with HLA class I-restricted CDK4 TCR+Zap70-Siglec11-degron CRASH-IT switch, where the degron sequence includes the parent zinc finger degron (double hybrid degron shown in Figures 1 and 3) or the parent zinc finger degron with the following mutations: G14N / K21A, G14M / N15R, L17I / K21L, E4R / Q12L, Q12R / K13T, or Q12R / K13V. As a control, primary human T cells engineered with HLA class I-restricted CDK4 TCR+vector control were used. Cells were pretreated with 5 nM lenalidomide or left untreated. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP intermediate, CD8+ T cells when co-cultured with NKIRTIL006 melanoma cells in the continued absence (E-H) or presence (A-D) of 5 nM lenalidomide. Dashed horizontal lines indicate cytokine production and degranulation levels of cells engineered with the CRASH-IT switch containing the parental zinc finger degron. Error bars represent standard deviation (n=2). SynFinger and vector control groups were compared to the parental zinc finger group using a one-tailed t-test: *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, ns=not significant. Data are representative of two independent experiments.

[0024] [Figure 6-1]SynFingers containing a CRASH-IT switch allow restoration of cytokine production and degranulation at lower IMiD concentrations compared to non-inventive zinc finger degrons. (A-D) Primary human T cells engineered with an HLA class I-restricted CDK4 TCR+Zap70-Siglec11-degron CRASH-IT switch, where the degron sequence comprises a prior art zinc finger degron (single hybrid, dual zinc finger degron shown in FIG. 1), a parent zinc finger degron (double hybrid degron shown in FIG. 1 and FIG. 3), or a parent zinc finger degron with the following mutations: G14N / K21A, L17I / K21L, or Q12R / K13V, were pretreated with the indicated concentrations of lenalidomide or left untreated. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP intermediate, CD8+ T cells upon co-culture with NKIRTIL006 melanoma cells in the continued presence or absence of the indicated concentrations of lenalidomide (A-D). Error bars represent standard deviation (n=3). Data are representative of two independent experiments. (E) Table shows EC50 lenalidomide (nM) values ​​for cells in (A-D) and EC50 fold change between the prior art zinc finger degron and SynFinger containing Q12R / K13V substitutions. [Figure 6-2]SynFingers containing a CRASH-IT switch allow restoration of cytokine production and degranulation at lower IMiD concentrations compared to non-inventive zinc finger degrons. (A-D) Primary human T cells engineered with an HLA class I-restricted CDK4 TCR+Zap70-Siglec11-degron CRASH-IT switch, where the degron sequence comprises a prior art zinc finger degron (single hybrid, dual zinc finger degron shown in FIG. 1), a parent zinc finger degron (double hybrid degron shown in FIG. 1 and FIG. 3), or a parent zinc finger degron with the following mutations: G14N / K21A, L17I / K21L, or Q12R / K13V, were pretreated with the indicated concentrations of lenalidomide or left untreated. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP intermediate, CD8+ T cells upon co-culture with NKIRTIL006 melanoma cells in the continued presence or absence of the indicated concentrations of lenalidomide (A-D). Error bars represent standard deviation (n=3). Data are representative of two independent experiments. (E) Table shows EC50 lenalidomide (nM) values ​​for cells in (A-D) and EC50 fold change between the prior art zinc finger degron and SynFinger containing Q12R / K13V substitutions.

[0025] [Figure 7]Combining double substitution sets can further increase the lenalidomide sensitivity of SynFinger degrons. (A-D) Primary human T cells engineered with an HLA class I-restricted CDK4 TCR+Zap70-Siglec11-degron CRASH-IT switch, in which the degron sequence comprises the parent zinc finger degron (double hybrid degron shown in Figures 1 and 3) or the parent zinc finger degron with the following mutations: G14N / K21A, Q12R / K13V, or Q12R / K13V / G14N / K21A, were pretreated with the indicated concentrations of lenalidomide or left untreated. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP intermediate, CD8+ T cells upon co-culture with NKIRTIL006 melanoma cells in the continued presence or absence of the indicated concentrations of lenalidomide (A-D). Error bars represent standard deviation (n=3). Data are representative of two independent experiments.

[0026] explanation definition A portion of this disclosure contains material that is subject to copyright protection, such as, but not limited to, drawings, device photographs, or any other aspects of this application in which copyright protection is or may be available in any jurisdiction. The copyright owner has no objection to the facsimile reproduction of either the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0027] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms should be given their ordinary meaning in the art to which the invention belongs unless otherwise indicated. Other specifically defined terms should be interpreted consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the testing of the present invention, the preferred materials and methods are described herein. For purposes of the present invention, the following terms are defined below.

[0028] As used herein, the singular terms "a," "an," and "the" include plural references unless the context dictates otherwise. Thus, for example, reference to a "degron tag" includes a combination of two or more degron tags, and the like. For example, a method for administering a drug or fusion protein can include administration of multiple molecules (e.g., tens, hundreds, thousands, tens of thousands, hundreds of thousands, millions, or more molecules).

[0029] As used herein, "about" and "approximately," when referring to measurable values, e.g., amounts, durations, etc., are meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, where such variations are appropriate to practice the disclosed invention.

[0030] As used herein, "and / or" refers to a situation in which one or more of the stated cases may occur alone or in combination with at least one of the stated cases, up to and including all of the stated cases.

[0031] As used herein, "at least" a particular value means greater than or equal to that particular value. For example, "at least 2" is understood to be the same as "greater than or equal to 2", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc. As used herein, the term "at most / up to" a particular value means less than or equal to that particular value. For example, "at most / up to 5" is understood to be the same as "less than or equal to 5", i.e., 5, 4, 3, ...-10, -11, etc.

[0032] As used herein, "comprising" or "to comprise" is intended to be inclusive and open ended, not exclusive. Specifically, this term and variations thereof mean that the specified features, steps or components are included. These terms should not be interpreted to exclude the presence of other features, steps or components. It also encompasses the more restrictive "consisting of."

[0033] As used herein, "prior art" or "methods known to those skilled in the art" refers to situations in which it would be clear to one of ordinary skill in the art how to carry out the prior art used in the methods of the present invention. The practice of prior art techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical therapy, pharmacology, immunology and related fields is well known to those of skill in the art and is discussed in various handbooks and reference works.

[0034] As used herein, "exemplary" or "for example" means "serving as an example, instance, or illustration," and should not be interpreted as excluding other configurations, including those disclosed herein.

[0035] In the context of the present invention, in case of discrepancies between the sequences disclosed herein and those in the sequence listing, the sequences shown herein take precedence, unless it is clear that something else is intended. When referring to proteins or parts thereof, in a preferred embodiment these relate to human proteins or parts thereof.

[0036] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and should not be construed as a limitation on the scope of the invention. The description of a range should be considered to specifically disclose each and every numerical value within that range, including both integers and non-integer numbers, as well as all possible subranges. For example, the description of a range such as 1-6 should be considered to specifically disclose each and every number within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range.

[0037] As used herein, the term "fusion protein" refers to any polypeptide not normally found in nature, in particular a polypeptide in which one or more portions of the amino acid sequence are not naturally associated with each other. For example, a fusion protein may comprise an N-terminal portion consisting of a first sequence of amino acids and a C-terminal portion consisting of a second sequence of amino acids that are not naturally associated with each other and / or are not naturally associated with each other in this order. A fusion protein may, for example, result from the transcription and translation of a fusion gene of a nucleic acid. Such a fusion gene may be made by linking portions of two different genes / nucleic acid sequences. In the context of the present invention, a fusion protein may, for example, comprise a degron tag according to the present invention, or a hybrid zinc finger polypeptide according to the present invention, fused to a protein of interest.

[0038] As used herein, the term "host cell" refers to a cell into which an exogenous nucleic acid (polynucleotide) and / or an exogenous polypeptide has been introduced, including the progeny of such a cell. Host cells may include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells include in vitro host cells and in vivo host cells.

[0039] As used herein, "immunomodulatory drug", "immunomodulatory imid drug" or "IMiD" refers to compounds known in the art. IMiDs include thalidomide, pomalidomide, lenalidomide, iberdomide (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 each been approved for the treatment of various diseases, while other IMiDs or cereblon modulators are under investigation. The compounds may be in the form of a free acid or free base, or a pharmaceutically acceptable salt.

[0040] As used herein, "in vivo" refers to events that occur within a subject's body. "In vitro" refers to events that occur outside a subject's body. For example, an in vitro assay or method includes any assay or method that is performed outside a subject's body. In vitro assays or methods include cell-based assays that use live or dead cells. In vitro assays also include cell-free assays that do not use intact cells.

[0041] As used herein, when referring to a polynucleotide (nucleic acid) or polypeptide (protein), the term "isolated" refers to a protein or nucleic acid that is in a non-natural environment, e.g., separated from a naturally occurring environment. For example, an isolated protein or polypeptide according to the present invention relates to a protein that is no longer in its natural environment, e.g., in vitro or in a recombinant host cell. The term also refers to a protein or nucleic acid that is artificially or synthetically produced following isolation from a naturally occurring source. It will be clear to the skilled artisan whether a reference to a protein, polypeptide, nucleic acid, or polynucleotide in the context of the present invention includes a reference to an "isolated" protein, polypeptide, nucleic acid, or polynucleotide.

[0042] As used herein, the term "linker" when used in reference to a portion of a protein refers to a stretch of amino acids that links two portions of a protein together, for example in a fusion protein. Generally, such molecules have no specific biological activity other than binding or preserving some minimum distance or other spatial relationship between the proteins. However, in certain embodiments, the linker may be selected to affect some property of the linker and / or the protein, such as the folding, net charge, or hydrophobicity of the linker.

[0043] As used herein, the term "non-natural" refers to a polypeptide, polynucleotide, or domain contained in such a polypeptide or polynucleotide that is not known to occur in nature, e.g., not known to occur in a (human) cell, i.e., one or more portions of the polypeptide, polynucleotide, or domain are not naturally associated with each other. An example is a fusion protein as defined herein. As described herein, the term "non-natural" in particular refers to the fact that the first hybrid zinc finger domain is composed of a first portion and a second portion, the first portion being derived from a first Cys2-His2 zinc finger domain, the second portion being derived from a second Cys2-His2 zinc finger domain, and the first and second Cys2-His2 zinc finger domains being different from each other. A non-natural first hybrid zinc finger domain is thus a fusion protein composed of said first portion and said second portion. Alternatively, a non-natural first hybrid zinc finger domain may thus be referred to as a first hybrid zinc finger domain. As one of skill in the art will appreciate, the same analogy is applicable to the term "non-naturally occurring second hybrid zinc finger domain," which may therefore also be referred to as a "second hybrid zinc finger domain."

[0044] As used herein, the term "nucleic acid" or "polynucleotide" refers to any polymer or oligomer of (contiguous) nucleotides. Nucleic acids may be DNA or RNA or mixtures thereof and may exist permanently or transiently in single-stranded or double-stranded form (including homoduplexes, heteroduplexes, and hybrid states).

[0045] As used herein, the term "pharmaceutical composition" refers to a composition formulated into a pharma- ceutically acceptable or physiologically acceptable composition for administration to a cell or subject. The compositions of the present invention may also be administered in combination with other agents, so long as the additional agents do not adversely affect the ability of the composition to provide the intended treatment. Pharmaceutical compositions often contain one or more pharma- ceutically acceptable carriers (or excipients) in addition to the pharma- ceutically active agent. Pharmaceutical compositions are specifically formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, e.g., by subcutaneous, intramuscular or intravenous injection, e.g., as a sterile solution or suspension; (3) topical application, e.g., as a cream, ointment or spray applied to the skin; (4) intravaginally or rectally, e.g., as a pessary, cream or foam; or (5) as an aerosol, e.g., an aqueous aerosol, liposomal preparation or solid particles containing the compound. The drugs, therapeutic agents, medicaments and pharmaceutical compositions according to the present invention may be formulated for administration by several routes, including but not limited to parenteral, intravenous, intraarterial, intramuscular, intratumoral and oral. The drugs, therapeutic agents, medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection into a selected area of ​​the human or animal body.

[0046] As used herein, "protein" or "polypeptide" are used interchangeably and refer to a molecule consisting of a chain of amino acids, regardless of a particular mode of action, size, three-dimensional structure, or origin. Thus, a "fragment" or "portion" or "domain" of a protein may still be referred to as a "protein". A protein as defined herein and used in any method as defined herein may be an isolated protein, a naturally occurring protein, or a non-naturally occurring protein. An "isolated protein" is used to refer to a protein that is no longer in its natural environment, e.g., in vitro or in a recombinant bacterial or plant host cell. In certain embodiments of the invention, the protein is a protein involved in regulating cellular behavior, and in particular a protein that can regulate cellular behavior in the context of treating a disease. In other words, the term "portion" or "domain" refers to an amino acid sequence that is less than the complete protein sequence of any protein referred to herein. The term "domain" is also used more specifically herein to refer to functional domains known in the art, such as zinc finger domains, extracellular domains, intracellular domains, signaling domains, intracellular signaling domains, cytoplasmic domains, and transmembrane domains.

[0047] As used herein, "sequence" or "nucleotide sequence" may refer to the order of nucleotides of a nucleic acid or the order of nucleotides within a nucleic acid. In other words, any order of nucleotides in a nucleic acid may be referred to as a sequence or nucleotide sequence. The term "amino acid sequence" refers to the order of amino acids of or within a polypeptide (or protein). In other words, any order of amino acids in a polypeptide may be referred to as a sequence or amino acid sequence.

[0048] As used herein, "subject" refers, for example, to an organism to be treated, to which administration is intended. The subject may be any subject according to the present invention, including, but not limited to, a human, male, female, infant, child, adolescent, adult, young adult, middle-aged adult or elderly adult, and / or other primates or mammals. Preferably, the subject is a human patient. The subject may have been diagnosed with cancer or may be suspected of having cancer.

[0049] As used herein, "treatment," "treating," "alleviating," "relieving," and "ameliorating" in the context of a subject being treated all refer to an effort to obtain a beneficial or desired result, including but not limited to a therapeutic benefit. Therapeutic benefit refers to the eradication or amelioration of the underlying disease being treated. Therapeutic benefit is also achieved by the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient may still be affected by the underlying disease.

[0050] As used herein, the terms "construct", "nucleic acid construct", "vector", and "expression vector" may be used interchangeably and are defined as artificial nucleic acid molecules resulting from the use of recombinant DNA technology. Thus, these constructs and vectors do not include naturally occurring nucleic acid molecules, although nucleic acid constructs may include (parts of) naturally occurring nucleic acid molecules. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The invention is defined herein and particularly in the appended claims.

[0052] It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. An embodiment discussed in the context of a method, use and / or composition of the present invention can be used with respect to any other method, use or composition described herein. Thus, an embodiment related to one method, use or composition can also be applied to other methods, uses and compositions of the present invention.

[0053] Any reference in the discussion to methods of treatment also refers to the compounds, pharmaceutical compositions and medicaments of the invention for use in that method of treatment of the human (or animal) body by therapy.

[0054] The present invention provides compositions comprising degron tags and / or hybrid zinc finger polypeptides and methods for target-specifically modulating protein abundance via degron tags and / or hybrid zinc finger polypeptides.

[0055] The present invention can target endogenous and exogenous (e.g., therapeutic) proteins. As disclosed herein, degron tags and / or hybrid zinc finger polypeptides are peptides that, when fused to a target protein of interest (POI), can convert the POI into a substrate for cereblon (CRBN)-dependent ubiquitination and degradation induced by administration of an immunomodulatory drug (IMiD). Without intending to be bound by any theory, it is believed that the IMiD binds to cereblon to form a complex with binding specificity for the degron tag and / or hybrid zinc finger polypeptide. As a result, the fusion protein with the POI becomes a substrate for cereblon-dependent ubiquitination and degradation. Thus, the degron tag and / or hybrid zinc finger polypeptide of the present invention can be useful for targeted degradation of the POI.

[0056] As embodied and broadly described herein, the present invention relates to the surprising discovery that zinc finger degrons and / or zinc finger polypeptides can be provided that exhibit improved sensitivity to small molecules, particularly IMiDs such as those disclosed herein, that modulate protein degradation / stability by interacting with these degrons and / or polypeptide sequences.

[0057] Thus, the zinc finger degrons and / or zinc finger polypeptides according to the invention allow the use of low concentrations of these small molecules, in particular IMiDs, to control the proteolysis, expression and / or stability of a protein of interest (and therewith, e.g., the cellular activity (total activity in a cell) of a protein of interest that is fused to, e.g., a degron tag, zinc finger degron and / or zinc finger polypeptide according to the invention).

[0058] Thus, a zinc finger degron (degron tag) and / or zinc finger polypeptide according to the invention may provide better control of proteolysis, expression and / or stability of a protein of interest (e.g., contained in a fusion protein comprising a protein of interest and a zinc finger degron or zinc finger polypeptide according to the invention) at similar or reduced concentrations of small molecules, particularly IMiDs, which may allow for more sensitive regulation of the degradation, expression, levels and / or stability of the POI.

[0059] In other words, in one embodiment of the present invention, the zinc finger degrons (degron tags) and / or zinc finger polypeptides according to the present invention allow the control of the levels (expression) of a protein of interest expressed, e.g., in a cell or a subject, by regulating its degradation and / or stability at low concentrations of small molecules, in particular IMiDs, that regulate protein degradation / stability by interacting with these degrons and / or polypeptide sequences.

[0060] As will become apparent from the present disclosure, for example, it has surprisingly been found that improved zinc finger degrons and / or zinc finger polypeptides according to the present invention can be obtained by providing a degron tag and / or polypeptide comprising first and second zinc finger domains, each zinc finger domain generally comprising a beta hairpin derived from a first Cys2-His2 zinc finger domain (which may also be referred to in the context of the present invention as the "first" or "third" portion) and an alpha helix derived from a second Cys2-His2 zinc finger domain (which may also be referred to in the context of the present invention as the "second" or "fourth" portion).

[0061] In particular, it has been found that the first zinc finger domain is a hybrid zinc finger domain, i.e., the first zinc finger domain is composed of a first portion and a second portion that do not occur together in nature (non-natural), for example, the first zinc finger domain is a non-natural hybrid zinc finger domain obtained by combining a beta hairpin derived from a first Cys2-His2 zinc finger domain and an alpha helix derived from a second Cys2-His2 zinc finger domain, wherein the first and second Cys2-His2 zinc finger domains are neither identical nor the same.

[0062] With regard to the second zinc finger domain, it has surprisingly been found that preferably the second zinc finger domain comprised in a zinc finger degron and / or zinc finger polypeptide according to the present invention is also a hybrid zinc finger domain as described for the first hybrid zinc finger domain above. The second hybrid zinc finger domain may be the same as or different from the first hybrid zinc finger domain. Preferably, the second hybrid zinc finger domain is different from the first hybrid zinc finger domain.

[0063] At the same time, it has surprisingly been found that when the first hybrid zinc finger domain comprised in the zinc finger degron of the invention and / or the first hybrid zinc finger domain comprised in the zinc finger polypeptide according to the invention further comprises at least two amino acid substitutions as disclosed in detail herein, the sensitivity of the zinc finger degron (degron tag) of the invention and / or the zinc finger polypeptide of the invention to small molecules, in particular IMiDs, that modulate protein degradation / stability by interacting with these degrons and / or polypeptide sequences is even further improved.

[0064] In summary, it has surprisingly been found that improved zinc finger domains and degrons (e.g. degron tags) can be produced by providing a non-natural hybrid zinc finger polypeptide comprising a first hybrid zinc finger domain (as defined herein) and comprising at least two amino acid substitutions. In a preferred embodiment, in which the hybrid zinc finger polypeptide is preferably comprised in a degron tag according to the invention (as a first hybrid zinc finger domain), the degron tag according to the invention comprises two non-natural hybrid zinc finger domains (see FIG. 2 ).

[0065] In the context of the present invention, the degron tag according to the invention may also be referred to as a "double hybrid degron".

[0066] In the context of the present invention, a degron comprising a first hybrid zinc finger domain and a second, non-hybrid zinc finger domain may also be referred to as a "single-hybrid, dual zinc finger degron."

[0067] In the present invention, a degron tag that comprises a first hybrid zinc finger domain and no second zinc finger domain may also be referred to as a "single-hybrid, single-zinc finger degron."

[0068] In some embodiments, the non-natural hybrid zinc finger polypeptides according to the invention are "single-hybrid, single-zinc finger degrons" that contain two amino acid substitutions as described herein. In embodiments where the non-natural hybrid zinc finger polypeptides according to the invention further comprise a second non-hybrid zinc finger domain in addition to the first hybrid zinc finger domain, such molecules may also be referred to as single-hybrid, dual-zinc finger degrons that contain at least two amino acid substitutions as described herein. In embodiments where the non-natural hybrid zinc finger polypeptides according to the invention further comprise a second hybrid zinc finger domain in addition to the first hybrid zinc finger domain, such molecules may also be referred to as "double-hybrid degrons" that contain at least two amino acid substitutions as described herein.

[0069] According to the invention disclosed herein and according to a first aspect, there is provided a degron tag comprising a (non-natural) first hybrid zinc finger domain and a (non-natural) second hybrid zinc finger domain, (1) a first hybrid zinc finger domain comprising a first portion and a second portion; (2) the first portion is the amino acid sequence of a first Cys2-His2 zinc finger domain, X1X2C3X4X5C6X7X8X9X 10 X 11 wherein X represents any amino acid; (3) the second portion comprises the amino acid sequence X of a second Cys2-His2 zinc finger domain; 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23wherein X represents any amino acid, and the second Cys2-His2 zinc finger domain is distinct from the first Cys2-His2 zinc finger domain and (4) the second portion is C-terminal to the first portion; (5) the second hybrid zinc finger domain is C-terminal to the first hybrid zinc finger domain; (6) the second hybrid zinc finger domain comprises a third portion and a fourth portion; (7) the third portion has the amino acid sequence of a third Cys2-His2 zinc finger domain, (Z)C(Z)C(Z) 5-6 wherein Z represents any amino acid; (8) a fourth second portion having the amino acid sequence of a fourth Cys2-His2 zinc finger domain, (Z)H(Z) 3-4 H, where Z represents any amino acid, and the fourth Cys2-His2 zinc finger domain is distinct from the third Cys2-His2 zinc finger domain, (9) the fourth portion is C-terminal to the third portion; (10) The first hybrid zinc finger domain comprises at least two amino acid substitutions, the amino acid substitutions in the first portion of the first hybrid zinc finger domain being relative to the first portion of the first Cys2-His2 zinc finger domain and the amino acid substitutions in the second portion of the first hybrid zinc finger domain being relative to the second portion of the second Cys2-His2 zinc finger domain, the substitutions being at positions C3, C6, X7, H 19 , or H 23 does not exist in either of the above.

[0070] The present invention provides a system that may include a zinc finger degron, also referred to herein as a degron tag. Generally, a degron or degron tag is a peptide sequence or protein element that is involved in regulating the degradation rate of a protein that includes such a degron or degron tag. A degron tag according to the present invention, in some embodiments, includes two zinc finger domains. The term zinc finger domain is well understood by those skilled in the art in the context of the present invention. Thus, in an embodiment, the degron is a zinc finger degron that includes at least two zinc finger domains and can be controlled with an IMiD, such as thalidomide, lenalidomide, pomalidomide, and / or analogs thereof. Preferably, the degron tag can promote or control the degradation of a POI in the presence of such an IMiD, for example, when fused to the degron tag. Preferably, one or both of the hybrid zinc finger domains in a degron tag according to the present invention is a non-natural hybrid zinc finger.

[0071] The first hybrid zinc finger domain included in the degron tag according to the present invention includes a first portion and a second portion. The first portion and the second portion each independently consist of an amino acid sequence. The first portion and the second portion included in the first hybrid zinc finger domain may or may not be directly adjacent to each other. In some embodiments, the first portion and the second portion are connected via a linker peptide, for example, of 1, 2, 3, 4, 5, or more amino acids. Thus, in some embodiments, the first portion and the second portion may be connected to each other via an additional linker peptide, for example, comprising 1, 2, 3, 4, 5, 6, 7, or more amino acids. However, in some embodiments, there is no additional linker peptide between the first portion of the first hybrid zinc finger domain and the second portion of the first hybrid zinc finger domain. In such embodiments, the first and second portions are directly adjacent to each other. Preferably, the first and second portions are directly adjacent to each other.

[0072] The first portion of the first hybrid zinc finger domain may be N-terminal or C-terminal from the second portion of the first hybrid zinc finger domain, hi a preferred embodiment, the first portion of the first hybrid zinc finger domain is N-terminal to the second portion of the first hybrid zinc finger domain.

[0073] In preferred embodiments, the first portion consists of 8 to 30 amino acids, and with increasing preference 11 to 30, 10 to 20, 11 to 20, 10 to 14, 11 to 14, 10 to 11, and most preferably 11 amino acids.

[0074] In preferred embodiments, the second portion consists of 8 to 30 amino acids, with increasing preference 12 to 30, 11 to 20, 12 to 20, 11 to 14, 12 to 14, and most preferably 12 amino acids.

[0075] In some embodiments, the first hybrid zinc finger domain, preferably non-naturally occurring, consists of about 16-60 amino acids, preferably about 20-36 amino acids, about 20-30 amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. In certain embodiments, the first hybrid zinc finger domain comprises a Cys2 His2 (C2H2) domain, and the hybrid zinc finger domain comprises at least two subdomains (referred to herein as portions), each subdomain being derived from a different wild-type zinc finger. For example, a first portion of the first hybrid zinc finger domain is derived from a first wild-type zinc finger, and a second portion of the first hybrid zinc finger domain is derived from a second different wild-type zinc finger domain. Based on the disclosure herein, a person skilled in the art is fully capable of selecting a first portion of the first hybrid zinc finger domain derived from a first wild-type zinc finger and / or a second portion of the first hybrid zinc finger domain derived from a second wild-type zinc finger. For example, a person skilled in the art may appropriately select such a portion derived from a wild-type zinc finger domain (particularly a wild-type Cys2-His2 (C2H2) zinc finger domain), which is available from various scientific publications and publicly known gene and protein databases, for example (Sievers et al. Science. 2018 Nov 2; 362 (6414): eaat0572), which is known or predicted to destabilize a protein (e.g., a wild-type protein comprising such a wild-type zinc finger domain) in the presence of an IMiD, such as thalidomide, thereby controlling the degradation of such a protein.With respect to the second zinc finger, second hybrid zinc finger and / or third or fourth portion described herein, in some embodiments these may or may not themselves (i.e. in the absence of the first hybrid zinc finger domain) be sensitive to an IMiD, i.e. may or may not destabilize a protein in the presence of an IMiD such as thalidomide, thereby controlling the degradation of such a protein. Preferably, they are not themselves sensitive to an IMiD.

[0076] In certain embodiments, the first portion of the first hybrid zinc finger domain has the sequence X1X2C3X4X5C6X7X8X9X 10 X 11 and each X (X1, X2, etc.) independently represents any amino acid, and more specifically, each X represents any natural or proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). An alternative way of representing the amino acid sequence contained in the first portion of the first hybrid zinc finger domain is (X)2C(X)2C(X). However, as amino acid substitutions at particular positions in the first portion of the first hybrid zinc finger domain, among others, are part of the invention, the following is a representation of the individual amino acids and their positions: X1X2C3X4X5C6X7X8X9X. 10 X 11 It is preferred to use:

[0077] One of skill in the art will understand that, for example, X1 can represent a different amino acid than, for example, X2, or can represent the same amino acid. One of skill in the art will understand that C3 indicates the presence of a cysteine ​​at the third position in this amino acid sequence contained in the first portion of the first hybrid zinc finger domain. Similarly, C6 indicates the presence of a cysteine ​​at the sixth position. In a preferred embodiment, the first portion of the first hybrid zinc finger domain has the amino acid sequence shown above (X1X2C3X4X5C6X7X8X9X 10 X 11 ) and the amino acid sequence is that of a first Cys2-His2 zinc finger domain (e.g. a wild type Cys2-His2 zinc finger domain). In some embodiments, the indicated amino acid sequence of the first part of the first hybrid zinc finger domain may comprise an additional short stretch of amino acids at its N-terminus, e.g. 1-10, preferably 1-5, e.g. 1, 2, 3, 4 or 5 amino acids, of said first or second Cys2-His2 zinc finger domain. For example, FIG. 3 provides an example of a degron tag according to the invention (positions of amino acid substitutions are shown), where at the N-terminus of the first hybrid zinc finger domain there is a short stretch of amino acids GERP (in this case from IKZF1). In some other embodiments the stretch may be from another wild type zinc finger domain, e.g. GEKP). Experiments have confirmed that such short stretches may be included without substantially affecting the results obtained with the degron tag (and / or the hybrid zinc finger polypeptides according to the invention).

[0078] As described in detail elsewhere herein, the first hybrid zinc finger domain comprised in a degron tag according to the present invention is, in a preferred embodiment, a hybrid zinc finger comprising a Cys2 His2 (C2H2) zinc finger domain, the hybrid zinc finger domain comprising at least two subdomains, i.e. a first portion and a second portion, wherein the amino acid sequence of the first portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2 His2 (C2H2) zinc finger.

[0079] In certain embodiments, the second portion of the first hybrid zinc finger domain comprises the sequence X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 Each X (X 12 , X 13 Each X independently represents any amino acid, and more specifically, each X represents any natural or proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). An alternative way of representing the amino acid sequence contained in the second portion of the first hybrid zinc finger domain is (X)7H(X)3H. However, since amino acid substitutions at particular positions in the second portion of the first hybrid zinc finger domain, among others, are part of the invention, the X's representing individual amino acids and their positions are not intended to be limiting. 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X21 X 22 H 23、 It is also contemplated that in some embodiments, the second portion of the first hybrid zinc finger domain is (X)6H(X)3H.

[0080] Those skilled in the art will recognize that, for example, X 12 For example, X 13 It is understood that H may represent a different amino acid than H, or may represent the same amino acid. 19 indicates the presence of a histidine at position 19 in this amino acid sequence contained in the second portion of the first hybrid zinc finger domain. 23 indicates the presence of a histidine cysteine ​​at position 23 in this sequence. In a preferred embodiment, the second portion of the first hybrid zinc finger domain has the amino acid sequence shown above (X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 ) and the amino acid sequence is that of a second Cys2-His2 zinc finger domain.

[0081] In some embodiments, the indicated amino acid sequence of the second portion of the first hybrid zinc finger domain may include an additional short stretch of amino acids at its C-terminus, e.g., 1-10, preferably 1-5, e.g., 1, 2, 3, 4, or 5 amino acids, of said second Cys2-His2 zinc finger domain. In preferred embodiments, the first portion of the first hybrid zinc finger domain is N-terminal to the second portion of the first hybrid zinc finger domain. In such embodiments, the second portion is C-terminal to the first portion.

[0082] In other words, in a preferred embodiment, the first hybrid zinc finger domain included in the degron tag according to the present invention is a hybrid zinc finger domain including a Cys2 His2 (C2H2) domain, and the hybrid zinc finger domain includes at least two subdomains, i.e., a first portion and a second portion, in which the amino acid sequence of the first portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2 His2 (C2H2) zinc finger (i.e., a first Cys2-His2 zinc finger domain), and the amino acid sequence of the second portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2 His2 (C2H2) zinc finger (i.e., a first Cys2-His2 zinc finger domain), and The first portion is derived from a His2 (C2H2) zinc finger (i.e., a second Cys2-His2 zinc finger domain), and preferably the second Cys2-His2 zinc finger domain is different from the first Cys2-His2 zinc finger domain (e.g., the first Cys2-His2 zinc finger domain is a wild-type zinc finger that is different from the second Cys2-His2 zinc finger domain). 10 X 11 and / or the amino acid sequence of the second portion is X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 Contains or is.

[0083] As described above, the first portion and the second portion together form a first hybrid zinc finger domain, where the first portion is preferably a first Cys2-His2 zinc finger domain, e.g., a wild-type naturally occurring Cys2-His2 zinc finger domain, and the second portion is preferably a second, different Cys2-His2 zinc finger domain, e.g., a different wild-type naturally occurring Cys2-His2 zinc finger domain. In such embodiments, the first hybrid zinc finger domain formed by the first portion and the second portion may be referred to as a hybrid Cys2-His2(C2H2) zinc finger domain, e.g., a wild-type zinc finger protein and / or a non-naturally occurring Cys2-His2(C2H2) zinc finger domain.

[0084] The C2H2 zinc finger domain structure has been found to be a key determinant of binding, and may be even more important than the primary amino acid sequence of the zinc finger domain (e.g., Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572). The C2H2 zinc finger domain binds to the ubiquitin ligase CRL4. CRBN It is known as a repeat motif that mediates drug-dependent (e.g., IMiD) interactions with (e.g., An et al., Nat Commun. 8:15398 (2017), doi:10.1038 / ncommsl5398; Koduri et al., PNAS 116(7)2539-2544 (2019), doi:10.1073 / pnas.l818109116; International Publication No. 2019089592; International Publication No. 2020132039; International Publication No. 2021188286; or International Publication No. 202108042).

[0085] Typically, a C2H2 zinc finger domain, including, for example, a first hybrid zinc finger domain and / or a second (hybrid) zinc finger domain according to the invention, comprises a beta hairpin subdomain and an alpha helix subdomain. Typically, a 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. A C2H2 zinc finger domain typically comprises an N-terminal beta hairpin containing two conserved cysteine ​​residues followed by an alpha helix containing two conserved histidine residues at its C-terminus (see, for example, Fedotova et al., ActaNaturae, 2017 Apr-Jun;9(2):47-58). This motif is also present in zinc finger domains according to the invention, including the first and second hybrid zinc finger domains.

[0086] In addition to the first hybrid (non-natural) zinc finger domain, a degron tag according to the invention may also comprise a second (non-natural) hybrid zinc finger domain. In a degron tag according to the invention, the second hybrid zinc finger domain is C-terminal to the first hybrid zinc finger domain. In such an embodiment, the first hybrid zinc finger domain in a degron tag according to the invention is N-terminal to the second hybrid zinc finger domain.

[0087] In some embodiments, the first and second hybrid zinc finger domains are directly adjacent to each other and no peptide linker is present. In some embodiments, the first and second hybrid zinc finger domains are not directly adjacent to each other but are connected to each other via a short peptide linker. In embodiments where the first and second hybrid zinc finger domains are connected via a linker peptide, such a linker peptide may be composed of, for example, 1, 2, 3, 4, 5, 6, 7 or more amino acids.

[0088] Thus, in some embodiments, a first hybrid zinc finger domain may be connected to a second hybrid zinc finger domain via an additional linker peptide, e.g., comprising 1, 2, 3, 4, 5, 6, 7 or more amino acids, although in some embodiments, no additional linker peptide is present.

[0089] For example, in some embodiments, the linker may be a short stretch of amino acids derived from a wild-type zinc finger domain, e.g., derived from or adjacent to the N-terminal portion of the wild-type zinc finger domain. Such an amino acid sequence may be, for example, the SGEKP sequence (see FIG. 3) (in this case, the SGEKP sequence derived from the IKZF1 protein is the endogenous linker between IKZF1 ZF2 and IKZF1 ZF3 in the IKZF1 protein, in the same manner, other endogenous linkers may be used, for example, as linkers within the context of the present invention, e.g., equivalent endogenous linkers from other C2H2 zinc finger proteins), in some other embodiments, the stretch may be derived from another wild-type zinc finger domain, e.g., GERP). Experiments have confirmed that such short stretches may be included, but do not substantially affect the results obtained with the degron tag (and / or the hybrid zinc finger polypeptide according to the present invention), and that the skilled person is fully capable of providing suitable additional linkers within the context of the present invention.

[0090] With respect to the second hybrid zinc finger domain, it has been found that the inclusion of such a second hybrid zinc finger domain may further improve the sensitivity of the degron tag of the invention in controlling protein degradation, protein levels and / or cellular activity. Thus, according to this aspect of the invention, there is provided a degron tag comprising a first and a second hybrid zinc finger domain as detailed herein.

[0091] The second hybrid zinc finger domain comprises a third portion and a fourth portion. The third portion and the fourth portion each independently consist of an amino acid sequence. The third portion and the fourth portion included in the second hybrid zinc finger domain may or may not be directly adjacent to each other. In some embodiments, the third portion and the fourth portion are connected to each other via a linker peptide, for example, of 1, 2, 3, 4, 5, or more amino acids. Thus, in some embodiments, the third portion and the fourth portion may be connected to each other via an additional linker peptide, for example, of 1, 2, 3, 4, 5, 6, 7, or more amino acids. However, in some embodiments, there is no additional linker peptide between the third portion of the second hybrid zinc finger domain and the fourth portion of the second hybrid zinc finger domain. In such embodiments, the third and fourth portions are directly adjacent to each other. Preferably, the third and fourth portions are directly adjacent to each other.

[0092] The third portion of the second hybrid zinc finger domain may be N-terminal or C-terminal from the fourth portion of the second hybrid zinc finger domain. In a preferred embodiment, the third portion is N-terminal to the fourth portion.

[0093] In preferred embodiments, the third portion consists of 8 to 30 amino acids, and with increasing preference 11 to 30, 10 to 20, 11 to 20, 10 to 14, 11 to 14, 10 to 11, and most preferably 11 amino acids.

[0094] In preferred embodiments, the fourth portion consists of 8 to 30 amino acids, and with increasing preference 12 to 30, 11 to 20, 12 to 20, 11 to 14, 12 to 14, and most preferably 12 amino acids.

[0095] In some embodiments, the second hybrid zinc finger domain, preferably non-naturally occurring, consists of about 16-60 amino acids, preferably about 20-36 amino acids, about 20-30 amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. In certain embodiments, the second hybrid zinc finger domain comprises a Cys2 His2 (C2H2) domain, and the hybrid zinc finger domain comprises at least two subdomains (referred to herein as portions), each subdomain being derived from a different wild-type zinc finger. For example, a third portion of the second hybrid zinc finger domain is derived from a third wild-type zinc finger, and a fourth portion of the second hybrid zinc finger domain is derived from a fourth different wild-type zinc finger domain. Based on the disclosure herein, a person skilled in the art is fully capable of selecting a third portion of the second hybrid zinc finger domain derived from a third wild-type zinc finger and / or a fourth portion of the second hybrid zinc finger domain derived from a fourth wild-type zinc finger. For example, a person skilled in the art may appropriately select such a portion derived from a wild-type zinc finger domain (particularly a wild-type Cys2-His2 (C2H2) zinc finger domain), which is available from various scientific publications and public and well-known gene and protein databases, for example (Sievers et al. Science. 2018 Nov 2; 362 (6414): eaat0572), which is known or predicted to destabilize a protein (e.g., a wild-type protein comprising such a wild-type zinc finger domain) in the presence of an IMiD such as thalidomide, thereby controlling the degradation of such a protein.

[0096] In certain embodiments, the third portion of the second hybrid zinc finger domain has the sequence (Z)C(Z)C(Z). 5-6where 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 portion of the second hybrid zinc finger domain may, for example, be represented by 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 proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). One of skill in the art will appreciate that, for example, Z1 may represent a different amino acid than, for example, Z2, or may represent the same amino acid. One of skill in the art will appreciate that C3 indicates the presence of a cysteine ​​at the third position in this amino acid sequence contained in the third portion of the second hybrid zinc finger domain. Similarly, C6 indicates the presence of a cysteine ​​at the sixth position. In a preferred embodiment, the third portion of the second hybrid zinc finger domain has the amino acid sequence (Z)2C(Z)2C(Z) shown above. 5-6, and even more preferably (Z)2C(Z)2C(Z)6, ​​wherein the amino acid sequence is that of a third Cys2-His2 zinc finger domain (e.g. a wild-type Cys2-His2 zinc finger domain). In some embodiments, the indicated amino acid sequence of the third part of the second hybrid zinc finger domain may comprise an additional short stretch of amino acids at its N-terminus, e.g. 1-10, preferably 1-5, e.g. 1, 2, 3, 4 or 5 amino acids, of said third or fourth (wild-type) Cys2-His2 zinc finger domain. For example, FIG. 3 provides an example of a degron tag according to the invention (positions of amino acid substitutions are shown), wherein at the N-terminus of the second hybrid zinc finger domain there is a short stretch of amino acids SGEKP, as described above. Experiments have confirmed that such short stretches may be included without substantially affecting the results obtained with the degron tag (and / or the hybrid zinc finger polypeptides according to the invention).

[0097] As described in detail herein, and comparable to the first hybrid zinc finger domain comprised in the degron tag according to the present invention, the second hybrid zinc finger domain is in a preferred embodiment also a hybrid zinc finger comprising a Cys2 His2 (C2H2) zinc finger domain, which comprises at least two subdomains, i.e. a third portion and a fourth portion, and the amino acid sequence of the third portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2 His2 (C2H2) zinc finger.

[0098] In certain embodiments, the fourth portion of the second hybrid zinc finger domain has the sequence (Z)H(Z) 3-4 H. Thus, (Z)6 represents 6 amino acids, and (Z) 3-4 represents 3 or 4 amino acids. For example, the fourth portion of the second hybrid zinc finger domain may be, for example, the amino acid sequence Z13 Z 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 Z 16 Z 17 Z 18 H 19 Z 20 Z 21 Z 22 Z 23 H 24 and each Z(Z 13 , Z 14 Each Z independently represents any amino acid, and more specifically, each Z represents any natural or proteinogenic amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). 13 But for example, Z 14 It is understood that H may represent a different amino acid than H, or may represent the same amino acid. 19 indicates the presence of a histidine at position 19 in this amino acid sequence contained in the second portion of the first hybrid zinc finger domain. 23 Or H 24 is ((Z) 3-4 indicates the presence of a histidine cysteine ​​at position 23 in this sequence or at position 24 in this sequence (depending on whether Z is associated with 3 or 4 amino acids). For example, in some embodiments, it is explained elsewhere herein that there may be a linker between the first and second (hybrid) zinc finger domains, e.g., Z 13Note that the sequence (Z)6H(Z) does not indicate a position relative to the first hybrid zinc finger domain. It is also described herein that the length of the third portion of the second (hybrid) zinc finger may vary, for example, be 11 amino acids long. In a preferred embodiment, the fourth portion of the second hybrid zinc finger domain has the amino acid sequence (Z)6H(Z) shown above. 3-4 H and the amino acid sequence is that of a second Cys2-His2 zinc finger domain, ie, more specifically, a wild-type Cys2 His2 (C2H2) zinc finger.

[0099] In other words, in a preferred embodiment, the second hybrid zinc finger domain included in the degron tag according to the present invention is a hybrid zinc finger domain including a Cys2 His2 (C2H2) domain, and the hybrid zinc finger domain includes at least two subdomains, i.e., a third portion and a fourth portion, in which the amino acid sequence of the third portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2 His2 (C2H2) zinc finger (i.e., a third Cys2-His2 zinc finger domain), and the amino acid sequence of the fourth portion is derived from a wild-type zinc finger, more specifically a wild-type Cys2 His2 (C2H2) zinc finger (i.e., a third Cys2-His2 zinc finger domain), and Preferably, the amino acid sequence of the third portion is derived from a His2(C2H2) zinc finger (i.e., a fourth Cys2-His2 zinc finger domain), and preferably the third Cys2-His2 zinc finger domain is different from the fourth Cys2-His2 zinc finger domain (e.g., the third Cys2-His2 zinc finger domain is a wild-type zinc finger different from the fourth Cys2-His2 zinc finger domain). 5-6 and / or the amino acid sequence of the fourth portion is (Z)H(Z) 3-4 Contains or is H.

[0100] In a preferred embodiment, the fourth portion is C-terminal to the third portion.

[0101] As will be appreciated by one of skill in the art, in addition to the above domains, moieties and linkers, a degron tag according to the present invention may comprise additional amino acids, for example, N-terminal from the first hybrid zinc finger domain or C-terminal from the second hybrid zinc finger domain.

[0102] In a preferred embodiment, a degron tag according to the invention is further characterized by the presence of at least two amino acid substitutions in the first hybrid zinc finger domain, the amino acid substitutions in the first portion of the first hybrid zinc finger domain being relative to the first portion of the first Cys2-His2 zinc finger domain and the amino acid substitutions in the second portion of the first hybrid zinc finger domain being relative to the second portion of the second Cys2-His2 zinc finger domain, the substitutions being relative to positions C3, C6, X7, H 19 , or H 23 In some embodiments, X7 is not present in any of the following:

[0103] As discussed herein, in embodiments, the first hybrid zinc finger domain is composed of a first portion derived from a first Cys2-His2 zinc finger domain, in particular a first wild-type Cys2-His2 zinc finger domain, the first portion having the amino acid sequence X1X2C3X4X5C6X7X8X9X 10 X 11 (i.e., as present in the first wild-type Cys2-His2 zinc finger domain). As discussed herein, in embodiments, the first hybrid zinc finger domain is comprised of a second portion derived from a second Cys2-His2 zinc finger domain, in particular derived from a second wild-type Cys2-His2 zinc finger domain, the second portion comprising the amino acid sequence X 12 X 13 X 14 X 15 X 16 X 17 X18 H 19 X 20 X 21 X 22 H 23 (i.e., as present in the second wild-type Cys2-His2 zinc finger domain).

[0104] In embodiments where the degron tag according to the invention is further characterized by the presence of at least two amino acid substitutions in the first hybrid zinc finger domain, and the amino acid substitutions are present in the first portion of the first hybrid zinc finger domain, the amino acid substitutions are amino acid substitutions relative to the first portion of the first Cys2-His2 zinc finger domain from which the sequence was derived. For example, if X2 is a valine in the first (wild-type) Cys2-His2 zinc finger domain from which the sequence was derived, and this valine is replaced, for example, with a lysine in the degron tag according to the invention, then in the degron tag according to the invention, X2 is a 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 it was derived. In a similar manner, the amino acid substitutions in the second portion of the first hybrid zinc finger domain are defined herein. For example, X2 is a valine in the first (wild-type) Cys2-His2 zinc finger domain from which the sequence was derived, and this valine is replaced, for example, with a lysine in the degron tag according to the invention. 20 is a proline in the second (wild type) Cys2-His2 zinc finger domain from which the sequence for the second portion was derived, and this proline is replaced, for example, with a leucine in a degron tag according to the invention, then in the degron tag according to the invention, X 20 is a leucine, and the amino acid substitution to leucine is for the proline contained in the wild-type Cys2-His2 zinc finger domain from which it was derived.

[0105] As disclosed above, the first hybrid zinc finger domain comprises at least two amino acid substitutions relative to portions derived from the wild-type first and second Cys2-His2 zinc finger domains, and a person skilled in the art is fully capable of providing, preparing and recognizing such amino acid substitutions relative to the wild-type first and second Cys2-His2 zinc finger domains, for example, by comparing the amino acid sequence of the first and / or second portions of the first hybrid zinc finger domain therewith.

[0106] In some embodiments, the at least two amino acid substitutions (i.e., substitution of an amino acid from the wild-type sequence with another, different amino acid) are both in the first portion. In some embodiments, the at least two amino acid substitutions are both 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. In some preferred embodiments, the first hybrid zinc finger domain comprises no more than two amino acid substitutions. In some preferred embodiments, the first hybrid zinc finger domain comprises more than two amino acid substitutions, e.g., 3, 4, 5, or 6 amino acid substitutions, preferably 2, 3, or 4 amino acid substitutions. In some preferred embodiments, the first zinc finger domain comprises (exactly) two amino acid substitutions, i.e., no more than two amino acid substitutions. In some preferred embodiments, the first zinc finger domain comprises (exactly) three amino acid substitutions, i.e., no more than three amino acid substitutions. In some preferred embodiments, the first zinc finger domain comprises (exactly) four amino acid substitutions, i.e., no more than four amino acid substitutions. In embodiments where the first hybrid zinc finger domain comprises more than two amino acid substitutions, e.g., three or four amino acid substitutions, in preferred embodiments, for example, the third and / or fourth amino acid substitutions are also selected or are selected from the substitutions disclosed herein, e.g., the substitutions listed in Table 1. In embodiments where the first hybrid zinc finger domain comprises, e.g., four (or six) amino acid substitutions, in preferred embodiments, the four (or six) amino acid substitutions are selected from the substitution pairs disclosed herein, e.g., as listed in Table 2 (e.g., Q12R K13V G14N K21A) and / or the substituted positions and amino acid pairs as listed in Table 4.In a preferred embodiment as discussed herein and comprising four amino acid substitutions in the first hybrid zinc finger domain, the first hybrid zinc finger domain according to the invention comprises the amino acid sequence set forth in SEQ ID NO: 135 (LQCEICGFTCRRVNNLLRHIALH; i.e., the quadruple substitutions Q12R / K13V / G14N / K21A) and / or the degron tag according to the invention comprises the amino acid sequence set forth in SEQ ID NO: 136 (LQCEICGFTCRRVNNLLRHIALHSGEKPFKCHLCNYACRRKDSVVAHKAKSH).

[0107] In a preferred embodiment, the substitutions are at positions C3, C6, X7, H 19 , or H 23 In some embodiments, X7 is G7.

[0108] Surprisingly, it has been found that by introducing at least two amino acid substitutions into the first and / or second portion of the first hybrid zinc finger domain relative to the first portion of the (wild-type) first Cys2-His2 zinc finger domain and the second portion of the (wild-type) second Cys2-His2 zinc finger domain, a degron tag can be provided that has a highly enhanced or increased sensitivity to an IMiD molecule, such as thalidomide or an analogue, relative to the wild-type zinc finger domain and the hybrid zinc finger domain, but composed of the wild-type subdomain (described herein) and not comprising at least two amino acid substitutions. Interestingly, it has been found that introducing one amino acid substitution can also enhance or increase sensitivity to an IMiD molecule, but unexpectedly, introducing further amino acid substitutions can even further enhance or increase sensitivity to an IMiD molecule, as shown in the examples and described herein.

[0109] Thus, according to this aspect of the invention there is provided a degron tag comprising at least a first hybrid zinc finger domain which comprises at least two amino acid substitutions relative to a wild-type zinc finger domain (or a portion thereof) of which the hybrid zinc finger domain is comprised, and which has enhanced or increased sensitivity to an IMiD molecule.

[0110] In some embodiments, at least two amino acid substitutions are located immediately adjacent to one another, while in other embodiments, the positions of the amino acid substitutions are separated from one another by at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 or more amino acids (i.e., unsubstituted amino acids), e.g., as shown in the accompanying tables, figures, and examples.

[0111] In principle, any amino acid substitution is acceptable, but preferably the amino acid substitutions on the specific positions in the first hybrid zinc finger domain are those described herein (either for the substituted amino acid or for the substituted amino acid). A person skilled in the art can provide such amino acid substitutions and degron tags according to the invention without any real burden, based on the information disclosed herein.

[0112] Without wishing to be bound by theory, the inventors believe that the enhanced or increased sensitivity to IMiD molecules observed using the degron tags and / or hybrid zinc finger polypeptides according to the invention is due at least in part to an altered or improved interaction between the degron tag, cereblon and the immunomodulatory drug (IMiD). Thus, in a preferred embodiment, a degron tag according to the invention and / or a hybrid zinc finger polypeptide according to the invention is provided, which is capable of binding to a complex formed between cereblon (CRBN) and an immunomodulatory drug (IMiD).

[0113] Also provided is a degron tag and / or hybrid zinc finger polypeptide according to the invention, wherein the first portion is a beta hairpin loop of a first Cys2-His2 zinc finger domain, the second portion is an alpha helical region of a second Cys2-His2 zinc finger domain, the third portion is a beta hairpin loop of a third Cys2-His2 zinc finger domain, and / or the fourth portion is an alpha helical region of a fourth Cys2-His2 zinc finger domain. As explained above, the term "first, second, third or fourth Cys2-His2 zinc finger domain" refers to a Cys2-His2 zinc finger domain found in nature, i.e. a Cys2-His2 zinc finger domain derived from a wild-type zinc finger protein. As explained above, in the degron tag and / or hybrid zinc finger polypeptide according to the invention, the first hybrid zinc finger domain may comprise at least two amino acid substitutions. As explained above, portions of these wild-type Cys2-His2 zinc finger domains are used in providing the degron tag and / or hybrid zinc finger polypeptide according to the invention. According to a preferred embodiment of the invention, the first portion used is the beta hairpin loop of the first Cys2-His2 zinc finger domain, the second portion used is the alpha helix region of the second Cys2-His2 zinc finger domain, the third portion used is the beta hairpin loop of the third Cys2-His2 zinc finger domain, and / or the fourth portion used is the alpha helix region of the fourth Cys2-His2 zinc finger domain. A zinc finger domain comprises a beta hairpin loop and an alpha helix region, and one of skill in the art knows how to provide the beta hairpin loop and / or alpha helix region portions of a (wild-type) Cys2-His2 zinc finger domain. Representative examples are well known to those of skill in the art and include, for example, those disclosed and described herein.Examples of zinc fingers that contain a beta hairpin loop and an alpha helix region include (human) IKZF1, IKZF2, IKZF3, SALL4, ZFP91, GZF1, ZNF653, ZNF692, ZNF827, ZBTB39, WIZ, ZNF98, ZNF654, ZNF787, ZNF276, ZNF582, ZNF517, and E4F1 ( Uniprot accession numbers: Q13422, Q9UKS7, Q9UKT9, Q9UJQ4, Q96JP5, Q9H116, Q96CK0, Q9BU19, Q17R98, O15060, O95785, A6NK75, Q8IZM8, Q6DD87, Q8N554, Q96NG8, Q6ZMY9, Q66K89). As disclosed above, a degron tag and / or hybrid zinc finger polypeptide according to the invention may comprise one or more amino acid residues N-terminal to the beta hairpin portion, one or more amino acid residues between the beta hairpin portion and the alpha helix portion, and one or more amino acid residues C-terminal to the alpha helix portion, provided that the degron tag and / or hybrid zinc finger polypeptide according to the invention is a substrate for the CRBN-IMiD complex and / or exhibits enhanced or increased sensitivity (e.g., relative to the wild-type situation) to an IMiD molecule. These additional amino acids may correspond to residues in a naturally occurring Cys2-His2 zinc finger domain or may differ, provided that the degron tag maintains a zinc finger-like fold and exhibits the properties as disclosed herein.

[0114] In an embodiment of the present invention, a degron tag and / or hybrid zinc finger polypeptide according to the present invention is provided, in which a first substitution is in the second portion and a second substitution is in the first portion or in the second portion. The at least two amino acid substitutions may both be in the first portion, or both in the second portion, or one in the first portion and the second in the second portion, but in a preferred embodiment, at least one of the amino acid substitutions is in the second portion. In particular, it has been found that a suitable degron tag and / or hybrid zinc finger polypeptide according to the present invention may be provided, in which at least one of the amino acid substitutions is in the second portion, i.e., in the alpha helix portion of the first hybrid zinc finger domain. The second amino acid substitution may be in the second portion or in the first portion (i.e., in the beta hairpin loop).

[0115] At least one substitution is X1, X4, X 12 , X 13 , X 14 , X 15 , X 17 , X 21 , and X 22 Also provided are degron tags and / or hybrid zinc finger polypeptides according to the invention, in which the amino acid substitution at one of these positions in the first hybrid zinc finger domain makes it possible to provide degron tags and / or hybrid zinc finger polypeptides according to the invention with increased or enhanced sensitivity to IMiD molecules. Without being bound by theory, it is believed that these positions in particular are key positions in the first hybrid zinc finger domain that allow the observed improved or increased sensitivity to IMiDs, as shown in the examples. Preferably, the substituted amino acids are as disclosed herein.

[0116] In a preferred embodiment, a degron tag and / or hybrid zinc finger polypeptide according to the invention is provided, wherein at least one of the substitutions present in the degron tag and / or hybrid zinc finger polypeptide according to the invention (or present in the first hybrid zinc finger domain) is selected from those listed in Table 1. Table 1 shows various positions X1 to X 22 In the present specification, the substitutions in the degron tag and / or hybrid zinc finger polypeptide according to the present invention may, in preferred embodiments, be present together with the replacement amino acid (i.e., the amino acid contained at that position in the degron tag and / or hybrid zinc finger polypeptide according to the present invention; one-letter code). Preferably, both amino acid substitutions (both the position and the replacement amino acid) are selected from those listed in Table 1. A person skilled in the art will understand that when both amino acid substitutions are selected from those listed in Table 1 (or any other table in the list provided herein), each amino acid substitution is at a different position. At the same time, a person skilled in the art will understand that the replacement amino acid is meant to indicate an amino acid that is different from a natural amino acid (i.e., different from that present in the wild-type Cys2-His2 zinc finger domain used for the first or second portion of the first hybrid zinc finger domain). In that respect, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, the second portion (and / or the third and fourth portions as discussed herein).

[0117] Similarly, the present invention also encompasses the use of the positions and substitutions listed in Table 1 (or any other table in the list provided herein) in any suitable first or second portion of a (wild-type) Cys2-His2 zinc finger domain that can be used in providing a first hybrid zinc finger domain according to the present invention. In other words, the positions and substitution amino acids listed in Table 1 (or any other table in the list provided herein) are applicable to any suitable first hybrid zinc finger domain, so long as the degron tag 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., susceptibility to IMiDs in the context of proteolysis).

[0118] However, in a preferred embodiment, the positions and substitutions are relative to the first hybrid zinc finger domain as used in the Examples herein, i.e., the first portion of the first hybrid zinc finger domain is the beta hairpin region of ZFP91ZF4 and the second portion of the first hybrid zinc finger domain is the alpha helix region of IKZF1 ZF2 (see Figure 3; the first hybrid zinc finger domain of the "parent degron tag" used in the Examples is represented diagrammatically).

[0119] [Table 1]

[0120] In a preferred embodiment, a degron tag and / or hybrid zinc finger polypeptide according to the present invention is provided, wherein the at least two substitutions are selected from those listed in Table 2. Table 2 lists preferred combinations of two amino acid substitutions at two different positions according to the present invention in the first hybrid zinc finger domain described herein (preferred combinations of two positions in the first hybrid zinc finger domain and the corresponding substituted amino acids are shown line by line, e.g., 4R and 12L indicate that in this combination, the amino acid at position 4 in the first hybrid zinc finger domain is substituted with arginine (R) and the amino acid at position 12 in the first hybrid zinc finger domain is substituted with leucine (L). In other words, the original amino acids of the portion obtained from the wild-type Cys2-His2 zinc finger domain are substituted with arginine at position 4 and leucine at position 12). In a preferred embodiment of the present invention, the at least two substitutions are selected from any of the combinations shown in Table 2.

[0121] [Table 2]

[0122] Those of skill in the art will appreciate that a substituted amino acid is meant to refer to an amino acid that is different from a naturally occurring amino acid (i.e., different from that present in the wild-type Cys2-His2 zinc finger domain used in the first or second portion of the first hybrid zinc finger domain). In that regard, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, the second portion (and / or the third and fourth portions as discussed herein).

[0123] Similarly, the present invention also encompasses the use of the positions and substitutions listed in Table 2 (or any other table in the list provided herein) in any suitable first or second portion of a (wild-type) Cys2-His2 zinc finger domain that can be used in providing a first hybrid zinc finger domain according to the present invention. In other words, the positions and substitution amino acids listed in Table 2 (or any other table in the list provided herein) are applicable to any suitable first hybrid zinc finger domain, so long as the degron tag 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., susceptibility to IMiDs in the context of proteolysis).

[0124] However, in a preferred embodiment, the positions and substitutions are relative to the first hybrid zinc finger domain as used in the Examples herein, i.e., the first portion of the first hybrid zinc finger domain is the beta hairpin region of ZFP91ZF4 and the second portion of the first hybrid zinc finger domain is the alpha helix region of IKZF1 ZF2 (see Figure 3; the first hybrid zinc finger domain of the "parent degron tag" used in the Examples is represented diagrammatically).

[0125] Furthermore, the primary amino acid substitutions Q12R, Q12K, N15R and L22R in the most frequently observed examples, when combined with the secondary amino acid substitutions listed in Table 8 (see below), further synergistically increased susceptibility to IMiDs (i.e. EI Q12R / K13V=0.006877267, whereas EI Q12R=0.167217557). Thus, in particularly preferred embodiments of the invention, combinations of amino acid substitutions as listed in Table 8 are used / provided, such as, for example, 12R and 13T or 22R and 14L.

[0126] In a preferred embodiment, a degron tag and / or a hybrid zinc finger polypeptide according to the invention is provided, wherein the amino acids in the first portion to be substituted and / or the amino acids in the second portion to be substituted are selected from those listed in Table 3.

[0127] Table 3 lists preferred amino acids to be substituted in the first hybrid zinc finger domain of a degron tag and / or hybrid zinc finger polypeptide according to the present invention. In other words, in a preferred embodiment of the present invention, the positions and / or positions and amino acids to be substituted are selected from those listed in Table 3. Thus, according to a preferred embodiment of the present invention, the at least two amino acid substitutions are selected from combinations of positions 1, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21 and 22, and the substituted amino acids are combinations of two amino acids listed in Table 3.

[0128] [Table 3]

[0129] Preferably, both substituted amino acids (both positions and replacement amino acids) are selected from those listed in Table 3. As will be appreciated by one of skill in the art, the amino acids, e.g., the preferred amino acids shown in Table 3, may be substituted with any suitable amino acid, so long as it provides a degron tag and / or hybrid zinc finger polypeptide according to the present invention. However, in a preferred embodiment, the amino acids, preferably the combinations of amino acids listed in Table 3, are replaced with the corresponding amino acids listed in Table 1 or the corresponding combinations of amino acids shown in Table 2 (where "corresponding" refers to the corresponding positions, e.g., in the first hybrid zinc finger domain disclosed herein).

[0130] One of skill in the art will understand that when both substituted amino acids are selected from those listed in Table 3 (or any other table in the list provided herein), each amino acid substitution is at a different position. At the same time, one of skill in the art will understand that a substituted amino acid is meant to indicate an amino acid that is replaced with another amino acid and is different from the natural amino acid (i.e., different from that present in the wild-type Cys2-His2 zinc finger domain used in the first or second portion of the first hybrid zinc finger domain). In that regard, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, the second portion (and / or the third and fourth portions as discussed herein).

[0131] 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 portion of a (wild-type) Cys2-His2 zinc finger domain that can be used in providing a first 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 of the lists provided herein) are applicable to any suitable first hybrid zinc finger domain, so long as the degron tag 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., susceptibility to IMiDs in the context of proteolysis).

[0132] However, in a preferred embodiment, the positions and substitutions are relative to the first hybrid zinc finger domain as used in the Examples herein, i.e., the first portion of the first hybrid zinc finger domain is the beta hairpin region of ZFP91 ZF4 and the second portion of the first hybrid zinc finger domain is the alpha helix region of IKZF1 ZF2 (see Figure 3; the first hybrid zinc finger domain of the "parent degron tag" used in the Examples is represented diagrammatically).

[0133] In a preferred embodiment, a degron tag and / or hybrid zinc finger polypeptide according to the invention is provided, wherein at least two amino acids in the first portion and / or the second portion to be substituted are selected from those listed in Table 4.

[0134] Table 4 lists preferred combinations of two amino acids substituted at two different positions according to the present invention in the first hybrid zinc finger domain described herein (each line shows a preferred combination of two positions in the first hybrid zinc finger domain and the corresponding substituted amino acid, e.g., E4 and Q12 indicates that in this combination, the amino acid at position 4 in the first hybrid zinc finger domain that is substituted is glutamic acid (E) and the amino acid at position 12 that is substituted is glutamine (Q). In other words, the original amino acids of the portion derived from the wild-type Cys2-His2 zinc finger domain are glutamic acid at position 4 and glutamine at position 12. In certain preferred embodiments, the at least two amino acids that are substituted are selected from any of the combinations shown in Table 4. As will be appreciated by one of skill in the art, the amino acids, e.g., the preferred amino acids shown in Table 4, may be substituted with any suitable amino acids, so long as they provide a degron tag and / or hybrid zinc finger polypeptide according to the present invention. However, in preferred embodiments, the amino acids, preferably the combinations of amino acids listed in Table 4, are replaced with the corresponding amino acids listed in Table 1 or the corresponding combinations of amino acids shown in Table 2 (where "corresponding" refers to the corresponding positions in, e.g., the first hybrid zinc finger domain disclosed herein).

[0135] [Table 4]

[0136] One of skill in the art will understand that a substituted amino acid is meant to refer to an amino acid that is different from the naturally occurring amino acid that is substituted (i.e., different from that present in the wild-type Cys2-His2 zinc finger domain used in the first or second portion of the first hybrid zinc finger domain). In that regard, it should be noted that the present invention is not particularly limited to the particular wild-type Cys2-His2 zinc finger domain used to provide the first portion, the second portion (and / or the third and fourth portions as discussed herein).

[0137] Similarly, the present invention also encompasses the use of the positions and substitutions listed in Table 4 (or any other table in the list provided herein) in any suitable first or second portion of a (wild-type) Cys2-His2 zinc finger domain that can be used in providing a first hybrid zinc finger domain according to the present invention. In other words, the positions and amino acids substituted and listed in Table 4 (or any other table in the list provided herein) are applicable to any suitable first hybrid zinc finger domain, so long as the degron tag 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., susceptibility to IMiDs in the context of proteolysis).

[0138] However, in a preferred embodiment, the positions and amino acids substituted are relative to the first hybrid zinc finger domain as used in the Examples herein, i.e., the first portion of the first hybrid zinc finger domain is the beta hairpin region of ZFP91 ZF4 and the second portion of the first hybrid zinc finger domain is the alpha helix region of IKZF1 ZF2 (see Figure 3; the first hybrid zinc finger domain of the "parent degron tag" used in the Examples is represented diagrammatically).

[0139] In a further embodiment, a degron tag and / or hybrid zinc finger polypeptide according to the invention is provided, (1) 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; (2) the second Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF2, IKZF3 ZF2, ZFP91 ZF4, ZNF654 ZF1, ZNF787 ZF5, ZNF653 ZF4, ZNF276 ZF4, ZNF692 ZF4, ZNF582 ZF9, ZNF517 ZF10, E4F1 ZF2, and ZNF827 ZF1, preferably IKZF1 ZF2; (3) 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 IKZF1 ZF3; and / or (4) The fourth Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, and ZNF692 ZF5, preferably ZFP91 ZF5.

[0140] The present invention is not particularly limited to the particular (wild-type) Cys2-His2 zinc finger domain that provides the first, second, third and / or fourth portion, respectively, in the first hybrid zinc finger domain and the second hybrid zinc finger domain, in particular the second hybrid zinc finger domain. A variety of wild-type or naturally occurring Cys2-His2 zinc finger domains have been described in the art that are suitable for use in degron systems, including the use of IMiDs (e.g., Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572). Therefore, those skilled in the art will understand that such a Cys2-His2 zinc finger domain containing a typical beta hairpin loop and an alpha helix can be suitably used in the present invention, for example, by providing a first, second, third and / or fourth portion in the first hybrid zinc finger domain and the second zinc finger domain, particularly in the second hybrid zinc finger domain, respectively, and at least two amino acids can be substituted in the first and / or second portions, as detailed herein. Thus, such a degron tag and / or hybrid zinc finger polypeptide according to the present invention that maintains a zinc finger-like fold (beta hairpin loop and alpha helix) and exhibits the properties disclosed herein (e.g., susceptibility to IMiD in the context of proteolysis) can be easily provided by those skilled in the art.

[0141] However, in a preferred embodiment, (1) 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; (2) the second Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF2, IKZF3 ZF2, ZFP91 ZF4, ZNF654 ZF1, ZNF787 ZF5, ZNF653 ZF4, ZNF276 ZF4, ZNF692 ZF4, ZNF582 ZF9, ZNF517 ZF10, E4F1 ZF2, and ZNF827 ZF1, preferably IKZF1 ZF2; (3) 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 IKZF1 ZF3; and / or (4) The fourth Cys2-His2 zinc finger domain is selected from the group consisting of IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, and ZNF692 ZF5, preferably ZFP91 ZF5.

[0142] In other words, the amino acid sequence of the Cys2-His2 zinc finger domain for selection of the first portion and / or the second portion (see below) may, in some embodiments, be selected from 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).

[0143] In other words, the amino acid sequence of the Cys2-His2 zinc finger domain for selection of the third portion and / or the fourth portion (see below) may, in some embodiments, be selected from 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).

[0144] According to this embodiment, the first Cys2-His2 zinc finger domain provides a first portion of the first hybrid zinc finger domain and is selected from those listed above.

[0145] According to this embodiment, the second Cys2-His2 zinc finger domain provides a second portion of the first hybrid zinc finger domain and is selected from those listed above.

[0146] In the degron tag and / or hybrid zinc finger polypeptides according to the invention, these first and second portions provide a first hybrid zinc finger domain and further comprise at least two amino acid substitutions relative to the first and / or second portions provided by the first and second Cys2-His2 zinc finger domains.

[0147] According to this embodiment, the third Cys2-His2 zinc finger domain provides a third portion of the second hybrid zinc finger domain and is selected from those listed above.

[0148] According to this embodiment, the fourth Cys2-His2 zinc finger domain provides the fourth portion of the second hybrid zinc finger domain and is selected from those listed above. As discussed herein, the second zinc finger in a degron tag and / or hybrid zinc finger polypeptide according to the invention may in certain embodiments be a hybrid zinc finger domain or a non-hybrid zinc finger domain. In the latter case, the second zinc finger may therefore be selected from either the third or fourth Cys2-His2 zinc finger domains listed above (providing both the first and second portions from the same Cys2-His2 zinc finger domain).

[0149] In a preferred embodiment, at least one, two, three or four of the first, second, third and fourth Cys2-His2 zinc finger domains listed above are selected. In a preferred embodiment, all of the first, second, third and fourth Cys2-His2 zinc finger domains listed above are selected. In a preferred embodiment, at least the first and second Cys2-His2 zinc finger domains are selected from those listed above. In a preferred embodiment, at least the third and fourth Cys2-His2 zinc finger domains are selected from those listed above.

[0150] In a highly preferred embodiment, the beta hairpin loop (or first portion) of the first hybrid zinc finger domain is from ZFP91 ZF4 and the alpha helix (or second portion) of the first hybrid zinc finger is from IKZF1 ZF2. In an even more highly preferred embodiment, the beta hairpin loop (or third portion) of the second hybrid zinc finger domain is from IKZF1 ZF3 and the alpha helix (or fourth portion) of the second hybrid zinc finger is from ZFP91 ZF5.

[0151] 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 derived from the same zinc finger protein (e.g., a ZF4 domain derived from ZFP91 and a ZF5 domain derived from ZFP91). In some embodiments, the first and second Cys2-His2 zinc finger domains are derived from two different zinc finger proteins and the third and fourth Cys2-His2 zinc finger domains are derived from the same two different zinc finger proteins (e.g., the first Cys2-His2 zinc finger domain and the fourth Cys2-His2 zinc finger domain are both derived from ZFP91 (zinc finger domains ZF4 and ZF5, respectively) and the second Cys2-His2 zinc finger domain and the third Cys2 The Cys2-His2 zinc finger domains are both derived from different zinc finger proteins, such as IKZF1 (zinc finger domains ZF2 and ZF3, respectively). When two different Cys2-His2 zinc finger domains derived from the same zinc finger protein are provided, the Cys2-His2 zinc finger domains that are N-terminal to the other in the wild-type zinc finger protein are preferably also N-terminal to the other in the degron tag and / or hybrid zinc finger polypeptide according to the invention.

[0152] The enumerated Cys2-His2 zinc finger domains, their sequences and structures are well known to those skilled in the art and available in various scientific publications and databases.

[0153] As already briefly discussed, in one embodiment of the present invention, there is also provided a degron tag and / or hybrid zinc finger polypeptide according to the invention, in which at least two amino acid substitutions are introduced into a first hybrid zinc finger domain, a first part of which has the amino acid sequence set forth in SEQ ID NO: 101 (LQCEICGFTCR-ZFP91 ZF4 (first part)) and / or a second part of which has the amino acid sequence set forth in SEQ ID NO: 102 (QKGNLLRHIKLH-IKZF1 ZF2 (second part)) and / or the first hybrid zinc finger domain has the amino acid sequence set forth in SEQ ID NO: 103 (LQCEICGFTCRQKGNLLRHIKLH-(ZFP91 ZF4 / IKZF1 ZF2 first hybrid zinc finger domain)). In a preferred embodiment, in these sequences at least two amino acid substitutions according to the present invention may be introduced.

[0154] In a further preferred embodiment, a degron tag and / or hybrid zinc finger polypeptide according to the present invention is provided, comprising at least two amino acid substitutions introduced therein (e.g., any of the sequences set forth in SEQ ID NOs: 101 to 103), a third portion thereof having the amino acid sequence set forth in SEQ ID NO: 104 (FKCHLCNYACRR-(IKZF1 ZF3(third portion)), and / or a fourth portion thereof having the amino acid sequence set forth in SEQ ID NO: 105 (KDSVVAHKAKSH(ZFP91 ZF5(fourth portion)), and / or a second hybrid zinc finger domain having the amino acid sequence set forth in SEQ ID NO: 106 (FKCHLCNYACRRKDSVVAHKAKSH-second hybrid zinc finger domain).

[0155] In a further preferred embodiment, a degron tag and / or hybrid zinc finger polypeptide according to the present invention is provided, in which at least two amino acid substitutions are introduced, and the degron tag and / or hybrid zinc finger polypeptide has the amino acid sequence set forth in SEQ ID NO: 107 (LQCEICGFTCRQKGNLLRHIKLHSGEKPFKCHLCNYACRRKDSVVAHKAKSH). In a preferred embodiment, in these sequences, at least two amino acid substitutions according to the present invention may be introduced.

[0156] In a preferred embodiment, a degron tag and / or hybrid zinc finger polypeptide according to the present invention is provided, wherein the first hybrid zinc finger domain comprising two substitutions is selected from those listed in Table 5. Table 5 lists preferred first hybrid zinc finger domains (or hybrid zinc finger polypeptides) according to the present invention. SEQ ID NO: 135 is also a preferred first hybrid zinc finger domain (or hybrid zinc finger polypeptide) according to the present invention. SEQ ID NO: 136 is included in a preferred degron tag according to the present invention. These preferred zinc finger domains show the most beneficial EI index (enrichment index) calculated according to the Examples section. The results are summarized in Table 6.

[0157] [Table 5]

[0158] [Table 6-1] [Table 6-2] [Table 6-3]

[0159] Indeed, it has been surprisingly found that at least two substitutions in the first hybrid zinc finger domain described herein provide a degron tag and / or hybrid zinc finger polypeptide according to the invention with enhanced or increased IMiD sensitivity, in particular, compared to, for example, those comprising only one substitution in the first hybrid zinc finger domain. For example, when the first hybrid zinc finger domain used in the examples comprises 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 (at least 5 times higher compared to SEQ ID NO: 100) (=indicating a degron tag and / or hybrid zinc finger polypeptide with reduced sensitivity to IMiDs). This therefore shows that, despite the introduction of a second or more, for example a third or fourth amino acid substitution in the first hybrid zinc finger domain, the sensitivity of the resulting first hybrid zinc finger domain (with at least two substitutions) is surprisingly and dramatically improved. Exemplary EI values ​​obtained with various single mutations in the first hybrid single domain used in the Examples herein are shown in Table 7.

[0160] [Table 7]

[0161] In preferred embodiments, a degron tag and / or hybrid zinc finger polypeptide according to the invention comprises a first hybrid zinc finger domain comprising any of the sequences listed in Table 5, however, it is also contemplated that the first hybrid zinc finger domain of a degron tag and / or hybrid zinc finger polypeptide according to the invention comprises any of the beta hairpins of the first hybrid zinc finger domains listed in Table 5 or any of the alpha helices of the first hybrid zinc finger domains listed in Table 5, each further comprising a different alpha helix and beta hairpin portion. For example, in preferred embodiments, it is contemplated that the first hybrid zinc finger domain is comprised of a beta hairpin portion of any one of the sequences listed in Table 5 in combination with an alpha helix portion of any one of the sequences listed in Table 5. In some embodiments, the first hybrid zinc finger domain is comprised of a beta hairpin portion of any one of the sequences listed in Table 5 in combination with an additional alpha helix portion not necessarily listed in Table 5. In some embodiments, the first hybrid zinc finger domain is composed of a beta hairpin portion, not necessarily listed in Table 5, in combination with an alpha helix portion of any one of the sequences listed in Table 5.

[0162] In some embodiments, the first hybrid zinc finger domain of a degron tag and / or zinc finger polypeptide according to the present invention is any one of those listed in Table 5, preferably any one of SEQ ID NOs: 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, or SEQ ID NO: 135. In another embodiment, the first hybrid zinc finger domain of a degron tag and / or zinc finger polypeptide according to the present invention is any one of SEQ ID NOs: 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, or 91-100, or SEQ ID NO: 135. In further embodiments, the first hybrid zinc finger domain (ZFP91 ZF4-IKZF1 ZF2) shown in FIG.

[0163] Although the present invention does not require the inclusion of a second zinc finger domain or a second hybrid zinc finger domain in a degron tag and / or zinc finger polypeptide according to the invention (as described below), it has surprisingly been found that the sensitivity of a degron tag and / or zinc finger polypeptide according to the invention can be further enhanced or increased by combining a first hybrid zinc finger domain (e.g., having at least two substitutions) with a further second zinc finger domain, preferably a second hybrid zinc finger domain as described herein. A variety of second zinc finger domains and / or second hybrid zinc finger domains may be used (as discussed elsewhere herein; e.g., IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, ZNF692 ZF3, ZNF71 ZF5 ... ZF5 (see also Uniprot accession numbers Q13422, Q9UKT9, Q96JP5, Q96CK0, Q8N554, Q17R98, Q9BU19, respectively), in a preferred embodiment a degron tag and / or hybrid zinc finger polypeptide according to the invention is provided, wherein a third portion of the second hybrid zinc finger domain comprises the amino acid sequence set forth in SEQ ID NO: 104 (FKCHLCNYACRR), a fourth portion of the second hybrid zinc finger domain comprises the amino acid sequence set forth in SEQ ID NO: 105 (KDSVVAHKAKSH), and / or the second hybrid zinc finger comprises the amino acid sequence set forth in SEQ ID NO: 106 (FKCHLCNYACRRKDSVVAHKAKSH). In a preferred embodiment of the second hybrid zinc finger, the second hybrid zinc finger is composed of a beta hairpin portion of IKZF1 ZF3 and an alpha helix portion of ZFP91 ZF5.

[0164] According to another aspect of the invention, the first and / or second portions of the first hybrid zinc finger domain are derived from a first and / or a second Cys2-His2 zinc finger domain, which are IMiD substrates in a naturally occurring protein having said first and / or second Cys2-His2 zinc finger domain. Preferably, the first and second portions of the first hybrid zinc finger domain are derived from a first and a second Cys2-His2 zinc finger domain, which are both IMiD substrates in a naturally occurring protein having said first or second Cys2-His2 zinc finger domain.

[0165] According to another aspect of the invention, the first and / or second part of the first hybrid zinc finger domain is preferably derived from a first and / or a second Cys2-His2 zinc finger domain, which is an IMiD substrate in a naturally occurring protein having said first and / or second Cys2-His2 zinc finger domain. Preferably, the first and second parts of the first hybrid zinc finger domain are derived from a first and a second Cys2-His2 zinc finger domain, which are both IMiD substrates in a naturally occurring protein having said first or second Cys2-His2 zinc finger domain. According to the invention, and according to this aspect, in preferred embodiments, the third and / or fourth portion of the second (hybrid) zinc finger domain ("(hybrid) zinc finger domain" refers herein to both options, i.e., either the second hybrid zinc finger domain or the second zinc finger domain) is a third Cys2-His2 zinc finger domain and / or a fourth Cys2-His2 zinc finger domain that is N-terminal or C-terminal, preferably C-terminal, to a Cys2-His2 zinc finger domain that is an IMiD substrate in a naturally occurring protein. In some embodiments, the third and / or fourth portion are derived from a Cys2-His2 zinc finger domain that is not an IMiD substrate in a naturally occurring protein that has said zinc finger domain.

[0166] Finally, as already discussed herein in relation to a linker peptide between a first hybrid zinc finger domain and a second hybrid zinc finger domain, there is provided a degron tag and / or hybrid zinc finger polypeptide according to the invention, wherein the first hybrid zinc finger domain and the second zinc finger domain, preferably the second hybrid zinc finger domain, are adjacent or connected via a linker peptide comprising, for example, 1, 2, 3, 4, 5, 6, 7 or more amino acids, however in some embodiments no additional linker peptide is present.

[0167] According to another aspect, there is provided a degron tag according to the invention comprising a first hybrid zinc finger domain, further comprising at least two amino acid substitutions, and comprising a second hybrid zinc finger domain. According to this aspect of the invention, there is also provided a non-natural hybrid zinc finger polypeptide comprising a first hybrid zinc finger domain comprising a first portion and a second portion, (1) the first portion is the amino acid sequence of the first Cys2-His2 zinc finger domain, X1X2C3X4X5C6X7X8X9X 10 X 11 wherein X represents any amino acid; (2) the second portion comprises the amino acid sequence X of a second Cys2-His2 zinc finger domain; 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 wherein X represents any amino acid, and the second Cys2-His2 zinc finger domain is distinct from the first Cys2-His2 zinc finger domain and (3) the second portion is C-terminal to the first portion; (4) the first and second portions together comprise at least two amino acid substitutions, the amino acid substitutions in the first portion being relative to a first portion of a first Cys2-His2 zinc finger domain and the amino acid substitutions in the second portion being relative to a second portion of a second Cys2-His2 zinc finger domain, the substitutions being at positions C3, C6, X7, H 19 , or H 23 does not exist in either of the above.

[0168] Although the experiments show the presence of a second zinc finger domain or a second hybrid zinc finger domain in the degron tag and / or zinc finger polypeptide according to the invention, the experiments also reveal that such a second zinc finger domain or a second hybrid zinc finger domain is not necessarily or required to provide enhanced or increased sensitivity to IMiDs. In other words, it has surprisingly been confirmed by the inventors that by providing a hybrid zinc finger polypeptide as described herein, an increased or enhanced sensitivity to IMiDs can also be provided. The hybrid zinc finger polypeptide comprises or consists of a first hybrid zinc finger domain, e.g., as already described in great detail herein in connection with the degron tag according to the invention. It will therefore be understood by the skilled artisan that all the aspects, preferences and features detailed herein (e.g., size, position and type of substitution (e.g., Tables 1 to 6), sequence, linker, etc.) also apply to this aspect of the invention and there is no need to repeat them in full detail.

[0169] In preferred embodiments of the invention, the hybrid zinc finger polypeptide comprises any one of the sequences listed in Table 5. In some embodiments, the hybrid zinc finger polypeptide according to the invention comprises any one of the sequences listed in Table 5, preferably any one of SEQ ID NOs: 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, or 135. In another embodiment, the zinc finger polypeptide according to the invention comprises any one of SEQ ID NOs: 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, or 91-100, or 135.

[0170] In an embodiment, a non-natural hybrid zinc finger polypeptide according to the invention is provided, the non-natural hybrid zinc finger comprising a first hybrid zinc finger domain as defined herein.

[0171] Also provided are non-natural hybrid zinc finger polypeptides according to the invention further comprising a second zinc finger domain, wherein the second zinc finger domain is a non-hybrid zinc finger domain. As described herein, it has been established that, although it may be beneficial to include a second zinc finger domain in a hybrid zinc finger polypeptide according to the invention, such a second zinc finger domain is not required to provide the observed enhanced or increased sensitivity to IMiDs. Furthermore, in some embodiments, a non-natural hybrid zinc finger polypeptide according to the invention further comprises 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, e.g., IKZF1 ZF3, IKZF3 ZF3, as well as ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, and / or ZNF827 ZF2, ZNF692 ZF5.

[0172] The second zinc finger domain is preferably oriented C-terminal to the first hybrid zinc finger domain and may be directly adjacent to or linked to the first hybrid zinc finger domain, as described elsewhere herein.

[0173] In some embodiments, the second zinc finger domain is a second hybrid zinc finger domain, such as those described elsewhere herein. In embodiments in which the second zinc finger domain in the hybrid zinc finger domain according to the present invention is a non-hybrid, e.g., a wild-type or naturally occurring zinc finger domain, or a mutant thereof, the second zinc finger domain may preferably be selected from, for example, IKZF1 ZF3 (SEQ ID NO: 109 (FKCHLCNYACRRRDALTGHLRTH) or IKZF3 ZF3 (SEQ ID NO: 110 (FKCHLCNYACQRRDALTGHLRTH) or comprises the amino acid sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110. Further examples of suitable second zinc finger domains include, for example, IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, ZNF692 ZF5, SEQ ID NOs: 109 to 115, etc.

[0174] According to a further aspect, there is provided a fusion protein comprising a degron tag according to the invention or a non-natural hybrid zinc finger polypeptide according to the invention and further comprising a protein of interest. This aspect of the invention relates to a fusion protein comprising a protein of interest (POI) and a degron tag or a hybrid zinc finger polypeptide according to the invention, which is capable of mediating degradation of the POI comprising the fusion protein in the context of the use of an IMiD.

[0175] In some embodiments, the degron tag of the hybrid zinc finger polypeptide may be located at the N-terminus of the POI, the C-terminus of the POI, or within the POI.

[0176] The fusion proteins according to the invention can be used in a method of degrading a protein of interest, comprising contacting a cell expressing the fusion protein with an effective amount of an IMiD. The protein of interest can be any suitable protein (or fragment thereof). In some embodiments, the protein of interest is an endogenous protein. In some embodiments, the protein of interest is an exogenous protein. In some embodiments, the protein of interest is a recombinant protein or a non-natural protein. In some embodiments, the protein of interest is a naturally occurring protein. Thus, the degron tag and / or hybrid zinc finger proteins of the invention can be utilized to generate and stably express, for example, an endogenous protein-degron tag fusion protein or an exogenous protein-degron tag fusion protein in a cell. An endogenous protein originates within an organism, tissue, or cell and is expressed by the same organism, tissue, or cell, whereas an exogenous protein originates from outside the organism, tissue, or cell and is introduced into the organism, tissue, or cell.

[0177] The protein of interest in the fusion protein according to the present invention is not particularly limited to any particular type of protein, but suitable examples of proteins of interest include, for example, Cbl-b, SOCS1, CISH, Tox, Eomes, IL12, and IL15. CBL-B is an E3 ubiquitin-protein ligase encoded by the CBLB gene in humans. SOCS family proteins form part of a classical negative feedback system that regulates cytokine signaling. SOCS1 is involved in the negative regulation of cytokines that signal through the JAK / STAT pathway. It inhibits their kinase activity through binding to JAK and IFNGR1. In vitro, it also suppresses Tec protein-tyrosine activity. Cytokine-inducible SH2-containing (CISH) protein is a protein encoded by the CISH gene in humans, which controls T cell receptor (TCR) signaling and contains an SH2 domain and a SOCS box domain. Thymocyte selection-associated high mobility group box protein TOX is a protein encoded by the TOX gene in humans. TOX drives T cell exhaustion and plays a role in innate lymphoid cell development. Eomesodermin, also known as T box brain protein 2 (Tbr2), is a protein encoded by the EOMES gene in humans. Eomesodermin / Tbr2 is highly expressed in CD8+ T cells but not in CD4+ T cells. Interleukin 12 (IL-12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigenic stimulation. Interleukin-15 (IL-15) is a cytokine that binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection with a virus and induces the proliferation of natural killer cells.

[0178] Other proteins of interest include, for example, chimeric polypeptides as described in WO2021080427, which allow for time- and / or dose-dependent modulation of T cell activity as a result of signaling through a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR), and time- and / or dose-dependent modulation of NK cell activity as a result of signaling through a NK cell receptor (NKR) and / or a chimeric antigen receptor (CAR). The chimeric polypeptides described therein are designed to interact with phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR / CD3 complex and / or the CAR and / or the NK cell receptor (NKR) complex, including signaling molecules with ITAMs, such as DAP12, the gamma chain of the immunoglobulin receptor FceRI, or the CD3 zeta chain (Lanier et al., Nat Immunol. 2008 May;9(5):495-502).

[0179] The chimeric polypeptides of WO2021080427 provide tight regulation of T cell and NK cell activity (e.g., cytotoxic activity and / or cytokine secretion) due to the presence of a small molecule regulatory protein stability domain that is utilized to regulate (e.g., decrease or increase) the expression of the chimeric polypeptide in a time- and / or dose-dependent manner. In a preferred embodiment of the invention, this / small molecule regulatory protein stability domain is a degron tag and / or a hybrid zinc finger polypeptide according to the invention. The protein of interest comprises a first portion comprising an SH2 domain from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), as disclosed in detail in WO2021080427, and preferably also comprises a second portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM). This protein is preferably combined with a third portion comprising a small molecule regulatory protein stability domain, which according to the invention is a degron tag or a hybrid zinc finger polypeptide as disclosed herein. In some embodiments, the ITAM is an ITAM contained in a T cell receptor (TCR) complex and / or an NK cell receptor (NKR) complex and / or a chimeric antigen receptor (CAR), preferably an ITAM derived from the CD3 zeta chain, CD3 epsilon chain, CD3 delta chain, CD3 gamma chain, gamma chain of the immunoglobulin receptor FceRI and DAP12. In some embodiments, the SH2 domain is derived from a protein selected from the group consisting of Zap70, Syk, and Lck. In some embodiments, the chimeric polypeptide comprises two or more SH2 domains derived from a protein that binds to phosphorylated immunoreceptor tyrosine-based activation motifs. In some embodiments, the ITIM and / or ITSM is derived from an inhibitory receptor protein, preferably an inhibitory immunoreceptor protein, preferably a protein selected from the group consisting of PD1, BTLA, SIRP alpha, SIGLEC5, SIGLEC9, SIGLEC11, PECAM1 or LY9.

[0180] Also provided is a non-naturally occurring nucleic acid encoding a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, or a fusion protein according to the invention.

[0181] Vectors containing such non-naturally occurring nucleic acids are also provided.

[0182] Also provided is a cell or host cell expressing a non-naturally occurring nucleic acid according to the invention. Preferably, the cell, host cell, protein of interest, and / or other sequences in the Cys2-His2 zinc finger domain or zinc finger domain, as well as the degron tag and / or hybrid zinc finger polynucleotide according to the invention, are human or of human origin or consist essentially of human sequences.

[0183] According to another aspect of the invention there is provided a method of providing a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, a fusion protein according to the invention, or a nucleic acid encoding same, the method comprising the steps of: (A) providing a hybrid zinc finger domain comprising a first portion and a second portion, (1) the first portion is the amino acid sequence of the first Cys2-His2 zinc finger domain, X1X2C3X4X5C6X7X8X9X 10 X 11 wherein X represents any amino acid; (2) the second portion comprises the amino acid sequence X of a second Cys2-His2 zinc finger domain; 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23wherein X represents any amino acid, and the second Cys2-His2 zinc finger domain is distinct from the first Cys2-His2 zinc finger domain and (3) the second portion is C-terminal to the first portion; and (B) introducing at least two different amino acid substitutions into the hybrid zinc finger domain, the substitutions being at positions C3, C6, X7, H 19 , or H 23 and (C) using the hybrid zinc finger domain obtained in step (B) or using a nucleic acid sequence encoding the hybrid zinc finger domain obtained in step (B) in preparing a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, a fusion protein according to the invention, or a nucleic acid encoding any of the above.

[0184] The skilled artisan is well aware of methods that make it possible to prepare the degron tag according to the invention, the non-natural hybrid zinc finger polypeptide according to the invention, the fusion protein according to the invention, or the nucleic acid encoding same, such methods being readily available in the prior art and including, for example, those described in the examples.

[0185] Those of skill in the art will also understand that the same considerations, features, and preferences described elsewhere herein apply with respect to the first portion, the second portion, and with respect to the first and second Cys2-His2 zinc finger domains.

[0186] The skilled artisan will also understand that with respect to the at least two different amino acid substitutions introduced in the methods of the present invention, this can be at any position in the hybrid zinc finger domain comprising the first and second portions, and the substitution can be to any amino acid (as long as the substituted amino acid is different from the replacing amino acid). The skilled artisan will also understand that in preferred embodiments, the positions, substituted amino acids, and / or replacement amino acids are described in relation to any one of Tables 1-6 and 8, including combinations of at least two substitutions, and the preferences indicated.

[0187] The method may comprise the step of testing a hybrid zinc finger domain comprising at least two substitutions obtained by a method of the invention and / or a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, a fusion protein according to the invention, or a nucleic acid encoding the same, for susceptibility to an IMiD, e.g. by determining an EI value or index as described herein, e.g. as described in the Examples.

[0188] Based on the EI value or index, it can be determined whether the resulting hybrid zinc finger domain comprising at least two substitutions and / or the degron tag according to the invention, the non-natural hybrid zinc finger polypeptide according to the invention, the fusion protein according to the invention, or the nucleic acid encoding same should be discarded or not. Thus, in a preferred embodiment, the method comprises the step of analysing the resulting hybrid zinc finger domain comprising at least two substitutions and / or the degron tag according to the invention, the non-natural hybrid zinc finger polypeptide according to the invention, the fusion protein according to the invention, or the nucleic acid encoding same, for susceptibility to an IMiD, e.g. as described in the Examples section, to determine its usefulness as a degron tag or the like.

[0189] Thus, in one embodiment, it is provided to validate the susceptibility of a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, a fusion protein according to the invention, e.g. obtained by a method according to the invention, to immunomodulatory imide drug (IMiD)-induced degradation, preferably the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iberdomide, salts and analogues thereof.

[0190] Thus, the degron tag according to the invention, the non-natural hybrid zinc finger polypeptide according to the invention, the fusion protein according to the invention can be selected for its ability to be induced by a particular small molecule, preferably an immunomodulatory inducer drug (IMiD). In one embodiment, the IMiD is thalidomide or one of its analogues, such as lenalidomide, pomalidomide, avadomide, or iveldmide.

[0191] According to yet another aspect there is provided a method for degrading a protein of interest or a method for controlling expression of a protein of interest comprising contacting a cell in vitro or in vivo with an effective amount of an IMiD, wherein the cell expresses a nucleic acid encoding a degron tag according to the invention, and / or a non-natural hybrid zinc finger polypeptide according to the invention, and / or a fusion protein according to the invention, and preferably the method is for modulating the activity of the protein of interest.

[0192] According to a further aspect, there is provided a method of degrading a protein of interest or a method of controlling expression of a protein of interest comprising administering an effective amount of an IMiD to a subject, preferably a human subject, where the subject has previously been treated via gene therapy, causing at least some cells in the subject to express a nucleic acid encoding a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, or preferably a fusion protein according to the invention comprising a protein of interest. In a preferred embodiment, there is provided a method, wherein the gene therapy comprises introducing into the subject cells (e.g. T cells, B cells or NK cells), the introduced cells expressing a nucleic acid encoding a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, or a fusion protein according to the invention, e.g. comprising a protein of interest.

[0193] These aspects of the invention relate to methods of degrading a protein of interest, the methods comprising, for example, contacting a transgenic cell with an effective amount of an IMiD, where the cell produces a fusion protein, for example comprising the protein of interest and at least one degron tag and / or hybrid zinc finger polynucleotide according to the invention.

[0194] The method can be performed in vivo or in vitro. The POI can be exogenous or endogenous. The ability to degrade a specific endogenous or exogenous protein of interest by creating a POI-degron tag fusion and administering an IMiD can be used to treat disorders by controlling cell behavior, for example as described in WO2021080427.

[0195] Thus, the degron tags, hybrid zinc finger polynucleotides and fusion proteins of the invention can be utilized to produce stably expressed endogenous or exogenous protein-degron tag fusion proteins, the levels or cellular activity of which can be regulated by providing an IMiD to or removing an IMiD from cells expressing such fusion proteins.

[0196] There is also provided the use of a nucleic acid encoding a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, or a fusion protein according to the invention in the treatment of a subject, preferably a human subject. The treatment of a patient in need may be as described herein or may be any other treatment.

[0197] Thus, they also provide the use of a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, a fusion protein according to the invention, or a nucleic acid encoding any of these, in the control of transcription factors such as Eomes, Tox, negative regulators of TCR signalling such as Cbl-b, negative regulators of cytokine receptors such as SOCS1, CISH, membrane bound cytokines such as IL12, IL15, antigen receptors such as TCR, CAR, NKR, checkpoint receptors such as PD1, LAG3, TIM3, nucleases such as Cas9, TALEN, or zinc finger nucleases.

[0198] Also provided is a first hybrid zinc finger domain and / or a second hybrid zinc finger domain disclosed herein.

[0199] Also provided is the use of a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, a fusion protein according to the invention, a first hybrid zinc finger domain according to the invention, a second hybrid zinc finger domain according to the invention, or a nucleic acid encoding any of these, as a medicament, preferably in combination with the use of an IMiD as a medicament. Pharmaceutical compositions comprising a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, a fusion protein according to the invention, a first hybrid zinc finger domain according to the invention, a second hybrid zinc finger domain according to the invention, or a nucleic acid encoding any of these, including in combination with an IMiD, are also provided.

[0200] In one aspect, a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, or a fusion protein according to the invention has enhanced or increased sensitivity to an IMiD molecule, e.g., a thalidomide analog, e.g., compared to a wild-type zinc finger domain.

[0201] In one aspect, a degron tag according to the invention, a non-natural hybrid zinc finger polypeptide according to the invention, or a fusion protein according to the invention has enhanced or increased sensitivity to one or more IMiD molecules compared to the wild-type Cys2-His2 zinc finger domain from which the beta hairpin and / or alpha helix subdomain is derived.

[0202] In one embodiment, enhanced or increased susceptibility to one or more IMiD molecules allows for a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more reduction in the amount of IMiD molecule administered to induce degradation. In one aspect, the amount of small molecule, e.g., IMiD molecule, administered is reduced by 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 110, 120, 130, 140, 150 fold or more.

[0203] The foregoing description of specific embodiments fully discloses the general nature of the invention so that others can readily modify and / or adapt such specific embodiments to various applications without departing from the general concept of the invention by applying knowledge within the skill of the art (including the contents of the references cited herein). Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0204] All references cited herein (including journal articles or abstracts, published or corresponding patent applications, patents, or any other references), including all data, tables, figures, and text presented in the cited references, are incorporated herein by reference in their entirety. In addition, the entire contents of the references cited within the references cited herein are also incorporated by reference in their entirety.

[0205] It is to be understood that the phrases or terms used herein are for purposes of description and not of limitation, and thus should be interpreted by one of ordinary skill in the art in light of the teachings and guidance presented herein, in combination with the knowledge of those skilled in the art.

[0206] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of an embodiment of the invention are equally applicable to any other aspect or embodiment of the invention, and therefore it is not necessary to separately detail all such details, embodiments, and preferences for every aspect.

[0207] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. Further aspects and embodiments will be apparent to those skilled in the art. EXAMPLES

[0208] Due to concerns related to the use of IMiDs at higher doses, the identification of novel zinc finger degron tag sequences that can be regulated at lower drug doses is desirable. In a previous study (Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572), a hybrid zinc finger sequence was described that combines a beta hairpin and an alpha helix from two different zinc finger degrons and shows increased susceptibility to IMiD-induced degradation compared to the parent zinc finger degron.

[0209] Specifically, a hybrid zinc finger degron comprising a ZFP91 ZF4 beta hairpin and an IKZF1 ZF2 alpha helix was found to exhibit increased susceptibility to IMiD-induced degradation compared to non-hybrid ZFP91 ZF4 and IKZF1 ZF2 zinc finger degrons. The inventors previously generated a ZFP91 ZF4 beta hairpin IKZF1 ZF2 alpha helix IKZF1 ZF3 degron (abbreviated as single-hybrid, dual zinc finger prior art degron, FIG. 1) and demonstrated that this CRASH-IT switch design comprising a single-hybrid, dual zinc finger prior art degron domain can be regulated by an IMiD molecule (WO2021080427). Here, we identify novel zinc finger-based degron domains that contain modifications in the first and preferably the second zinc finger motifs of the degron tag and show significantly improved response to lower drug concentrations. Based on these data, we propose these novel degron domains, called synthetic zinc fingers (SynFingers), as optimal building blocks for controlling the abundance of proteins of interest.

[0210] Example 1 Materials and Methods Cell lines and cell culture FLYRD18 (Sigma-Aldrich), NKIRTIL006 (Kvistborg et al. Oncoimmunology. 2012 Jul 1;1(4):409-418) and Jurkat cells were cultured in IMDM (ThermoFisher) supplemented with 8% FCS (ThermoFisher) and penicillin-streptomycin (100 IU / mL penicillin, 100 μg / mL streptomycin, Sigma-Aldrich). FLYRD18 and NKIRTIL006 cells were passaged every 2-3 days using trypsin-EDTA (ThermoFisher). All cell lines were tested for mycoplasma using a PCR-based screen and found to be negative.

[0211] Retrovirus production Retroviral particles were produced in FLYRD18 packaging cells. Briefly, 700,000 FLYRD18 packaging cells were seeded per 10 cm dish one day before transfection. The next day, the cell culture medium was refreshed with IMDM supplemented with 8% FCS without antibiotics. 25 μl of X-tremeGENE 9 (Roche) was mixed with 800 μl of Opti-MEM (ThermoFisher) and incubated for 5 min. The Optimem-X-tremeGENE 9 mixture was then added on top of 10 μg of retroviral plasmid DNA dissolved in water, incubated for 15 min, and the resulting transfection mixture was added dropwise to the packaging cells. The retrovirus-containing supernatant was harvested 48 h after transfection and either used immediately or flash frozen in liquid nitrogen.

[0212] Plasmids Retroviral vectors encoding a single-hybrid, dual zinc finger prior art degron (pMP71-Zap70 2xSH2-PD1-ZFP91 ZF4 beta hairpin IKZF1 ZF2 alpha helix-IKZF1 ZF3 iresEGFP, WO 2021080427(A1)), a CRASH-IT variant containing a single-hybrid, single zinc finger degron (pMP71-Zap70 2xSH2-PD1-(ZFP91 ZF4 beta hairpin-IKZF1 ZF2 alpha helix iresEGFP, WO 2021080427(A1)), and an HLA class I restricted CDK4 TCR (TCR 17, Stronen et al, Science. 2016 Jun 10;352(6291):1337-41) have been previously described.

[0213] The IKZF1 ZF3 alpha helix coding sequence in the pMP71-Zap70 2xSH2-PD1-ZFP91 ZF4 beta hairpin-IKZF1 ZF2 alpha helix-IKZF1 ZF3 iresEGFP vector was replaced by a gene synthesis product (IDT, Iowa, USA) encoding the ZFP91 ZF5 alpha helix sequence using the Gibson assembly method (Gibson et al. Nat Methods. 2009 May;6(5):343-5). The resulting degron is named "double hybrid degron" (Figure 3).

[0214] A high diversity SynFinger library (approximately 50200 mutants) was synthesized by Twist Biosciences (CA, USA) using the Combinatorial Mutant Library (CVL) service as described in Figure 3. pMP71-Zap70-Siglec11-FKBP12F36V iresEGFP was previously described (Sahillioglu et al. Hum Gene Ther. 2021 Oct;32(19-20):1029-1043). The FKBP12F36V coding sequence in this vector was replaced by a gene synthesis product encoding a double hybrid degron (Figure 3) or a double hybrid degron mutant containing the G14N / K21A, G14M / N15R, L17I / K21L, E4R / Q12L, Q12R / K1T or Q12R / K13V double substitutions, or the Q12R / K13V / G14N / K21A quadruple substitution (SEQ ID NO:135; SEQ ID NO:136) using the Gibson assembly method.

[0215] screening Quality control of the SynFinger library was performed by Twist Biosciences, and all but two SynFinger sequences out of approximately 50200 synthetic sequences as described in Figure 3 were observed in the cloned library. 150 billion Jurkat cells were transduced with the SynFinger library (SynFingers containing CRASH-IT switches) at a transduction rate of 12.8% and selected for high EGFP expression to enrich for transduced cells. For quality control of SynFinger-transduced Jurkat cells, SynFinger sequences were amplified and analyzed by NGS. Ten randomly selected SynFinger-encoding DNA sequences, or DNA sequences encoding the same SynFinger amino acid sequence but containing two nucleotide mismatches due to alternative codon usage, were used to evaluate the ability to distinguish true SynFinger sequences from noise in the NGS data. 10 / 10 SynFingers encoding perfect nucleotide sequence matches were observed within the NGS data, but no 2-nucleotide mismatch-containing sequences were detected.

[0216] Jurkat cells expressing the SynFinger library were treated with 50 nM lenalidomide (a suboptimal dose that activates only about 50% of cells expressing the CRASH-IT switch containing the parent double hybrid degron in Jurkat cells compared to the maximally effective 1000 nM lenalidomide dose) for 24 hours or mock treated, and then activated with anti-CD3 / anti-CD28 antibodies for 5 hours in the presence or absence of 50 nM lenalidomide. Cells were then stained with IR dye (1:400) and anti-CD69-PE (1:200). Cells were sorted for viability (IR dye negative), EGFP-high terminal, CD69-high (top 5%) or CD69-low (bottom 5%) expression using an Aria Fusion cytometer.

[0217] Subsequently, cells were washed with PBS and cell pellets were frozen at -20°C until DNA extraction. Genomic DNA was isolated using 5x10^6 cells per column using the DNeasy Blood&Tissue Kit (Qiagen) according to the manufacturer's instructions. DNA was eluted in 22μl RNAse-free water. Deep sequencing adapters and indexes were added to the SynFinger coding DNA sequences using PCR amplification. Briefly, 3μg genomic DNA (20μl), indexed forward and reverse primers (10μM, 2.5μl each) and 25μl NEBNext® High-Fidelity 2X PCR Master Mix were added to the PCR reaction. Optimized PCR reactions (98°C for 30 seconds, (98°C for 10 seconds, 56°C for 10 seconds, 72°C for 5 seconds) x 25 cycles, 72°C for 3 minutes) were performed using an Eppendorf PCR cycler.

[0218] Indexed PCR products were run on an agarose gel, relative DNA concentrations were estimated by measuring band intensity, and 12 indexed PCR reaction products were mixed per MiSeq chip. To increase sequence diversity, samples were mixed with 25% PhiX DNA. In total, two MiSeq chips were used (100 bp, single read). To distinguish bona fide SynFinger sequences from sequencing errors, the results were filtered using a reference list of actual sequences used in the SynFinger library design (100% codon match). Sequences with very low read counts (<500 reads in total across all samples) were excluded from the analysis.

[0219] To identify SynFingers that improve the sensitivity of the CRASH-IT Switch to lenalidomide while preserving or improving the stringency of switch-mediated control of T cell activation in the absence of lenalidomide, an enrichment index (EI) value was calculated for each sequence as described below, and SynFingers were ranked according to their EI, with a lower EI indicating a SynFinger with the desired properties.

number

[0220] CD69-HI and CD60-LO read counts from the top 100 SynFingers were compared to read counts from the parent double hybrid degron using a two-tailed Fisher's exact test to determine enrichment of CD69-HI cells in the presence of lenalidomide and enrichment of CD69-LO cells in the absence of lenalidomide. Results for SEQ ID NOs: 1-100 are shown in Tables 6 and 9.

[0221] [Table 8-1] [Table 8-2]

[0222] Example 2 result To our knowledge, the possibility of using a hybrid zinc finger as the second zinc finger as a means to improve the overall sensitivity of the dual zinc finger degron system has not been previously tested. To investigate whether the second zinc finger (IKZF1 ZF3) of a single hybrid, dual zinc finger prior art degron could be replaced with other zinc finger sequences while preserving or improving sensitivity to IMiDs, we generated a panel of CRASH-IT switches containing the zinc finger degrons shown in Figure 1. Primary human T cells were transduced to express CDK4 neoantigen-specific MHC class I-restricted TCRs together with the CRASH-IT variants encoding the indicated zinc finger degrons. Analysis of cytokine production (IFNγ, IL2, and TNFα) and T cell degranulation (LAMP-1 surface expression) upon coculture with NKIRTIL006 cells, which endogenously express the CDK4 neoantigen, revealed that the double-hybrid degron improved the sensitivity of the CRASH-IT switch to thalidomide compared with a CRASH-IT variant containing a single-hybrid, dual zinc finger prior art degron sequence ( Fig. 1 ).

[0223] Consistent with previous data, CRASH-IT switches containing a single-hybrid, single-zinc-finger degron showed reduced thalidomide susceptibility compared to single-hybrid, dual-zinc-finger prior art degron sequences (Figure 1).

[0224] To further improve the sensitivity of the double hybrid degron to IMiDs, we performed a high-throughput screen using a library of about 50200 CRASH-IT mutants encoding either single or double amino acid substitutions at the indicated positions of the first hybrid zinc finger sequence, thereby changing 18 amino acids of this zinc finger to any amino acid except cysteine ​​(Figure 3). Jurkat T cells expressing this synthetic zinc finger (SynFinger) containing the CRASH-IT mutant library were treated with low dose (50 nM) lenalidomide or left untreated for 24 hours, and then stimulated with aCD3 / aCD28 antibodies for 5 hours in the continued presence or absence of lenalidomide. 5% of Jurkat cells with the highest or lowest expression of the T cell activation marker CD69 were then isolated by FACS, and the SynFinger sequences present in these populations were amplified and analyzed by NGS. Analysis of the relative abundance of SynFinger sequences in CD69-HI and CD69-LO populations identified amino acid substitutions within zinc finger degrons that improve sensitivity to IMiDs. Enrichment index (EI) values, reflecting both high expression of CD69 in the presence of lenalidomide and low expression of CD69 in the absence of lenalidomide, were calculated for each SynFinger (see Methods). SynFingers were then ranked according to EI values ​​to identify novel zinc finger degrons with desirable properties such as improved sensitivity to IMiDs and efficient switch function (hence stable POI expression) in the absence of IMiDs.

[0225] The top 100 SynFingers identified by this method were all double amino acid substitutions (Table 6). Importantly, certain amino acid substitutions such as Q12R, Q12K, N15R and L22R (present in 11%, 8%, 9% and 6% of the top 100 SynFingers, respectively) were frequently observed among the top 100 SynFingers containing double substitution mutations (Figure 4). Notably, these mutations also ranked among the top hits when analyzed as single amino acid substitutions. Furthermore, the frequently observed primary amino acid substitutions Q12R, Q12K, N15R and L22R further synergistically increased susceptibility to IMiDs when combined with the secondary amino acid substitutions listed in Table 8 (i.e., EI Q12R / K13V=0.006877267, whereas EI Q12R=0.167217557). Thus, in particularly preferred embodiments of the invention, combinations of amino acid substitutions as set out in Table 8 are used / provided, such as 12R and 13T or 22R and 14L.

[0226] [Table 9]

[0227] To test whether the SynFingers showing improved drug sensitivity in this Jurkat SynFinger library screen also confer improved sensitivity to low IMiD concentrations when expressed in primary human T cells, human peripheral blood T cells were engineered to express the indicated SynFingers containing the CDK4 TCR and CRASH-IT switch mutants (Figure 5). Importantly, T cells expressing the G14N / K21A, G14M / N15R, L17I / K21L, E4R / Q12L, Q12R / K1T and Q12R / K13V double substitutions containing SynFingers showed improved cytokine production at very low (5 nM) lenalidomide concentration levels compared to T cells expressing the double hybrid degron containing the CRASH-IT switch that formed the starting point for the genetic screen (Figure 5). Furthermore, small molecule titration experiments revealed that the Q12R / K13V double substitution containing SynFinger exhibited 5.3-7.4-fold lower EC50 values ​​compared to the single-hybrid, dual zinc finger prior art degron containing the CRASH-IT switch for all T cell effector functions tested (Figure 6).

[0228] Upon identifying two sets of amino acid substitutions, such as Q12R / K13V and G14N / K21A, that improve the lenalidomide sensitivity of SynFinger degrons, we hypothesized that combinations of these sets might further increase drug sensitivity. To test this, we generated CRASH-IT switch mutants that contain the parent ZF or SynFinger degrons with double (Q12R / K13V or G14N / K21A) or quadruple (Q12R / K13V / G14N / K21A; SEQ ID NO: 135 and SEQ ID NO: 136) substitutions. Primary human T cells were transduced to express the CDK4 TCR together with the indicated CRASH-IT mutants. Analysis of cytokine production (IFNγ, IL2, and TNFα) and T cell degranulation (LAMP-1 surface expression) upon coculture with NKIRTIL006 cells, which endogenously express the CDK4 neoantigen, revealed that the combination of Q12R / K13V / G14N / K21A substitutions improved the sensitivity of SynFinger to lenalidomide compared to SynFinger degrons containing Q12R / K13V or G14N / K21A (Figure 7).

[0229] Thus, these data identify specific positions and mutations in zinc finger sequences that result in improved substrates for regulation by IMiDs, and also specific combinations of amino acid mutations that result in such improved substrates. Note that while the SynFinger validation experiments (Figures 5 and 6) used Siglec11 signaling domain containing CRASH-IT mutants, the high-throughput SynFinger library screens (Figures 3 and 4) demonstrated that the CRASH-IT mutants contained the PD1 signaling domain, and that optimized SynFingers improved control over cellular function independent of the POI effector domain used.

[0230] The synthetic zinc finger (SynFinger) sequences we identified in this study can be used in a range of applications beyond their use in the CRASH-IT Switch platform. For example, key regulators of cell function, such as receptors that determine the antigen specificity of immune cells, but also regulators that control antigen sensitivity, cytokine signaling, or cell differentiation, can be regulated by creating fusion proteins with such novel SynFinger sequences. The ability to induce protein degradation with reduced IMiD levels opens new possibilities for implementing this protein degradation technology in clinical settings, both in cell therapy and beyond.

Claims

1. 1. A degron tag comprising a first non-naturally occurring hybrid zinc finger domain and a second non-naturally occurring hybrid zinc finger domain, (1) the first hybrid zinc finger domain comprises a first portion and a second portion; (2) the first portion comprises a first Cys ２ -His ２ Zinc finger domain amino acid sequence X １ X ２ C ３ X ４ X ５ C ６ X ７ X ８ X ９ X １０ X １１ wherein X represents any amino acid; (3) the second portion is a second Cys ２ -His ２ Zinc finger domain 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 wherein X represents any amino acid, and said second Cys ２ -His ２ The zinc finger domain comprises the first Cys ２ -His ２ Unlike zinc finger domains, (4) the second portion is C-terminal to the first portion; (5) the second hybrid zinc finger domain is C-terminal to the first hybrid zinc finger domain; (6) the second hybrid zinc finger domain comprises a third portion and a fourth portion; (7) The third portion comprises a third Cys ２ -His ２ Amino acid sequence of zinc finger domain (Z) ２ C(Z) ２ C(Z) ５－６ wherein Z represents any amino acid; (8) The fourth second portion is a fourth Cys ２ -His ２ Amino acid sequence of zinc finger domain (Z) ６ H(Z) ３－４ H, wherein Z represents any amino acid, and the fourth Cys ２ -His ２ The zinc finger domain may comprise the third Cys ２ -His ２ Unlike zinc finger domains, (9) the fourth portion is C-terminal to the third portion; (10) The first hybrid zinc finger domain comprises at least two amino acid substitutions, the amino acid substitutions in the first portion of the first hybrid zinc finger domain being at least one of the first Cys ２ -His ２ the amino acid substitution in the second portion of the first hybrid zinc finger domain is to the second Cys ２ -His ２ to the second portion of the zinc finger domain, wherein the substitution is at position C ３ , C ６ , X ７ , H １９ , or H ２３ The degron tag is not present in any of the above.

2. 2. The degron tag of claim 1, wherein the degron tag is capable of binding to a complex formed between cereblon (CRBN) and an immunomodulatory drug (IMiD).

3. The first portion comprises a first Cys ２ -His ２ a beta hairpin loop of a zinc finger domain, said second portion comprising a second Cys ２ -His ２ the third portion is an alpha helix region of a zinc finger domain, the third portion comprising a third Cys ２ -His ２ a beta hairpin loop of a zinc finger domain, and / or the fourth portion is a fourth Cys ２ -His ２ The degron tag of claim 1 , which is an alpha helix region of a zinc finger domain.

4. The degron tag of claim 1 , wherein a first substitution is in the second portion and a second substitution is in the first portion or the second portion.

5. At least one substitution is X １ , X ４ , X １２ , X １３ , X １４ , X １５ , X １７ , X ２１ , and X ２２ The degron tag of claim 1, which is located at a position selected from the group consisting of:

6. 2. The degron tag of claim 1, wherein at least one substitution is selected from those listed in Table 1, and preferably, the at least two substitutions are each independently selected from those listed in Table 1.

7. The degron tag of claim 1 , wherein the at least two substitutions are selected from those listed in Table 2.

8. 2. The degron tag of claim 1, wherein the amino acids in the first portion that are substituted and / or the amino acids in the second portion that are substituted are selected from those listed in Table 3.

9. 2. The degron tag of claim 1, wherein the at least two amino acids in the first portion and / or the second portion that are substituted are selected from those listed in Table 4.

10. (1) the first Cys ２ -His ２ the 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; (2) the second Cys ２ -His ２ the 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; (3) the third Cys ２ -His ２ the 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 IKZF1 ZF3; and / or (4) the fourth Cys ２ -His ２ 2. The degron tag of claim 1, wherein the 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.

11. 2. The degron tag of claim 1, wherein the at least two amino acid substitutions are introduced into a first hybrid zinc finger domain, a first portion of which has the amino acid sequence set forth in SEQ ID NO: 101 and / or a second portion of which has the amino acid sequence set forth in SEQ ID NO: 102, and / or the first hybrid zinc finger domain has the amino acid sequence set forth in SEQ ID NO:

103.

12. The degron tag of claim 1 , wherein the first hybrid zinc finger domain comprising the at least two substitutions is selected from those listed in Table 5.

13. 2. The degron tag of claim 1 , wherein the third portion of the second hybrid zinc finger domain comprises the amino acid sequence set forth in SEQ ID NO: 104, the fourth portion of the second hybrid zinc finger domain comprises the amino acid sequence set forth in SEQ ID NO: 105, and / or the second hybrid zinc finger comprises the amino acid sequence set forth in SEQ ID NO:

106.

14. The degron tag of claim 1 , wherein the first hybrid zinc finger domain and the second hybrid zinc finger domain are adjacent or connected via a linker peptide.

15. 1. A non-natural hybrid zinc finger polypeptide comprising a first hybrid zinc finger domain comprising a first portion and a second portion, (1) the first portion comprises a first Cys ２ -His ２ Zinc finger domain amino acid sequence X １ X ２ C ３ X ４ X ５ C ６ X ７ X ８ X ９ X １０ X １１ wherein X represents any amino acid; (2) the second portion is a second Cys ２ -His ２ Zinc finger domain 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 wherein X represents any amino acid, and said second Cys ２ -His ２ The zinc finger domain comprises the first Cys ２ -His ２ Unlike zinc finger domains, (3) the second portion is C-terminal to the first portion; (4) the first and second portions together comprise at least two amino acid substitutions, and the amino acid substitution in the first portion is at least one amino acid substitution at the first Cys ２ -His ２ to the first portion of the zinc finger domain, and the amino acid substitution in the second portion is ２ -His ２ to the second portion of the zinc finger domain, wherein the substitution is at position C ３ , C ６ , X ７ , H １９ , or H ２３ A non-natural hybrid zinc finger polypeptide that is not present in any of the above.

16. The non-natural hybrid zinc finger polypeptide of claim 15, wherein the non-natural hybrid zinc finger comprises a first hybrid zinc finger domain of any one of claims 1 to 14.

17. 16. The non-natural hybrid zinc finger polypeptide of claim 15, further comprising a second zinc finger domain, wherein the second zinc finger domain is preferably a non-natural hybrid zinc finger domain selected from IKZF1 ZF3, IKZF3 ZF3, as well as ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, and / or ZNF827 ZF2, ZNF692 ZF5.

18. 16. The non-natural hybrid zinc finger polypeptide of claim 15, wherein the second zinc finger domain is C-terminal to the first hybrid zinc finger domain.

19. 16. The non-natural hybrid zinc finger polypeptide of claim 15, wherein the second zinc finger domain is selected from IKZF1 ZF3, IKZF3 ZF3, and / or the second zinc finger domain comprises the amino acid sequence set forth in SEQ ID NOs: 109-110.

20. 16. A fusion protein comprising the degron tag of claim 1 or the non-natural hybrid zinc finger polypeptide of claim 15, and further comprising a protein of interest.

21. 21. The fusion protein of claim 20, wherein the degron tag or the non-natural hybrid zinc finger polypeptide is located at the N-terminus or C-terminus of the protein of interest.

22. 21. The fusion protein of claim 20, wherein the protein of interest is selected from Cbl-b, SOCS1, CISH, Tox, Eomes, IL12, and IL15.

23. 16. A non-naturally occurring nucleic acid encoding the degron tag of claim 1 or the non-natural hybrid zinc finger polypeptide of claim 15.

24. 24. A vector comprising the non-naturally occurring nucleic acid of claim 23.

25. 25. A cell or host cell that expresses the non-naturally occurring nucleic acid of claim 24.

26. 19. A method for providing a degron tag of claim 1, a non-natural hybrid zinc finger polypeptide of claim 15, or a nucleic acid encoding the same, comprising: (A) providing a hybrid zinc finger domain comprising a first portion and a second portion, (1) the first portion comprises a first Cys ２ -His ２ Zinc finger domain amino acid sequence X １ X ２ C ３ X ４ X ５ C ６ X ７ X ８ X ９ X １０ X １１ wherein X represents any amino acid; (2) the second portion is a second Cys ２ -His ２ Zinc finger domain 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 wherein X represents any amino acid, and said second Cys ２ -His ２ The zinc finger domain comprises the first Cys ２ -His ２ Unlike zinc finger domains, (3) the second portion is C-terminal to the first portion; and (B) introducing at least two different amino acid substitutions into the hybrid zinc finger domain, wherein the substitutions are at positions C ３ , C ６ , X ７ , H １９ , or H ２３ and (C) using the hybrid zinc finger domain obtained in step (B), or using a nucleic acid sequence encoding the hybrid zinc finger domain obtained in step (B) in preparing the degron tag of claim 1, the non-natural hybrid zinc finger polypeptide of claim 15, or a nucleic acid encoding either or both.

27. 27. The method of claim 26, further comprising establishing the susceptibility of the degron tag of claim 1 and the non-natural hybrid zinc finger polypeptide of claim 15 to immunomodulatory imide drug (IMiD)-induced degradation, wherein preferably the IMiD is selected from thalidomide, lenalidomide, pomalidomide, avadomide, iberdomide, salts and analogs thereof.

28. A method for degrading a protein of interest or a method for controlling the expression of a protein of interest, comprising contacting a cell in vitro or in vivo with an effective amount of an IMiD, wherein the cell expresses a nucleic acid encoding a degron tag described in claim 1 and / or a non-natural hybrid zinc finger polypeptide described in claim 15.

29. 29. The method of claim 28, wherein the method is for modulating the activity of the protein of interest.

30. A pharmaceutical composition comprising an immunomodulatory imid drug (IMiD) for degrading or controlling expression of a protein of interest, wherein the subject has previously been treated via gene therapy to express a nucleic acid encoding a degron tag of claim 1 or a non-natural hybrid zinc finger polypeptide of claim 15 in at least some cells in the subject.

31. A pharmaceutical composition comprising an immunomodulatory imid drug (IMiD) for degrading a protein of interest or for controlling expression of a protein of interest, wherein the subject has previously been treated via gene therapy to express a nucleic acid encoding the fusion protein of claim 20 in at least some cells in the subject.

32. The pharmaceutical composition of claim 30, wherein the gene therapy comprises introducing cells into the subject, the introduced cells expressing a nucleic acid encoding the degron tag of claim 1 or the non-natural hybrid zinc finger polypeptide of claim 15.

33. A pharmaceutical composition as described in claim 30, wherein the gene therapy comprises introducing cells into the subject, and the introduced cells express a nucleic acid encoding the fusion protein of claim 20, which contains the target protein.

34. A method for ex vivo regulating transcription factors such as Eomes and Tox, negative regulators of TCR signaling such as Cbl-b, negative regulators of cytokine receptors such as SOCS1 and CISH, membrane-bound cytokines such as IL12 and IL15, antigen receptors such as TCR, CAR, and NKR, checkpoint receptors such as PD1, LAG3, and TIM3, and nucleases such as zinc finger nucleases Cas9 and TALEN, comprising: (1) The degron tag according to claim 1, (2) The non-natural hybrid zinc finger polypeptide of claim 15, or (3) A nucleic acid encoding either (1) or (2). How to use.

35. A method for ex vivo regulating transcription factors such as Eomes and Tox, negative regulators of TCR signaling such as Cbl-b, negative regulators of cytokine receptors such as SOCS1 and CISH, membrane-bound cytokines such as IL12 and IL15, antigen receptors such as TCR, CAR, and NKR, checkpoint receptors such as PD1, LAG3, and TIM3, and nucleases such as zinc finger nucleases Cas9 and TALEN, comprising: (1) The fusion protein according to claim 20, or (2) A nucleic acid encoding (1) How to use.

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