WO2023177296A1 - Zinc finger degron sequences - Google Patents

Zinc finger degron sequences Download PDF

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Publication number
WO2023177296A1
WO2023177296A1 PCT/NL2023/050136 NL2023050136W WO2023177296A1 WO 2023177296 A1 WO2023177296 A1 WO 2023177296A1 NL 2023050136 W NL2023050136 W NL 2023050136W WO 2023177296 A1 WO2023177296 A1 WO 2023177296A1
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Prior art keywords
zinc finger
finger domain
hybrid
amino acid
cys
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English (en)
French (fr)
Inventor
Ali Can SAHILLIOGLU
Antonius Nicolaas Maria Schumacher
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Netherlands Cancer Institute
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Netherlands Cancer Institute
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Application filed by Netherlands Cancer Institute filed Critical Netherlands Cancer Institute
Priority to IL315649A priority Critical patent/IL315649A/en
Priority to CA3255620A priority patent/CA3255620A1/en
Priority to AU2023233426A priority patent/AU2023233426A1/en
Priority to US18/845,540 priority patent/US20250206789A1/en
Priority to CN202380027768.5A priority patent/CN118922547A/zh
Priority to EP23714358.1A priority patent/EP4493696A1/en
Priority to JP2024555386A priority patent/JP2025509853A/ja
Publication of WO2023177296A1 publication Critical patent/WO2023177296A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • This invention pertains in general to novel zinc finger degron sequences wherein the zinc finger degron comprises 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 form a second zinc finger, preferably wherein the first portion comprises a beta- hairpin of a first zinc finger and the second portion comprises an alpha-helix of a second zinc finger.
  • the degron tag comprises a further, second, non-natural hybrid zinc finger domain.
  • the zinc finger domain comprises at least two amino acids substitutions.
  • fusion proteins comprising a degron tag according to the invention and for use of the degron tags according to the invention in methods that involve controlling protein-of- interest levels, cellular activity, expression and the like.
  • a POI can be fused with a degron domain that is inherently unstable and hence prone to proteolysis, and stabilization of the folded protein state by small molecule addition is used to prevent such proteolysis.
  • a POI can also be fused with a regulation domain that contains a constitutively active degron domain, a protease and the corresponding protease cleavage site in the linker between POI and degron tag.
  • the degron domain is proteolytically removed from the POI, thereby, for example, stabilizing POI expression.
  • protease inhibitor degradation of the fusion protein is induced.
  • a preferred protein stability regulator that is used in clinical cell therapy products should be composed of human sequences in order to minimize the risk of immune-mediated rejection, and the immunogenicity of e.g., the SMASh-tag degron system that is 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.
  • HCV hepatitis C virus
  • the small molecule regulators of protein stability control domains should be safe and preferably be clinically approved.
  • the absence of clinical approval of the dTAG-13 PROTAC that is used in the FKBP12F36V-tag degron system complicates the clinical development of FKBP12F36V-based regulation systems.
  • the large size of PROTAC molecules is considered as 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).
  • the small molecule concentration that is sufficient to induce biologically meaningful control over protein levels should preferably be compatible with clinical use, and in particular in the context of long-term small molecule administration, the side effects of such administration may form a concern.
  • the immunomodulatory imide drug (IMiD) inducible zinc finger degron system described by Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572, employs fusion of a POI with a short zinc finger degron tag (sometimes also referred to as zinc finger domain and/or zinc finger polypeptide) that facilitates recruitment of POI-zinc finger fusion degron protein to the IMiD/CRBN E3 ligase complex, e.g., in the presence of the small molecules known as IMiD.
  • IMiD immunomodulatory imide drug
  • IMiDs bind to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase and that CRBN can recruit (fusion)proteins containing a zinc finger degron tag through interaction with the zinc finger degron, which interaction is mediated by the 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.
  • 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.
  • this protein stability control system has demonstrated its value in clinically relevant applications, such as the regulation of 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, we recently reported the use of the IMiD/zinc finger system in the Chemically Regulated and SH2-delivered - Inhibitory Tail (CRASH-IT) switch platform that allows control over cellular activity levels of a variety of cell therapy platforms, such as CAR-T, TCR-T, and NK cells (Sahillioglu et al., W02021080427).
  • CRASH-IT Chemically Regulated and SH2-delivered - Inhibitory Tail
  • FIG. 1 Sensitivity of zinc finger degrons to IMiD can be improved by alteration of the second zinc finger sequence.
  • A Schematic representation of a panel of zinc finger degrons containing the ZFP91 ZF4 beta hairpin and IKZF1 ZF2 alpha helix as a hybrid first zinc finger.
  • the degrons contain second zinc finger sequences 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).
  • Figure 2 Schematic overview of non-limiting embodiments according to the invention.
  • FIG. 3 Design of the synthetic zinc finger (SynFinger) library.
  • the SynFinger library contains Zap70-PD1-degron CRASH-IT switch variants, in which the parental zinc finger degron contains single or dual amino acid substitutions, or no substitutions.
  • the figure depicts the sequence of the double hybrid degron that is used as the parental zinc finger sequence in the SynFinger library screen. Amino acids indicated with arrows are substituted to any of the other genetically encoded amino acids except cysteine. 2 cysteines and a conserved glycine in the ZFP91 ZF4 beta hairpin, as well as 2 histidines in the IKZF1 ZF2 alpha helix, were kept constant. Substitution mutations are numbered starting with the first amino acid of the ZFP91 ZF4beta hairpin (marked with asterisk).
  • FIG. 4 Enrichment of certain amino acid substitutions in the top 100 degrons. SynFinger degrons were ranked according to enrichment index (El) values following removal of SynFingers with low sequence reads (see Examples). The graph depicts the number of times each amino substitution was found in combination with other amino acid substitutions in the top 100 SynFinger degrons. For example, the Q12R mutation was observed in 11 dual amino acid substitution combinations, together with either K13T, L17M, K13V, E4L, L17Y, L22H, C10A, I20R, N15S, E4W and E4Q, amongst the top 100 SynFingers.
  • Figure 5 SynFingers fusion proteins enable sensitive control of immune cell functions.
  • a subset of SynFingers identified in the SynFinger library screen were individually validated.
  • A-H Primary human T cells were modified with the HLA class I restricted CDK4 TCR plus a Zap70-Siglec11-degron CRASH-IT switch, in which the degron sequence contains the parental zinc finger degron (double hybrid degron depicted in Figure 1 and 3), or the parental zinc finger degron with the following mutations: G14N/K21A, G14M/N15R, L17I/K21 L, E4R/Q12L, Q12R/K13T, or Q12R/K13V.
  • As a control primary human T cells modified with the HLA class I restricted CDK4 TCR plus vector control were used.
  • FIG. 6 SynFinger containing CRASH-IT switches enable restoration of cytokine production and degranulation at lower IMiD concentrations as compared to zinc finger degrons not according to the invention.
  • A-D Primary human T cells modified with the HLA class I restricted CDK4 TCR plus a Zap70-Siglec11-degron CRASH-IT switch, in which the degron sequence contains prior art zinc finger degrons (single hybrid, dual zinc finger degron depicted in Figure 1), the parental zinc finger degron (double hybrid degron depicted in Figure 1 and 3), or the parental zinc finger degron with the following mutations: G14N/K21A, L17I/K21 L, or Q12R/K13V, were pretreated with the indicated concentrations of lenalidomide or left untreated.
  • A-D Primary human T cells modified with the HLA class I restricted CDK4 TCR plus a Zap70-Siglec11-degron CRASH-IT switch, in which the degron sequence contains the parental zinc finger degron (double hybrid degron depicted in Figure 1 and 3), or the parental 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.
  • a portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.).
  • copyright protection such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.
  • the copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
  • a degron tag includes a combination of two or degron tags, and the like.
  • a method for administrating a drug or a fusion protein includes the administrating of a plurality of the molecules (e.g., 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).
  • At least a particular value means that particular value or more.
  • at least 2 is understood to be the same as “2 or more” i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc.
  • at most a particular value means that particular value or less.
  • at most 5" is understood to be the same as "5 or less” i.e., 5, 4, 3, ... .-10, -11 , etc.
  • fusion protein refers to any polypeptide which is not normally found in nature is a species, in particular a polypeptide in which one or more part of the amino acids sequence are not associated with each other in nature.
  • a fusion protein may comprise a N-terminal part consisting of a first sequence of amino acids and a C-terminal part consisting of a second sequence of amino acids that are not associated with each other in nature and/or are not associated with each other in nature in this order.
  • a fusion protein may for example be obtained from transcription and translation of a fusion gene of nucleic acid. Such fusion gene may be created by joining parts of two different genes/nucleic acid sequences.
  • the fusion protein may, for example, comprise a degron tag according to the invention, or a hybrid zinc finger polypeptide according to the invention, fused to a protein of interest.
  • host cell refers to a cell into which exogenous nucleic acid (polynucleotides) and/or exogenous polypeptide has been introduced, including the progeny of such cells.
  • Host cells may include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that has 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.
  • IMiDs 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).
  • CRBN modulators cereblon modulators
  • Thalidomide, lenalidomide, and pomalidomide have each been approved for treatment of various diseases while other IMiDs or cereblon modulators are under review.
  • the compounds may be in the form of a free acid or free base, or a pharmaceutically acceptable salt.
  • in vivo refers to an event that takes place in a subject's body
  • in vitro refers to an event that takes places outside of a subject's body.
  • an in vitro assay or method encompasses any assay or method conducted outside of a subject.
  • In vitro assays or methods encompass cell-based assays in which cells, alive or dead, are employed.
  • In vitro assays also encompass a cell-free assay in which no intact cells are employed.
  • isolated when referring to a polynucleotide (nuclei acid) or polypeptide (protein), refers to proteins or nucleic acids being present in a non-naturally occurring environment, e. g. are separated from their naturally occurring environment.
  • an isolated protein or polypeptide according to the invention relates to a protein which is no longer in its natural environment, for example, in vitro or in a recombinant host cell.
  • the terms, next to being isolated from naturally occurring source also refers to such protein or nucleic acid being artificially or synthetically produced.
  • a reference to a protein, polypeptide, nucleic acid, or polynucleotide includes reference to an “isolated” protein, polypeptide, nucleic acid, or polynucleotide.
  • linker as used in reference to a part of a protein refers to a stretch of amino acids which joins two part of the proteins together, for example in a fusion protein. Generally, such molecules have no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the proteins. However, in certain embodiments, the linker may be selected to influence some property of the linker and/or the protein such as the folding, net charge, or hydrophobicity of the linker.
  • non-natural refers to a polypeptide, polynucleotide, or domain comprised in such polypeptide or polynucleotide and that is not known to exist in nature, for example, that is not known to exist in (human) cells, i.e. , wherein one or more parts of the polypeptide, polynucleotide or domain are not associated with each other in nature.
  • a fusion protein as defined herein.
  • non-natural refers to the fact the first hybrid zinc finger domain is comprised of a first portion and a second portion and wherein the first portion is obtained from a first Cys 2 -His 2 zinc finger domain and the second portion is obtained form a second Cys 2 -His 2 zinc finger domain, and wherein the first and second Cys 2 -His 2 zinc finger domain are different from each other.
  • the non- natural first hybrid zinc finger domain is thus a fusion protein comprised of said first portion and said second portion.
  • the non-natural first hybrid zinc finger domain may thus be referred to as first hybrid zinc finger domain.
  • the same analogy is applicable to the term “non-natural second hybrid zinc finger domain”, which therefore may also be referred to as “second hybrid zinc finger domain”.
  • nucleic acid or “polynucleotide” refers to any polymers or oligomers of (contiguous) nucleotides.
  • the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
  • composition refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable compositions for administration to a cell or subject.
  • the compositions of the invention may be administered in combination with other agents as well, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.
  • the pharmaceutical composition often comprises, in addition to a pharmaceutical active agent, one or more pharmaceutical acceptable carriers (or excipients).
  • compositions be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes
  • parenteral administration for example, by subcutaneous, intramuscular or intravenous injection as, for example,
  • Drugs, therapeutic agents, medicaments and pharmaceutical compositions according to the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoral and oral. Drugs, therapeutic agents, medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body.
  • protein or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin.
  • a “fragment” or “portion” or “domain” of a protein may thus still be referred to as a “protein.”
  • a protein as defined herein and as used in any method as defined herein may be an isolated protein, a natural protein or a non-natural protein.
  • An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • the protein is a protein that is involved in regulating cell behavior, in particular that can regulate cell behavior in the context of the treatment of a disease.
  • portion or “domains” refer to amino acid sequences that are less than the full protein sequence of any protein mentioned herein.
  • domains is also more specifically used herein to refer to functional domains known in the art, e.g., zinc-finger domains, extracellular domains, intracellular domains, signaling domains, intracellular signaling domains, cytoplasmic domains and transmembrane domains.
  • sequence or “nucleotide sequence” may refer to the order of nucleotides of, or 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.
  • 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.
  • a "subject” is to indicate the organism to be treated e.g., to which administration is contemplated.
  • the subject may be any subject in accordance with the present invention, including, but not limited to humans, males, females, infants, children, adolescents, adults, young adults, middle-aged adults or senior adults and/or other primates or mammals.
  • Preferably the subject is a human patient.
  • a subject may have been diagnosed with a cancer or be suspected of having a cancer.
  • “treatment”, “treating”, “palliating”, “alleviating” and “ameliorating” in the context of a subject to be treated all refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder.
  • nucleic acid construct As used herein, the terms “construct”, “nucleic acid construct”, “vector”, and “expression vector” may be used interchangeably and are defined as a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. These constructs and vectors therefore do not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules.
  • any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein.
  • Embodiments discussed in the context of methods, use and/or compositions of the invention may be employed with respect to any other method, use or composition described herein.
  • an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.
  • Any references in the description to methods of treatment also refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.
  • compositions that include a degron tag and/or hybrid zinc finger polypeptide, and methods for modulating protein abundance in a target-specific manner via the degron tags and/or hybrid zinc finger polypeptide.
  • the invention may target endogenous and exogenous (e.g., therapeutic) proteins.
  • degron tags and/or hybrid zinc finger polypeptides are peptides that when fused to a target protein of interest (POI), may transform the POI into a substrate for cereblon (CRBN)-dependent ubiquitination and degradation, which is induced by the administration of immunomodulatory drugs (IMiDs).
  • IMSiDs immunomodulatory drugs
  • the present invention is directed to the surprising finding that zinc finger degrons and/or zinc finger polypeptides can be provided and that display improved sensitivity towards small molecules, in particular towards IMiDs, such as those disclosed herein, that regulate protein degradation/stability by interacting with these degron and/or polypeptide sequences.
  • the zinc finger degrons and/or zinc finger polypeptides according to the invention thus allow using reduced concentrations of these small molecules, in particular IMiDs, to control protein degradation, expression and/or stability of a protein of interest (and therewith, for example, cellular activity (total activity in a cell) of the protein of interest; for example, fused to the degron tags, zinc finger degrons and/or zinc finger polypeptides- according to the invention).
  • the zinc finger degrons (degron tag) and/or zinc finger polypeptides according to the invention may therefore provide for better control of protein degradation, expression and/or stability of a protein of interest (for example as comprised in a fusion protein comprising the protein of interest and the zinc finger degron or zinc finger polypeptide according to the invention) at similar or reduced concentrations of the small molecules, in particular IMiDs. At the same time, it may allow for more sensitive regulation of the degradation, expression, level and/or stability of the POI.
  • the zinc finger degrons (degron tag) and/or zinc finger polypeptides according to the invention allow for control of the level (expression) of a protein of interest, for example expressed in a cell or in a subject, by regulating degradation and/or stability thereof at reduced concentration of the small molecules, in particular IMiDs, that regulate protein degradation/stability by interacting with these degron and/or polypeptide sequences.
  • improved zinc finger degrons and/or zinc finger polypeptides according to the invention may obtained by providing for degron tags and/or polypeptides that comprise a first and a second zinc finger domain, wherein each zinc finger domain generally comprises of a beta hairpin (within the context of the current invention also sometimes referred to as a “first” or “third” portion) obtained from a first Cys2-His2 zinc finger domain and an alpha helix from a second Cys2-His2 zinc finger domain (within the context of the current invention also sometimes referred to as a “second” or “fourth” portion).
  • a beta hairpin (within the context of the current invention also sometimes referred to as a “first” or “third” portion) obtained from a first Cys2-His2 zinc finger domain and an alpha helix from a second Cys2-His2 zinc finger domain (within the context of the current invention also sometimes referred to as a “second” or “fourth” portion).
  • the first zinc finger domain is a hybrid zinc finger domain, i.e. , the first zinc finger domain is comprised of a first portion and a second portion that together do not exist in nature (non-natural).
  • the first zinc finger domain is a non-natural hybrid zinc finger domain obtained by combining a beta hairpin from a first Cys2-His2 zinc finger domain and an alpha helix from a second Cys2-His2 zinc finger domain, and wherein the first and second Cys2-His2 zinc finger domain are not identical or the same.
  • the second zinc finger domain comprised in the zinc finger degrons and/or zinc finger polypeptides according to the invention is a hybrid zinc finger domain, like 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.
  • the second hybrid zinc finger domain is different from the first hybrid zinc finger domain.
  • 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 a zinc finger polypeptide according to the invention further comprises at least two amino acid substitutions, as disclosed herein in detail, 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 regulate protein degradation/stability by interacting with these degron and/or polypeptide sequences, is even further improved.
  • improved zinc finger domains and degrons can be provided 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.
  • said hybrid zinc finger polypeptide is preferably comprised (as the first hybrid zinc finger domain) in a degron tag according to the invention, said degron tag according to the invention comprising two non-natural hybrid zinc finger domains (see Figure 2).
  • the degron tag according to the invention may also be referred to as ‘double hybrid degron’.
  • a degron comprising a first hybrid zinc finger domain and a second non-hybrid zinc finger domain may also be referred to as ‘single hybrid, dual zinc finger degron’
  • a degron tag comprising a first hybrid zinc finger domain and no second zinc finger domain may also be referred to as ‘single hybrid, single zinc finger degron’.
  • the non-natural hybrid zinc finger polypeptide according to the invention is such ‘single hybrid, single zinc finger degron’ comprising two amino acid substitutions as described herein.
  • the non-natural hybrid zinc finger polypeptide according to the invention in addition to the first hybrid zinc finger domain, further comprises a second non-hybrid zinc finger domain, such molecule may also be referred to as single hybrid, dual zinc finger degron comprising at least two amino acid substitutions as described herein.
  • non-natural hybrid zinc finger polypeptide in addition to the first hybrid zinc finger domain, further comprises a second hybrid zinc finger domain, such molecule may also be referred to as ‘double hybrid degron’ comprising at least two amino acid substitutions as described herein.
  • double hybrid degron comprising at least two amino acid substitutions as described herein.
  • the first hybrid zinc finger domain comprises a first portion and a second portion
  • the first portion comprises the amino acid sequence X 1 X 2 C 3 X 4 X 5 C 6 X 7 X 8 X 9 X 10 X 1 1 , wherein X represents any amino acid, of a first Cys 2 -His 2 zinc finger domain;
  • the second portion comprises the amino acid sequence X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 , wherein X represents any amino acid, of a second Cys 2 -His 2 zinc finger domain, wherein the second Cys 2 -His 2 zinc finger domain is different from the first Cys 2 -His 2 zinc finger domain;
  • the second portion is C-terminal with respect to the first portion
  • the second hybrid zinc finger domain is C-terminal with respect to the first hybrid zinc finger domain
  • the second hybrid zinc finger domain comprises a third portion and a fourth portion
  • the third portion comprises the amino acid sequence (Z) 2 C(Z) 2 C(Z) 5-6 wherein Z represents any amino acid, of a third Cys 2 -His 2 zinc finger domain;
  • the fourth second portion comprises the amino acid sequence (Z) 6 H(Z) 3-4 H, wherein Z represents any amino acid, of a fourth Cys 2 - HiS 2 zinc finger domain, wherein the fourth Cys 2 -His 2 zinc finger domain is different from the third Cys 2 -His 2 zinc finger domain;
  • the fourth portion is C-terminal with respect to the third portion
  • the first hybrid zinc finger domain comprises at least two amino acid substitutions, wherein an amino acid substitution in the first portion of the first hybrid zinc finger domain is relative to the first portion of the first Cys 2 -His 2 zinc finger domain and wherein an amino acid substitution in the second portion of the first hybrid zinc finger domain is relative to the second portion of the second Cys 2 -His 2 zinc finger domain, and wherein the substitution is not at any of positions C 3 , C 6 , X 7 , H 19 , or H 23 .
  • the current invention provides for systems that may comprise zinc finger degrons herein also referred to as degron tags.
  • a degron or degron tag is a peptide sequence or protein element that is involved in regulating the degradation rate of a protein that, for example, comprises such degron or degron tag.
  • the degron tag according to the invention comprises, in some embodiments, two zinc finger domains.
  • the term zinc finger domain is, within the context of the current invention, well understood by the skilled person.
  • the degron is therefore a zinc finger degron comprising at least two zinc finger domains and that can be controlled with an IMiD such as thalidomide, lenalidomide, pomalidomide, and/or analogs thereof.
  • the degron tag is able to promote or control degradation of a POI, for example when fused to the degron tag, in the presence of such IMiD.
  • a POI for example when fused to the degron tag, in the presence of such IMiD.
  • one or both of the hybrid zinc finger domains in the degron tag according to the invention is/are non-natural hybrid zinc finger.
  • the first hybrid zinc finger domain that is comprised in a degron tag according to the invention comprises 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 that are comprised in the first hybrid zinc finger domain may or may not be directly adjacent to each other.
  • the first portion and the second portion are connected via a linker peptide of, for example, one, two, three, four, five, or more amnio acids. Therefore, is some embodiments the first portion and the second portion may be connected to each other via a further linker peptide, for example comprising one, two, three, four, five, six, seven or more amino acids.
  • no additional linker peptide is present between the first portion of the first hybrid zinc finger domain and the second portion of the first hybrid zinc finger domain.
  • the first and second portion are directly adjacent to each other.
  • the first portion and the second portion are directly adjacent to each other.
  • the first portion of the first hybrid zinc finger domain may be N-terminal or may be C-terminal from the second portion of the first hybrid zinc finger domain. In a preferred embodiment the first portion of the first hybrid zinc finger domain is N- terminal of the second portion of the first hybrid zinc finger domain. [073] In a preferred embodiment the first portion consists of 8 - 30 amino acids, with increasing preference 11- 30, 10 - 20, 11 - 20, 10 - 14, 11- 14, 10 - 11 , most preferably 11 amino acids.
  • the second portion consists of 8 - 30 amino acids, with increasing preference 12- 30, 11 - 20, 12 - 20, 11 - 14, 12 - 14, most preferably 12 amino acids.
  • the, preferably non-natural, first hybrid zinc finger domain consist of about 16 - 60 amino acids, preferably about 20 - 36 amino acids, about 20 - 30 amino acids, for example 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • the first hybrid zinc finger domain comprises a Cys2 His2 (C2H2) domain, and wherein in the hybrid zinc finger domain comprises at least two subdomains (herein referred to as portions), and wherein each subdomain is from a different wild type zinc finger.
  • the first portion of the first hybrid zinc finger domain is from a first wild type zinc finger
  • the second portion of the first hybrid zinc finger domain is from a second, different, wild type zinc finger domain.
  • the skilled person is well capable of selecting the first portion of the first hybrid zinc finger domain from a first wild type zinc finger and/or well capable of selecting the second portion of the first hybrid zinc finger domain from a second wild type zinc finger.
  • he may appropriately select such portions from wild type zinc finger domains (in particular wild type Cys2-His2 (C2H2) zinc finger domains) that are available from various scientific publications and public and well-known gene- and protein databases, for example from (Sievers et al. Science.
  • the first portion of the first hybrid zinc finger domain comprises an amino acid sequence represented by the sequence X 1 X 2 C 3 X 4 X 5 C 6 X 7 X 8 X 9 X 10 X 1 1 , wherein each X (X 1 , X 2 , ... etc.) represents, independently, any amino acid, more in particular wherein each X represent any natural or proteinogenic amino acid (e.g.
  • amino acid sequence comprised in the first portion of the first hybrid zinc finger domain is (X) 2 C(X) 2 C(X) 5 .
  • Xi may represent another amino acid than, for example X 2 , or it may represent the same amino acid.
  • C 3 indicates the presence of a cysteine at the third position in this amino acid sequence that is comprised in the first portion of the first hybrid zinc finger domain.
  • C 6 indicates the presence of a cysteine at the sixth position.
  • the first portion of the first hybrid zinc finger domain has the above indicated 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 ) and wherein the amino acid sequence is of a first Cys 2 - HiS 2 zinc finger domain (e.g., of a wild type Cys 2 -His 2 zinc finger domain).
  • the indicated amino acid sequence of the first portion of the first hybrid zinc finger domain may comprise an additional short stretch of amino acids N- terminal thereof, for example 1 - 10, preferably 1 - 5, for example 1 , 2, 3, 4, or 5 amino acids, for example of said first or second Cys 2 -His 2 zinc finger domain.
  • Figure 3 provides an example of a degron tag according to the invention (with positions of amino acid substitutions indicated) and wherein N-terminal of the first hybrid zinc finger domain there is a short stretch of amino acids GERP (in this case originating from the IKZF1 ; in some other embodiments the stretch may be from another wild type zinc finger domain, and may, for example be GEKP).
  • GERP amino acids originating from the IKZF1 ; in some other embodiments the stretch may be from another wild type zinc finger domain, and may, for example be GEKP.
  • the first hybrid zinc finger domain comprised in the degron tag according to the invention is in preferred embodiments a hybrid zinc finger comprising a Cys2 His2 (C2H2) zinc finger domain and wherein the hybrid zinc finger domain comprises at least two subdomains, i.e., a first portion and a second portion, and wherein the amino acid sequence of the first portion is from a wild type zinc finger, more in particular a wild type Cys2 His2 (C2H2) zinc finger.
  • C2H2 Cys2 His2
  • the second portion of the first hybrid zinc finger domain comprises an amino acid sequence represented by 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 , wherein each X (X 12 , X 13 , ... etc.) represents, independently, any amino acid, more in particular wherein each X represent 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.
  • 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.
  • An alternative way of representing the amino acid sequence comprised in the second portion of the first hybrid zinc finger domain is (X) 7 H(X) 3 H.
  • amino acids substitutions at particular positions in, amongst others, the second portion of the first hybrid zinc finger domain are part of the invention it is preferred to use X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 ,, indicating the individual amino acids and positions thereof.
  • the second portion of the first hybrid zinc finger domain is (X) 6 H(X) 3 H.
  • X 12 may represent another amino acid than, for example X 13 , or it may represent the same amino acid.
  • H 19 indicates the presence of a histidine at the nineteenth position in this amino acid sequence that is comprised in the second portion of the first hybrid zinc finger domain.
  • H 23 indicates the presence of a histidine cysteine at the twenty-third position in this sequence.
  • the second portion of the first hybrid zinc finger domain has the above indicated amino acid sequence (X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 ) and wherein the amino acid sequence is of a second Cys 2 -His 2 zinc finger domain.
  • the indicated amino acid sequence of the second portion of the first hybrid zinc finger domain may comprise an additional short stretch of amino acids C-terminal thereof, for example 1 - 10, preferably 1 - 5, for example 1 , 2, 3, 4, or 5 amino acids, for example of said second Cys 2 -His 2 zinc finger domain.
  • the first portion of the first hybrid zinc finger domain is N- terminal from the second portion of the first hybrid zinc finger domain.
  • the second portion is C-terminal with respect to the first portion.
  • the first hybrid zinc finger domain comprised in the degron tag according to the invention is in preferred embodiments a hybrid zinc finger domain comprising a Cys2 His2 (C2H2) domain and wherein the hybrid zinc finger domain comprises at least two subdomains, i.e. a first portion and a second portion, and wherein the amino acid sequence of the first portion is from a wild type zinc finger, more in particular a wild type Cys2 His2 (C2H2) zinc finger (i.e. the first Cys 2 -His 2 zinc finger domain), and wherein the amino acid sequence of the second portion is from a wild type zinc finger, more in particular a wild type Cys2 His2 (C2H2) zinc finger (i.e.
  • the amino acid sequence of the first portion comprises or is X 1 X 2 C 3 X 4 X 5 C 6 X 7 X 8 X 9 X 10 X 11 and/or the amino acid sequence of the second portion comprises or 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 .
  • the first portion and the second portion together form the first hybrid zinc finger domain, and wherein the first portion is preferably of a first Cys 2 -His 2 zinc finger domain, e.g., of a wild type, naturally occurring Cys 2 -His 2 zinc finger domain and the second portion is preferably of a second, different, Cys 2 -His 2 zinc finger domain, e.g., of a different wild type, naturally occurring Cys 2 -His 2 zinc finger domain.
  • the first hybrid zinc finger domain that is formed by the first portion and the second portion may be referred to as a hybrid Cys 2 -His 2 (C2H2) zinc finger domain, e.g., a Cys 2 -His 2 (C2H2) zinc finger domain that does not occur in wild type zinc finger proteins and/or in nature.
  • C2H2 Cys 2 -His 2
  • the C2H2 zinc finger domain structure has been found to be an important determinant in binding and may even be a more important determining factor than the primary amino acid sequence of the zinc finger domain (see, for example Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572).
  • the C2H2 zinc finger domains are known as recurrent motifs that mediate drug-dependent (e.g., IMiDs) interactions with ubiquitin ligase CRL4 CRBN (see, for example, An et al, Nat Commun.
  • a C2H2 zinc finger domain comprising e.g., the first hybrid zinc finger domain and/or the second (hybrid) zinc finger domain according to the invention, comprise beta-hairpin and alpha-helix subdomains.
  • a C2H2 zinc finger domain consist of about 20 - 36 amino acids, about 20 - 30 amino acids, for example 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 comprising two conserved cysteine residues, followed by an alpha helix comprising two conserved histidine residues at its C-terminus (see, for example Fedotova et al., ActaNaturae, 2017 Apr- Jun; 9(2): 47-58). Said motif is also present in the zinc finger domains according to the invention, including the first and the second hybrid zinc finger domain.
  • the degron tag according to the invention may also comprises a second (non-natural) hybrid zinc finger domain.
  • said second hybrid zinc finger domain is C-terminal with respect to the first hybrid zinc finger domain.
  • the first hybrid zinc finger domain in the degron tag according to the invention is N-terminal with respect to the second hybrid zinc finger domain.
  • first hybrid zinc finger domain and the second hybrid zinc finger domain are directly adjacent to each other, with no peptide linker being present.
  • the first hybrid zinc finger domain and the second hybrid zinc finger domains are not directly adjacent to each other but connected to each other via a short peptide linker.
  • linker peptide may be comprised of, for example, one, two, three, four, five, six, seven, or more amino acids.
  • the first hybrid zinc finger domain may be connected to the second hybrid zinc finger domain via a further linker peptide, for example comprising one, two, three, four, five, six, seven or more amino acids. However, in some embodiments no additional linker peptide is present.
  • the linker may be a short stretch of amino acids that originate from a wild-type zinc finger domain, for example from the N- terminal part or adjacent to the N-terminal part of a wild type zinc finger domain.
  • Such amino acid sequence may, for example, be a SGEKP sequence (see Figure 3) (in this case originating from the IKZF1 protein - SGEKP sequence is the endogenous linker between IKZF1 ZF2 and IKZF1 ZF3 in the IKZF1 protein; in the same manner other endogenous linker may, for example, be used as linker within the context of the current invention, for example comparable endogenous linkers from other C2H2 zinc finger proteins); in some other embodiments the stretch may be from another wild type zinc finger domain, and may, for example be GERP).
  • the 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. According to this aspect of the invention there is therefore provided for a degron tag wherein the degron tag comprises a first and a second hybrid zinc finger domain as detailed herein.
  • 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 that are comprised in the second hybrid zinc finger domain may or may not be directly adjacent to each other.
  • the third portion and the fourth portion are connected via a linker peptide of, for example, one, two, three, four, five, or more amino acids. Therefore, is some embodiments the third portion and the fourth portion may be connected to each other via a further linker peptide, for example comprising one, two, three, four, five, six, seven or more amino acids.
  • no additional linker peptide is present between the third portion of the second hybrid zinc finger domain and the fourth portion of the second hybrid zinc finger domain.
  • the third and fourth portion are directly adjacent to each other.
  • the third portion and the fourth portion are directly adjacent to each other.
  • the third portion of the second hybrid zinc finger domain may be N-terminal or may be C-terminal from the fourth portion of the second hybrid zinc finger domain.
  • the third portion is N-terminal of the fourth portion.
  • the third portion consists of 8 - 30 amino acids, with increasing preference 11- 30, 10 - 20, 11 - 20, 10 - 14, 11- 14, 10 - 11 , most preferably 11 amino acids.
  • the fourth portion consists of 8 - 30 amino acids, with increasing preference 12- 30, 11 - 20, 12 - 20, 11 - 14, 12 - 14, most preferably 12 amino acids.
  • the, preferably non-natural, second hybrid zinc finger domain consist of about 16 - 60 amino acids, preferably about 20 - 36 amino acids, about 20 - 30 amino acids, for example 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • the second hybrid zinc finger domain comprises a Cys2 His2 (C2H2) domain, and wherein in the hybrid zinc finger domain comprises at least two subdomains (herein referred to as portions), and wherein each subdomain is from a different wild type zinc finger.
  • the third portion of the second hybrid zinc finger domain is from a third wild type zinc finger
  • the fourth portion of the second hybrid zinc finger domain is from a fourth, different, wild type zinc finger domain.
  • the skilled person is well capable of selecting the third portion of the second hybrid zinc finger domain from a third wild type zinc finger and/or well capable of selecting the fourth portion of the second hybrid zinc finger domain from a fourth wild type zinc finger.
  • he may appropriately select such portions from wild type zinc finger domains (in particular wild type Cys2-His2 (C2H2) zinc finger domains) that are available from various scientific publications and public and well-known gene- and protein databases, for example from (Sievers et al. Science.
  • the third portion of the second hybrid zinc finger domain comprises an amino acid sequence represented by the sequence (Z) 2 C(Z) 2 C(Z) 5-6 , wherein Z represents any amino acid.
  • (Z) 2 thus represent two amino acids whereas (Z) 5-6 indicates 5 or 6 amino acids.
  • the third portion of the second hybrid zinc finger domain may, for example, comprise an amino acid sequence Z 1 Z 2 C 3 Z 4 Z 5 C 6 Z 7 Z 8 Z 9 Z 10 Z 11 or Z 1 Z 2 C 3 Z 4 Z 5 C 6 Z 7 Z 8 Z 9 Z 10 Z 11 Z 12 and wherein each Z (Z 1 , Z 2 , ... etc.) represents, independently, any amino acid, more in particular wherein each Z represent 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.
  • each Z represents, independently, any amino acid, more in particular wherein each Z represent any natural or proteinogenic amino acid (e.g. Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine,
  • Z1 may represent another amino acid than, for example Z 2 , or it may represent the same amino acid.
  • C 3 indicates the presence of a cysteine at the third position in this amino acid sequence that is comprised in the third portion of the second hybrid zinc finger domain.
  • C 6 indicates the presence of a cysteine at the sixth position.
  • the third portion of the second hybrid zinc finger domain has the above indicated amino acid sequence (Z) 2 C(Z) 2 C(Z) 5-6 , even more preferably (Z) 2 C(Z) 2 C(Z) 6 and wherein the amino acid sequence is of a third Cys2-His2 zinc finger domain (e.g., of a wild type Cys2-His2 zinc finger domain).
  • the indicated amino acid sequence of the third portion of the second hybrid zinc finger domain may comprise an additional short stretch of amino acids N- terminal thereof, for example 1 - 10, preferably 1 - 5, for example 1 , 2, 3, 4, or 5 amino acids, for example of said third or fourth (wildtype) Cys2-His2 zinc finger domain.
  • Figure 3 provides an example of a degron tag according to the invention (with positions of amino acid substitutions indicated) and wherein N- terminal of the second hybrid zinc finger domain there is a short stretch of amino acids SGEKP, and as discussed above. Experiments confirmed that although such short stretch may be included, it does not substantially affect the results obtained with the degron tags (and/or hybrid zinc finger polypeptides according to the invention).
  • the second hybrid zinc finger domain is in preferred embodiments a hybrid zinc finger comprising a Cys2 His2 (C2H2) zinc finger domain and wherein the hybrid zinc finger domain comprises at least two subdomains, i.e. a third portion and a fourth portion, and wherein the amino acid sequence of the third portion is from a wild type zinc finger, more in particular a wild type Cys2 His2 (C2H2) zinc finger.
  • C2H2 Cys2 His2
  • the fourth portion of the second hybrid zinc finger domain comprises an amino acid sequence represented by the sequence (Z) 6 H(Z) 3 - 4 H.
  • (Z) 6 thus represent six amino acids whereas (Z) 3-4 indicates 3 or 4 amino acids
  • the fourth portion of the second hybrid zinc finger domain may, for example, comprise an amino acid sequence Z 13 Z 14 Z 15 Z 16 Z 17 Z 18 H 19 Z 20 Z 21 Z 22 H 23 or Z 1 3 Z 1 4 Z 1 5 Z 1 6 Z 1 7 Z 1 8 H 1 9 Z 20 Z 21 Z 22 Z 23 H 24 , wherein each Z (Z 13, Z 1 4 , ... etc.) represents, independently, any amino acid, more in particular wherein each Z represent any natural or proteinogenic amino acid (e.g.
  • Z 13 may represent another amino acid than, for example Z 14 , or it may represent the same amino acid.
  • H 19 indicates the presence of a histidine at the nineteenth position in this amino acid sequence that is comprised in the second portion of the first hybrid zinc finger domain.
  • H 23 or H 24 indicates the presence of a histidine cysteine at the twenty-third position in this sequence or the twenty-fourth position in this sequence (depending on whether (Z) 3-4 relates to 3 or 4 amino acids).
  • Z 13 does not indicate a position relative to the first hybrid zinc finger domain, since it is, for example, explained herein elsewhere that in some embodiments there may be linkers between the fist hybrid zinc finger domain and the second (hybrid) zinc finger domain.
  • the length of the third portion of the second (hybrid) zinc finger may vary, and may, for example be 11 amino acids in lengths.
  • the fourth portion of the second hybrid zinc finger domain has the above indicated amino acid sequence (Z) 6 H(Z) 3-4 H and wherein the amino acid sequence is of a second Cys2-His2 zinc finger domain, i.e., more in particular a wild type Cys2 His2 (C2H2) zinc finger.
  • the second hybrid zinc finger domain comprised in the degron tag according to the invention is in preferred embodiments a hybrid zinc finger domain comprising a Cys2 His2 (C2H2) domain and wherein the hybrid zinc finger domain comprises at least two subdomains, i.e.
  • the amino acid sequence of the third portion is from a wild type zinc finger, more in particular a wild type Cys2 His2 (C2H2) zinc finger (i.e. the third Cys2-His2 zinc finger domain), and wherein the amino acid sequence of the fourth portion is from a wild type zinc finger, more in particular a wild type Cys2 His2 (C2H2) zinc finger (i.e. the fourth Cys2-His2 zinc finger domain), and wherein, preferably, the third Cys2-His2 zinc finger domain is different from the fourth Cys2-His2 zinc finger domain (for example, wherein the third Cys2-His2 zinc finger domain is a different wild type zinc finger than the fourth Cys2-His2 zinc finger domain).
  • the amino acid sequence of the third portion comprises or is (Z) 2 C(Z) 2 C(Z) 5-6 and/or the amino acid sequence of the fourth portion comprises or is (Z) 6 H(Z) 3-4 H.
  • the fourth portion is C-terminal with respect to the third portion.
  • the degron tag according to the invention may comprise further amino acid, for example N-terminal from the first hybrid zinc finger domain or C-terminal form the second hybrid zinc finger domain.
  • 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 wherein an amino acid substitution in the first portion of the first hybrid zinc finger domain is relative to the first portion of the first Cys2- HiS2 zinc finger domain and wherein an amino acid substitution in the second portion of the first hybrid zinc finger domain is relative to the second portion of the second Cys 2 -His 2 zinc finger domain, and wherein the substitution is not at any of positions C 3 , C 6 , X 7 , H 19 , or H 23 .
  • X 7 is a glycine.
  • the first hybrid zinc finger domain is comprised from a first portion that is from a first Cys 2 -His 2 zinc finger domain, in particular from a first wild type Cys 2 -His 2 zinc finger domain, and wherein the first portion comprises the 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 (i.e., as present in the first wild type Cys 2 -His 2 zinc finger domain).
  • the first hybrid zinc finger domain is comprised from a second portion that is from a second Cys 2 -His 2 zinc finger domain, in particular from a second wild type Cys 2 -His 2 zinc finger domain, and wherein the second portion comprises the amino acid sequence X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 (i.e., as present in the second wild type Cys 2 -His 2 zinc finger domain).
  • 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 wherein an amino acid substitution is in the first portion of the first hybrid zinc finger domain, this amino acid substitution is an amino acid substitution relative to the first portion of the first Cys 2 -His 2 zinc finger domain from which the sequence was obtained.
  • X 2 is a Valine in the first (wildtype) Cys 2 -His 2 zinc finger domain from which the sequence was obtained, and this Valine is substituted with, for example, a Lysine in the degron tag according to the invention
  • X 2 will be a Lysine and the amino acid substitution to a Lysine is relative to the Valine that was comprised in the wild type Cys 2 -His 2 zinc finger domain from which it was obtained.
  • an amino acid substitution in the second portion of the first hybrid zinc finger domain is defined herein.
  • X 20 is a Proline in the second (wildtype) Cys 2 -His 2 zinc finger domain from which the sequence for the second portion was obtained, and this Proline is substituted with, for example, a Leucine in the degron tag according to the invention
  • X 20 will be a Leucine and the amino acid substitution to a Leucine is relative to the Proline that was comprised in the wild type Cys 2 -His 2 zinc finger domain from which it was obtained.
  • the first hybrid zinc finger domain comprises at least two amino acid substitutions.
  • the skilled person is well capable of providing, preparing and recognizing such amino acid substitutions relative to the wild type first Cys 2 -His 2 zinc finger domain and second Cys 2 -His 2 zinc finger domain, for example by comparing the amino acid sequence of the first portion and or the second portion of the first hybrid zinc finger domain therewith.
  • the at least two amino acids substitutions 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 embodiment one amino acid substitution is in the first portion and one amino acid substitutions is in the second portion.
  • 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, for example, 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.
  • 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 those embodiments wherein the first hybrid zinc finger domain comprises more than two amino acid substitutions, for example three or four amino acid substitutions, in preferred embodiments, the for example third and/or fourth amino acid substitution is selected or also selected from the substitutions as disclosed herein, for example as listed in Table 1.
  • the first hybrid zinc finger domain comprise, for example, four amino acid substitutions (or six)
  • the four amino acid substitutions (or six) are selected from the pair of substitutions disclosed herein, for example as listed in Table 2 (for example Q12R K13V G14N K21A) and/or the pair of position and amino acids to be substituted as listed in Table 4.
  • the first hybrid zinc finger domain according to the invention comprises an amino acid sequence according to SEQ ID NO: 135 (LQCEICGFTCRRVNNLLRHIALH; i.e. quadruple substitutions
  • Q12R/K13V/G14N/K21A and/or the degron tag according to the invention comprises an amino acid sequence according to SEQ ID NO: 136 (LQCEICGFTCRRVNNLLRHIALHSGEKPFKCHLCNYACRRKDSVVAHKAKSH).
  • the substitution is not in any of the positions C 3 , C 6 , X 7 , H 19 , or H 23 .
  • X 7 is G 7 .
  • thalidomide or analogue relative to a wild-type zinc finger domain and relative to a hybrid zinc finger domain but that is comprised of a wild- type subdomains (as described herein) and do not comprise the at least two amino acid substitutions.
  • introducing one amino acid substitutions may also enhanced or increased sensitivity to an ImiD molecule, unexpectedly introducing a further amino acid substitution may even further enhance or increase sensitivity to an ImiD molecule, as is shown in the examples and described herein.
  • degron tags comprising at least a first hybrid zinc finger domain comprising at least two amino acid substitutions relative to the wild type zinc finger domains (portions thereof) from which the hybrid zinc finger domain is composed, and that has enhanced or increased sensitivity to an ImiD molecule.
  • the at least two amino acid substitutions are at position that are directly adjacent to each other, in other embodiments, the positions of the amino acid substitutions are separated from each other by at least 1 , 2, 3, 4, 5, 6, 7, 8, 10, 11 , 12, 13 or more amino acids (i.e., non-substituted amino acids), for example as shown in the accompanying tables, figures and examples.
  • amino acid substitutions on a particular position in the first hybrid zinc finger domain is one as described herein (with respect to either the amino acid substituted or with respect to the substituting amino acid).
  • the skilled person is, based on the information disclosed herein, and without due burden, able to provide for such amino acid substitutions, and degron tags according to the invention.
  • the current inventors belief that the enhanced or increased sensitivity to an ImiD molecule that is observed with the degron tags and/or hybrid zinc finger polypeptides according to the invention is at least in part due to altered or improved interaction of the degron tag, cereblon and the immunomodulatory drug (IMiD). Therefore, in preferred embodiments, there is provided for a degron tag according to the invention and/or a hybrid zinc finger polypeptide according to the invention and wherein the degron tag and/or the hybrid zinc finger polypeptide is able to bind a complex formed between cereblon (CRBN) and an immunomodulatory drug (IMiD).
  • CRBN cereblon
  • IiD immunomodulatory drug
  • a degron tag and/or hybrid zinc finger polypeptide wherein the first portion is a beta-hairpin loop of a first Cys 2 -His 2 zinc finger domain, the second portion is an alpha-helix region of a second Cys 2 -His 2 zinc finger domain, the third portion is a beta-hairpin loop of a third Cys 2 - HiS 2 zinc finger domain, and/or the fourth portion is an alpha-helix region of a fourth Cys 2 -His 2 zinc finger domain.
  • first, second, third or fourth Cys 2 -His 2 zinc finger domain refer to Cys 2 -His 2 zinc finger domains as these are found in nature, i.e.
  • Cys 2 -His 2 zinc finger domains from wild type zinc finger proteins.
  • the first hybrid zinc finger domain may comprise at least two amino acid substitutions.
  • 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.
  • the first portion that is used is a beta-hairpin loop of a first Cys 2 -His 2 zinc finger domain
  • the second portion that is used is an alpha-helix region of a second Cys 2 -His 2 zinc finger domain
  • the third portion that is used is a beta-hairpin loop of a third Cys 2 -His 2 zinc finger domain
  • the fourth portion that is used is an alpha-helix region of a fourth Cys 2 -His 2 zinc finger domain.
  • Zinc finger domains include a beta-hairpin loop and an alpha-helix region, and the skilled person knows how to provide for a beta-hairpin loop portion and/or an alpha-helix region portion of (wildtype) Cys 2 -His 2 zinc finger domains. Representative examples are well-known to the skilled person and include, for example, those disclosed and described herein.
  • Examples of zinc finger comprising a beta-hairpin loop and an alpha-helix region include but are not limited (human) IKZF1 , IKZF2, IKZF3, SALL4, ZFP91 , GZF1 , ZNF653, ZNF692, ZNF827, ZBTB39, WIZ, ZNF98, ZNF654, ZNF787, ZNF 276, ZNF582, ZNF517 and E4F1 (see also Uniprot Accession no: Q13422, Q9UKS7, Q9UKT9, Q9UJQ4, Q96JP5, Q9H116, Q96CK0, Q9BU19, Q17R98, 015060, 095785, A6NK75, Q8IZM8, Q6DD87, Q8N554, Q96NG8, Q6ZMY9, Q66K89).
  • the degron tag and/or hybrid zinc finger polypeptide according to the invention may include one or more amino acid residues N- terminal with respect 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 with respect to the alpha-helix portion provided that the degron tag and/or hybrid zinc finger polypeptide according to the invention is a substrate for a CRBN- IMiD complex, and/or shows enhanced or increased sensitivity to an IMiD molecule (relative to e.g. to wild type situation).
  • These additional amino acids may correspond to residues in the native Cys 2 -His 2 zinc finger domains or be different provided that the degron tag maintains a zinc finger-like fold and exhibits the properties as disclosed herein.
  • the degron tag and/or hybrid zinc finger polypeptide according to the invention wherein the first substitution is in the second portion and the second substitution is in the first portion or in the second portion.
  • the at least two amino acid substitutions may both be comprised in the first portion, or may both be comprised in the second portion, or one may be comprised in the first portion and the second may be comprised in the second portion, in a preferred embodiment, at least one of the amino acid substitutions is present in the second portion.
  • suitable degron tags and/or hybrid zinc finger polypeptides according to the invention can be provided and wherein at least one of the amino acid substitutions is in the second portion, i.e. , is in the alpha helix portion of the first hybrid zinc finger domain.
  • the second amino acid substitutions may be present in either the second portion or may be present in the first portion (i.e., in the beta hairpin loop).
  • degron tag and/or hybrid zinc finger polypeptide wherein at least one substitution is at a position selected from the group consisting of X 1 , X 4 , X 12 , X 13 , X 14 , X 15 , X 17 , X 21 , and X 22 .
  • an amino acid substitution on one of these position in the first hybrid zinc finger domain allows for providing degron tag and/or hybrid zinc finger polypeptide according to the invention with increased or enhanced sensitivity to an IMiD molecule.
  • the substituting amino acid is as disclosed herein.
  • a degron tag and/or hybrid zinc finger polypeptide according to the invention wherein at least one of the substitutions that is present in the degron tag and/or hybrid zinc finger polypeptide according to the invention (or that is present in the first hybrid zinc finger domain) is selected from those listed in Table 1 .
  • Table 1 list the various position X 1 - X 22 and where a substitution in the degron tag and/or hybrid zinc finger polypeptide according to the invention may, in preferred embodiments, be present, together with the substituting amino acid (i.e. , the amino acid that is included at that position in the degron tag and/or hybrid zinc finger polypeptide according to the invention; one letter code).
  • both amino acid substitutions are selected from those listed in Table 1.
  • each amino acid substitution is at a different position.
  • a substituting amino acid is meant to indicate an amino acid that is different from the native amino acid (i.e., that is different from that as present in the wild type Cys 2 -His 2 zinc finger domain used for the first or second portion of the first hybrid zinc finger domain).
  • the current invention is not in particular limited to a particular wild type Cys 2 -His 2 zinc finger domain used for providing the first portion, second portion (and/or third and fourth portion as discussed herein).
  • the current invention also encompasses the use of the positions and substitutions as listed in Table 1 (or any other Table of list provided herein)- in any suitable first or second portion of a (wildtype) Cys 2 -His 2 zinc finger domain that may be used in providing for the first hybrid zinc finger domain according to the invention.
  • the positions and substituting amino acids listed in Table 1 are applicable to any suitable first hybrid zinc finger domain as long the degron tag and/or hybrid zinc finger polypeptide according to the invention maintains a zinc finger-like fold and exhibits the properties as disclosed herein (e.g., sensitivity to IMiD in the context of protein degradation).
  • the position and substitutions are relative to the first hybrid zinc finger domain as used in the examples herein, i.e., wherein the first portion of the first hybrid zinc finger domain is a beta-hairpin region of ZFP91ZF4, and the second portion of the first hybrid zinc finger domain is an alpha-helix region of IKZF1 ZF2 (see Figure 3; wherein the first hybrid zinc finger domain of the ‘parental degron tag’ used in the examples is schematically represented).
  • a degron tag and/or hybrid zinc finger polypeptide according to the invention, and 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 position according to the invention in the first hybrid zinc finger domain as described herein (showing per row a preferred combination of two positions within the first hybrid zinc finger domain and the corresponding substituting amino acids; for example 4R and 12L indicates that in this combination the amino acid at position 4 in the first hybrid zinc finger domain is substituted with an Arginine (R) and the amino acid at position 12 in the first hybrid zinc finger domain is substituted with a Leucine (L) - in other words the original amino acids in the portions obtained from the wild type Cys2-His2 zinc finger domains are substituted with an Arginine in position 4 and an Leucine at position 12).
  • the at least two substitutions are selected from any of the combinations shown in Table 2.
  • a substituting amino acid is meant to indicate an amino acid that is different from the native amino acid (i.e., that is different from that as present in the wild type Cys 2 -His 2 zinc finger domain used for the first or second portion of the first hybrid zinc finger domain).
  • the current invention is not in particular limited to a particular wild type Cys 2 -His 2 zinc finger domain used for providing the first portion, second portion (and/or third and fourth portion as discussed herein).
  • the current invention also encompasses the use of the positions and substitutions as listed in Table 2 (or any other Table of list provided herein)- in any suitable first or second portion of a (wildtype) Cys 2 -His 2 zinc finger domain that may be used in providing for the first hybrid zinc finger domain according to the invention.
  • the positions and substituting amino acids listed in Table 2 are applicable to any suitable first hybrid zinc finger domain as long the degron tag and/or hybrid zinc finger polypeptide according to the invention maintains a zinc finger-like fold and exhibits the properties as disclosed herein (e.g., sensitivity to IMiD in the context of protein degradation).
  • the position and substitutions are relative to the first hybrid zinc finger domain as used in the examples herein, i.e. , wherein the first portion of the first hybrid zinc finger domain is a beta-hairpin region of ZFP91ZF4, and the second portion of the first hybrid zinc finger domain is an alpha-helix region of IKZF1 ZF2 (see Figure 3; wherein the first hybrid zinc finger domain of the ‘parental degron tag’ used in the examples is schematically represented).
  • Table 3 list preferred amino acids that are substituted in the first hybrid zinc finger domain of the degron tag and/or hybrid zinc finger polypeptide according to the invention.
  • the position and/or the position and the amino acid that is substituted is selected from those listed in Table 3.
  • the at least two amino acid substitutions are selected from a combination of the position 1 , 4, 5, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 20, 21 and 22, and wherein the amino acids that are substituted are a combination of two amino acids as listed in Table 3.
  • both amino acids that are substituted are selected from those listed in Table 3.
  • the amino acids for example the preferred amino acids as shows in Table 3 may be substituted with any suitable amino acid as long as it provides for a degron tag and/or hybrid zinc finger polypeptide according to the invention.
  • the amino acids preferably a combination of amino acids listed in Table 3 are replaced with a corresponding amino acid as listed in Table 1 or corresponding combination of amino acids as shown in Table 2 (wherein corresponding refers, for example, to the corresponding position in the first hybrid zinc finger domain as disclosed herein).
  • the current invention also encompasses the use of the positions and substitutions as listed in Table 3 (or any other Table or list provided herein) in any suitable first or second portion of a (wildtype) Cys2-His2 zinc finger domain that may be used in providing for the first hybrid zinc finger domain according to the invention.
  • the positions and amino acids that are substituted that are listed in Table 3 are applicable to any suitable first hybrid zinc finger domain as long the degron tag and/or hybrid zinc finger polypeptide according to the invention maintains a zinc finger-like fold and exhibits the properties as disclosed herein (e.g., sensitivity to IMiD in the context of protein degradation).
  • the position and substitutions are relative to the first hybrid zinc finger domain as used in the examples herein, i.e., wherein the first portion of the first hybrid zinc finger domain is a beta-hairpin region of ZFP91 ZF4 and the second portion of the first hybrid zinc finger domain is an alpha-helix region of IKZF1 ZF2 (see Figure 3; wherein the first hybrid zinc finger domain of the ‘parental degron tag’ used in the examples is schematically represented).
  • a degron tag and/or hybrid zinc finger polypeptide according to the invention, and wherein the at least two amino acids in the first portion and/or in the second portion that are substituted are selected from those listed in Table 4.
  • Table 4 lists preferred combinations of two amino acids that are substituted at two different position according to the invention in the first hybrid zinc finger domain as described herein (showing per row a preferred combination of two positions within the first hybrid zinc finger domain and the corresponding substituting amino acids; for example 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 that the amino acid at position 12 that is substituted is Glutamine (Q) - in other words the original amino acids in the portions obtained from the wild type Cys2-His2 zinc finger domains are Glutamic acid as position 4 and Glutamine at position 12.
  • the at least two amino acids that are substituted are selected from any of the combinations shown in Table 4.
  • the amino acids for example the preferred amino acids as shows in Table 4 may be substituted with any suitable amino acid as long as it provides for a degron tag and/or hybrid zinc finger polypeptide according to the invention.
  • the amino acids preferably the combination of amino acids listed in Table 4 are replaced with a corresponding amino acid as listed in Table 1 or corresponding combination of amino acids as shown in Table 2 (wherein corresponding refers, for example, to the corresponding position in the first hybrid zinc finger domain as disclosed herein).
  • the current invention also encompasses the use of the positions and substitutions as listed in Table 4 (or any other Table of list provided herein) in any suitable first or second portion of a (wildtype) Cys2-His2 zinc finger domain that may be used in providing for the first hybrid zinc finger domain according to the invention.
  • the positions and amino acids that are substituted and listed in Table 4 are applicable to any suitable first hybrid zinc finger domain as long the degron tag and/or hybrid zinc finger polypeptide according to the invention maintains a zinc finger-like fold and exhibits the properties as disclosed herein (e.g., sensitivity to I Mi D in the context of protein degradation).
  • the position and amino acids that are substituted are relative to the first hybrid zinc finger domain as used in the examples herein, i.e. wherein the first portion of the first hybrid zinc finger domain is a beta- hairpin region of ZFP91 ZF4 and the second portion of the first hybrid zinc finger domain is an alpha-helix region of IKZF1 ZF2 (see Figure 3; wherein the first hybrid zinc finger domain of the ‘parental degron tag’ used in the examples is schematically represented).
  • the first Cys 2 -His 2 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;
  • the second Cys 2 -His 2 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;
  • the third Cys 2 -His 2 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
  • the fourth Cys 2 -His 2 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.
  • the current invention is not in particular limited to specific (wild type) Cys 2 -His 2 zinc finger domains that provide for the first, second, third and/or fourth portion in, respectively, the first hybrid zinc finger domain and the second zinc finger domain, in particular second hybrid zinc finger domain.
  • various wild-types or naturally occurring Cys 2 -His 2 zinc finger domains have been described and that are suitable in degron systems that include the use of IMiDs (see for example those described by (Sievers et al. Science. 2018 Nov 2;362(6414):eaat0572)).
  • Cys 2 -His 2 zinc finger domains comprising the typical beta hairpin loop and the alpha helix
  • degron tags and/or hybrid zinc finger polypeptides according to the invention that maintain a zinc finger-like fold (beta-hairpin loop and alpha helix) and exhibits the properties as disclosed herein (e.g., sensitivity to IMiD in the context of protein degradation) can be easily provided for by the skilled person.
  • the first Cys 2 -His 2 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;
  • the second Cys 2 -His 2 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;
  • the third Cys 2 -His 2 zinc finger domain is selected from the group consisting of IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5,
  • the fourth Cys 2 -His 2 zinc finger domain is selected from the group consisting of IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5,
  • amino acid sequence of the Cys2-His2 zinc finger domains for selection of a first portion and/or second portion is in some embodiments selected from the group consisting of: SEQ ID NO: 116 (IKZF1 ZF2 -
  • amino acid sequence of the Cys2-His2 zinc finger domains for selection of a third portion and/or fourth portion is in some embodiments selected from the group consisting of: SEQ ID NO: 128 (IKZF1 ZF3 -
  • the first Cys 2 -His 2 zinc finger domain provides for the first portion of the first hybrid zinc finger domain and is selected from those listed above.
  • the second Cys 2 -His 2 zinc finger domain provides for the second portion of the first hybrid zinc finger domain and is selected from those listed above.
  • these first and second portion provides for the first hybrid zinc finger domain and further comprises at least two amino acid substitutions relative to the first and/or second portion provided by the first and second Cys 2 -His 2 zinc finger domain.
  • the third Cys 2 -His 2 zinc finger domain provides for the third portion of the second hybrid zinc finger domain and is selected from those listed above.
  • the fourth Cys 2 -His 2 zinc finger domain provides for the fourth portion of the second hybrid zinc finger domain and is selected from those listed above.
  • the second zinc finger in the degron tag and/or hybrid zinc finger polypeptide according to the invention may in certain embodiment be a hybrid zinc finger domain or may be a non-hybrid zinc finger domain.
  • the second zinc finger may therefor also be selected from any of the third or fourth Cys 2 -His 2 zinc finger domain zinc finger domains listed above (providing both the first portion and the second portion from the same Cys 2 -His 2 zinc finger domain).
  • At least one, two, three or four of the first, second, third and fourth Cys 2 -His 2 zinc finger domain listed above are selected. In a preferred embodiment all of the first, second, third and fourth Cys 2 -His 2 zinc finger domain listed above are selected. In a preferred embodiment at least the first and the second Cys2- HiS2 zinc finger domain is selected from those listed above. In a preferred embodiment at least the third and fourth Cys 2 -His 2 zinc finger domain is selected from those listed above.
  • 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.
  • 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.
  • one of the first and second Cys 2 -His 2 zinc finger domain and one of the third and fourth Cys 2 -His 2 zinc finger domain are from the same zinc finger protein (e.g., a ZF4 domain from ZFP91 and a ZF5 domain from ZFP91).
  • the first and second Cys 2 -His 2 zinc finger domain are from two different zinc finger protein
  • the third and fourth Cys 2 -His 2 zinc finger domain are from the same two different zinc finger proteins
  • the first Cys 2 -His 2 zinc finger domain and the fourth Cys 2 -His 2 zinc finger domain are both from ZFP91 (respectively zinc finger domain ZF4 and ZF5)
  • the second Cys 2 -His 2 zinc finger domain and the third Cys 2 -His 2 zinc finger domain are both from a different zinc finger protein, for example IKZF1 (respectively zinc finger domain ZF2 and ZF3).
  • the Cys 2 -His 2 zinc finger domain that in the wild type zinc finger protein is N-terminal orientated relative to the other is preferably also N-terminal from the other in the degron tags and/or hybrid zinc finger polypeptides according to the invention.
  • the degron tags and/or hybrid zinc finger polypeptides according to the invention wherein the at least two amino acid substitutions are introduced in a first hybrid zinc finger domain the first portion thereof having an amino acid sequence according to SEQ ID NO: 101 (LQCEICGFTCR - ZFP91 ZF4 (first portion)), and/or the second portion thereof having an amino acid sequence according to SEQ ID NO: 102 (QKGNLLRHIKLH - IKZF1 ZF2 (second portion)), and/or the first hybrid zinc finger domain having an amino acid sequence according to SEQ ID NO: 103 (LQCEICGFTCRQKGNLLRHIKLH - (ZFP91 ZF4/IKZF1 ZF2 first hybrid zinc finger domain)).
  • the at least two amino acid substitutions according to the invention may be introduced.
  • the degron tags and/or hybrid zinc finger polypeptides according to the invention wherein the at least two amino acid substitutions are introduced (e.g. having any of the sequences according to SEQ ID NO: 101 - 103), and wherein the third portion thereof having an amino acid sequence according to SEQ ID NO: 104 (FKCHLCNYACRR - (IKZF1 ZF3 (third portion)), and/or the fourth portion thereof having an amino acid sequence according to SEQ ID NO: 105 (KDSVVAHKAKSH (ZFP91 ZF5 (fourth portion)), and/or the second hybrid zinc finger domain having an amino acid sequence according to SEQ ID NO: 106 (FKCHLCNYACRRKDSVVAHKAKSH - Second hybrid zinc finger domain).
  • the at least two amino acid substitutions are introduced (e.g. having any of the sequences according to SEQ ID NO: 101 - 103), and wherein the third portion thereof having an amino acid sequence according to SEQ ID NO: 104 (FKCHLCNYACRR
  • degron tags and/or hybrid zinc finger polypeptides wherein the at least two amino acid substitutions are introduced, and wherein the degron tags and/or hybrid zinc finger polypeptides has an amino acid sequence according to SEQ ID NO: 107 (LQCEICGFTCRQKGNLLRHIKLHSGEKPFKCHLCNYACRRKDSVVAHKAKSH).
  • SEQ ID NO: 107 LQCEICGFTCRQKGNLLRHIKLHSGEKPFKCHLCNYACRRKDSVVAHKAKSH.
  • a degron tag and/or hybrid zinc finger polypeptide comprising the two substitutions is selected from those listed in Table 5.
  • Table 5 list preferred first hybrid zinc finger domains (or hybrid zinc finger polypeptides) according to the invention.
  • SEQ ID NO: 135 is a preferred first hybrid zinc finger domain (or hybrid zinc finger polypeptides) according to the invention.
  • SEQ ID NO: 136 is comprised in a preferred degron tag according to the invention.
  • These preferred zinc finger domains are those that showed the most beneficial El index (enrichment index) as calculated according to the example section. The results thereof are summarized in Table 6.
  • degron tags and/or hybrid zinc finger polypeptides according to the invention are provided that have enhanced or increased sensitivity of IMiDs, in particular in comparison to, for example, those that comprise only one substitution in the first hybrid zinc finger domain.
  • This thus shows that despite introducing a second substitution, or more, for example a third or fourth amino acid substitution, in the first hybrid zinc finger domain, sensitivity of the resulting first hybrid zinc finger domain (having at least two substitutions) is surprisingly and dramatically improved.
  • Exemplary El values obtained with various single mutations in the first hybrid single domain used in the examples herein are show in Table 7.
  • the degron tags and/or hybrid zinc finger polypeptides according to the invention comprise a first hybrid zinc finger domain comprising the sequence of any of those listed in Table 5, it is also contemplated the first hybrid zinc finger domain of the degron tags and/or hybrid zinc finger polypeptides according to the invention comprises either the beta hairpin of the first hybrid zinc finger domains listed in Table 5 or the alpha helix of the first hybrid zinc finger domains listed in Table 5, and further comprises a different alpha helix and beta hairpin portion respectively.
  • 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.
  • 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 a further alpha helix portion not necessarily listed in Table 5.
  • the first hybrid zinc finger domain is comprised 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.
  • the first hybrid zinc finger domain of the degron tag and/or zinc finger polypeptide according to the invention is any one of those listed in Table 5, preferably any one SEQ ID 1 - 90, SEQ ID 1 - 80, SEQ ID 1 - 70, SEQ ID 1 - 60, SEQ ID 1 - 50, SEQ ID 1 - 40, SEQ ID 1 - 30, SEQ ID 1 - 20, SEQ ID 1 - 10, or SEQ ID NO: 135.
  • the first hybrid zinc finger domain of the degron tag and/or zinc finger polypeptide according to the invention is any one of SEQ ID 1 - 10, SEQ ID 11 - 20, SEQ ID 21 - 30, SEQ ID 31 - 40, SEQ ID 41 - 50, SEQ ID 51 - 60, SEQ ID 61 - 70, SEQ ID 71 - 80, SEQ ID 81 - 90, or SEQ ID 91 - 100, or or SEQ ID NO: 135.
  • the first hybrid zinc finger domain as shown in Figure 3 (ZFP91 ZF4 - IKZF1 ZF2) is replaced by any one of the sequences listed in Table 5.
  • second zinc finger domains and/or second hybrid zinc finger domains may be used (as discussed herein elsewhere; for example those obtained from IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, ZNF692 ZF5 (see also Uniprot Accession no: Q13422, Q9UKT9, Q96JP5, Q96CK0, Q8N554, Q17R98, Q9BU19, respectively), in preferred embodiments there is provided for a degron tag and/or hybrid zinc finger polypeptide according to the invention, and wherein the third portion of the second hybrid zinc finger domain comprises an amino acid sequence according to SEQ ID NO: 104 (FKCHLCNYACRR), the fourth portion of the second hybrid zinc finger domain comprises an amino acid sequence according to SEQ ID NO: 105 (KDSVVAHKAKSH), and/or wherein the second hybrid zinc finger comprises an amino acid sequence according to SEQ ID NO: 106 (FKCHLC
  • the first portion and/or the second portion of the first hybrid zinc finger domain are obtained from a first Cys2- His2 zinc finger domain and/or a second Cys 2 -His 2 zinc finger domain that is a I Mi D substrate in the native protein having said first Cys2-His2 zinc finger domain and/or second Cys 2 -His 2 zinc finger domain.
  • the first portion and the second portion of the first hybrid zinc finger domain are obtained from a first Cys2-His2 zinc finger domain and a second Cys 2 -His 2 zinc finger domain that both are IMiD substrates in the native protein having said first Cys2-His2 zinc finger domain or second Cys 2 -His 2 zinc finger domain.
  • the first portion and/or the second portion of the first hybrid zinc finger domain are preferably obtained from a first Cys2-His2 zinc finger domain and/or a second Cys 2 -His 2 zinc finger domain that is a IMiD substrate in the native protein having said first Cys2-His2 zinc finger domain and/or second Cys 2 -His 2 zinc finger domain.
  • the first portion and the second portion of the first hybrid zinc finger domain are obtained from a first Cys2-His2 zinc finger domain and a second Cys 2 -His 2 zinc finger domain that both are IMiD substrates in the native protein having said first Cys2-His2 zinc finger domain or second Cys 2 -His 2 zinc finger domain.
  • the third portion and/or the fourth portion of the second (hybrid) zinc finger domain are a third Cys2-His2 zinc finger domain and/or a fourth Cys 2 -His 2 zinc finger domain that is N-terminal or C-terminal of a Cys2-His2 zinc finger domain that is a I Mi D substrate in the native protein, preferably C-terminal.
  • the third portion and/or the fourth portion are obtained from a Cys 2 -His 2 zinc finger domain that is not an IMiD substrate in the native protein having said zinc finger domain.
  • linker peptide between the first hybrid zinc finger domain and the second hybrid zinc finger domain
  • a degron tag and/or hybrid zinc finger polypeptide according to the invention, and wherein the first hybrid zinc finger domain and the second zinc finger domain, preferably second hybrid zinc finger domain, are adjacent or are connected via a linker peptide, for example comprising one, two, three, four, five, six, seven or more amino acids.
  • linker peptide for example comprising one, two, three, four, five, six, seven or more amino acids.
  • no additional linker peptide is present.
  • a degron tag comprising a first hybrid zinc finger domain, further comprising at least two amino acid substitutions and comprising a second hybrid zinc finger domain.
  • a non-natural hybrid zinc finger polypeptide comprising a first hybrid zinc finger domain comprising a first portion and a second portion wherein
  • the first portion comprises the amino acid sequence X 1 X 2 C 3 X 4 X 5 C 6 X 7 X 8 X 9 X 10 X 1 1 , wherein X represents any amino acid, of a first Cys 2 -His 2 zinc finger domain;
  • the second portion comprises the amino acid sequence X 12 X 13 X 14 X 15 X 16 X 17 X 18 H 19 X 20 X 21 X 22 H 23 , wherein X represents any amino acid, of a second Cys 2 -His 2 zinc finger domain, wherein the second Cys 2 -His 2 zinc finger domain is different from the first Cys 2 -His 2 zinc finger domain;
  • the first and second portion together comprises (the) at least two amino acid substitutions, wherein an amino acid substitution in the first portion is relative to the first portion of the first Cys 2 -His 2 zinc finger domain and wherein an amino acid substitution in the second portion is relative to the second portion of the second Cys 2 -His 2 zinc finger domain, and wherein the substitution is not at any of positions C 3 , C 6 , X 7 , H 19 , or H 23 .
  • the hybrid zinc finger polypeptide comprises any one of the sequences listed in Table 5.
  • the hybrid zinc finger polypeptide according to the invention comprises any one of the sequences listed in Table 5, preferably any one SEQ ID 1 - 90, SEQ ID 1 - 80, SEQ ID 1 - 70, SEQ ID 1 - 60, SEQ ID 1 - 50, SEQ ID 1 - 40, SEQ ID 1 - 30, SEQ ID 1 - 20, SEQ ID 1 - 10, or SEQ ID NO: 135.
  • the zinc finger polypeptide according to the invention comprises any one of SEQ ID 1 - 10, SEQ ID 11 - 20, SEQ ID 21 - 30, SEQ ID 31 - 40, SEQ ID 41 - 50, SEQ ID 51 - 60, SEQ ID 61 - 70, SEQ ID 71 - 80, SEQ ID 81 - 90, or SEQ ID 91 - 100 or SEQ ID NO: 135
  • non-natural hybrid zinc finger polypeptide according to the invention and wherein the non-natural hybrid zinc finger comprises a first hybrid zinc finger domain as defined herein.
  • non-natural hybrid zinc finger polypeptide according to the invention further comprising a second zinc finger domain, wherein the second zinc finger domain is a non-hybrid zinc finger domain.
  • the non-natural hybrid zinc finger polypeptide according to the invention further comprising a second zinc finger domain, wherein the second zinc finger domain is a non-hybrid zinc finger domain.
  • the second zinc finger domain may be a wild type Cys 2 -His 2 zinc finger domain, for example, such as those disclosed herein, for example IKZF1 ZF3, IKZF3 ZF3, but also ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, and/or ZNF827 ZF2, ZNF692 ZF5.
  • the second zinc finger domain is preferably oriented C-terminal relative to the first hybrid zinc finger domain and may be directly adjacent or may be linked to the first hybrid zinc finger domain, as described herein elsewhere.
  • the second zinc finger domain is a second hybrid zinc finger domain, such as those described herein elsewhere.
  • the second zinc finger domain in the hybrid zinc finger domain according to the invention is a non-hybrid, e.g., wild type or naturally occurring zinc finger domain, or variation thereof
  • the second zinc finger domain may preferably be selected from e.g. IKZF1 ZF3 (SEQ ID NO: 109 (FKCHLCNYACRRRDALTGHLRTH) or IKZF3 ZF3 (SEQ ID NO: 110 (FKCHLCNYACQRRDALTGHLRTH) or comprises an amino acid sequence according to SEQ ID NO: 109 or SEQ ID NO: 110.
  • suitable second zinc finger domains include, for example, IKZF1 ZF3, IKZF3 ZF3, ZFP91 ZF5, ZNF653 ZF5, ZNF276 ZF5, ZNF827 ZF2, ZNF692 ZF5, SEQ ID NO: 109 - 115, and the like.
  • a fusion protein comprising a degron tag according to the invention, or a non-natural hybrid zinc finger polypeptide according to the invention, further comprising a protein of interest.
  • This aspect of the invention is directed to a fusion protein including a protein of interest (POI) and a degron tag or hybrid zinc finger polypeptide according to the invention and that can mediate degradation of the POI comprising fusion protein in connection to the use of an IMiD.
  • POI protein of interest
  • the degron tag of hybrid zinc finger polypeptide may be located N-terminal to the POI, C-terminal to the POI or within the POI.
  • the fusion protein according to the invention may be used in methods of degrading a protein of interest, and that includes contacting a cell expressing the fusion protein with an effective amount of an IMiD.
  • the protein of interest may be any suitable protein (of fragment thereof).
  • the protein of interest is an endogenous protein.
  • the protein of interest is an exogenous protein.
  • the protein of interest is a recombinant protein or non-natural protein.
  • the protein of interest is a natural occurring protein.
  • the degron tags and/or hybrid zinc finger proteins of the current invention can be utilized to produce an endogenous protein- degron tag fusion protein or exogenous protein-degron tag fusion protein, for example stably expressed in a cell.
  • Endogenous proteins originate within an organism, tissue or cell and is expressed by that same organism, tissue or cell, whereas exogenous proteins originate outside of an organism, tissue or cell and are introduced into the organism, tissue or cell.
  • protein of interest in the fusion proteins according to the invention is not in particular limited to any particular type of protein
  • suitable example of protein of interest include, for example, Cbl-b, SOCS1 , CISH, Tox, Eomes, IL12, IL15.
  • CBL-B is an E3 ubiquitin-protein ligase that in humans is encoded by the CBLB gene.
  • SOCS family proteins form part of a classical negative feedback system that regulates cytokine signal transduction. SOCS1 is involved in negative regulation of cytokines that signal through the JAK/STAT pathway. Through binding to JAKs and IFNGR1 , inhibits their kinase activity. In vitro, also suppresses Tec protein-tyrosine activity.
  • Cytokine-inducible SH2-containing (CISH) protein is a protein that in humans is encoded by the CISH gene
  • CISH controls T cell receptor (TCR) signaling and contains a SH2 domain and a SOCS box domain.
  • Thymocyte selection-associated high mobility group box protein TOX is a protein that in humans is encoded by the TOX gene. 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 that in humans is encoded by the EOMES gene. Eomesodermin/Tbr2 is highly expressed in CD8+ T cells, but not CD4+ T cells.
  • Interleukin 12 is an interleukin that is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) in response to antigenic stimulation.
  • Interleukin-15 is a cytokine that binds to and signals through a complex composed of 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 by virus(es) and induces the proliferation of natural killer cells.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the chimeric polypeptide described therein is designed to interact with phosphorylated immunoreceptor tyrosine-based activation motifs (ITAM) in the TCR/CD3 complex and/or CAR and/or NK cell receptor (NKR) complexes that contain ITAM bearing signaling molecules such as DAP12, gamma chain of the immunoglobulin receptor FceRI or CD3 zeta chain (Lanier et al, Nat Immunol. 2008 May; 9(5): 495-502).
  • ITAM phosphorylated immunoreceptor tyrosine-based activation motifs
  • the chimeric polypeptides of WO2021080427 provides for 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-regulated protein stability domain that is utilized to modulate (e.g., reduce or increase) in a time and/or dose dependent manner the expression of the chimeric polypeptide.
  • this/the small molecule-regulated protein stability domain is a degron tag and/or hybrid zinc finger polypeptide according to the invention.
  • the protein of interest is as is disclosed in detail in WO2021080427, comprising a first part comprising a SH2-domain from a protein which binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), and preferably also comprising a second part comprising an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).
  • ITAM phosphorylated immunoreceptor tyrosine-based activation motif
  • ITSM immunoreceptor tyrosine-based switch motif
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • This protein is preferably combined with a third part comprising a small molecule-regulated protein stability domain, which is, in accordance with the current invention a degron tag or a hybrid zinc finger polypeptide as disclosed herein.
  • the ITAM is an ITAM comprised in a T cell receptor (TCR) complex and/or a NK cell receptor (NKR) complex and/or a chimeric antigen receptor (CAR), preferably an ITAM derived from a CD3 zeta chain, a CD3 epsilon chain, a CD3 delta chain, CD3 gamma chain, gamma chain of the immunoglobulin receptor FceRI and DAP12.
  • the SH2-domain is from a protein selected from the group consisting of Zap70, Syk, and Lek.
  • the chimeric polypeptide comprises more than one SH2-domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif.
  • the ITIM and/or ITSM is from an inhibitory receptor protein, preferably an inhibitory immune receptor protein, preferably from a protein selected from the group consisting of PD1 , BTLA, SIRPalpha, SIGLEC5, SIGLEC9, SIGLEC11 , PECAM1 or LY9.
  • 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.
  • a cell or host cell which expresses the non-naturally occurring nucleic acid according to the invention.
  • the cell, the host cell, the protein of interest, and/or the Cys 2 -His 2 zinc finger domain or zinc finger domains, as well as other sequences in the degron tags and/or hybrid zinc finger polynucleotides according to the invention are human or are of human origin or consist essential of human sequences.
  • the first portion comprises the amino acid sequence X 1 X 2 C 3 X 4 X 5 C 6 X 7 X 8 X 9 X 10 X 1 1 , wherein X represents any amino acid, of a first Cys 2 -His 2 zinc finger domain;
  • the second portion comprises the amino acid sequence X 1 2 X 1 3 X 1 4 X 1 5 X 1 6 X 1 7 X 1 8 H 1 9 X 20 X 21 X 22 H 23 , wherein X represents any amino acid, of a second Cys 2 -His 2 zinc finger domain, wherein the second Cys 2 -His 2 zinc finger domain is different from the first Cys 2 -His 2 zinc finger domain;
  • step (C) using the hybrid zinc finger domain obtained in step (B), or the 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 any to invention, or a nucleic acid encoding these.
  • the skilled person will also understand that with respect to the at least two different amino acid substitutions that are introduced in the method of the invention, this may be at any position in the hybrid zinc finger domain comprising a first portion and a second portion, and the substitution may be into any amino acid (as long at the substituted amino acid is different from the substituting amino acid).
  • the position, the substituted amino acid, and/or the substituting amino acid are those as described in relation to any one of Tables 1 - 6, and 8, including combinations of the at least two substitutions, as well as preferences indicated.
  • the method may include the step of testing the hybrid zinc finger domain comprising the at least two substitutions, and/or the 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 these, obtained with the method of the invention of sensitivity for IMiDs, for example as described in the examples, for example by determining the El value or index as described herein.
  • the obtained hybrid zinc finger domain comprising the at least two substitutions, and/or the 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 these should be discarded or not.
  • the method includes a step of analyzing the obtained hybrid zinc finger domain comprising the at least two substitutions, and/or the 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 these for sensitivity to an IMiD, for example as described in the example section, in order to determine its usefulness as a degron tag and the like.
  • IMiD immunomodulatory imide drug
  • 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 may thus be selected for its ability to be induced by a particular small molecule, preferably an immunomodulatory inducing drug (IMiD).
  • the IMiD is a thalidomide or one of its analogues, for example, lenalidomide, pomalidomide, avadomide, or iberdomide.
  • a method of degrading a protein of interest or 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, a fusion protein according to the invention, preferably wherein the method is for regulating the activity of the protein of interest.
  • a method of degrading a protein of interest or for controlling expression of a protein of interest comprising administering an effective amount of an IMiD to a subject, preferably a human subject, wherein the subject 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 a fusion protein according to the invention, preferably comprising the protein of interest.
  • the method is a method wherein gene therapy comprises introducing cells to the subject (e.g., T cells, B cells or NK cells), wherein the introduced cells express 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 the protein of interest.
  • cells e.g., T cells, B cells or NK cells
  • the introduced cells express 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 the protein of interest.
  • These aspects of invention are directed to a method of degrading a protein of interest and that include, for example, contacting a transgenic cell with an effective amount of an IMiD, wherein the cell, for example, produces a fusion protein including a protein of interest and at least one degron tag and/or hybrid zinc finger polynucleotide according to the invention.
  • the methods may be conducted in vivo or in vitro.
  • the POIs may be exogenous or endogenous.
  • the ability to degrade a particular endogenous or exogenous protein of interest by creating POI-degron tag fusions and administering an IMiD can be used to treat disorders by controlling cellular behavior, for example as described in WO2021080427.
  • the degron tags, hybrid zinc finger polynucleotides and fusion proteins of the present invention can be utilized to produce a stably expressed endogenous protein-degron tag fusion protein or exogenous protein-degron tag fusion protein, and which levels or cellular activity may be regulated by providing or removing IMiDs to or from cells expressing such fusion proteins.
  • 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 the patient in need thereof may be as described herein or may be any other treatment.
  • 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 controlling transcription factors such as Eomes, Tox, negative regulators of TCR signaling, 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 nuclease.
  • transcription factors such as Eomes, Tox, negative regulators of TCR signaling, 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, check
  • first hybrid zinc finger domain and/or a second hybrid zinc finger domain as disclosed herein.
  • 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.
  • 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 are also provided, including those in combination with an IMiD.
  • 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., thalidomide analogue relative to, for example, wild-type zinc finger domains.
  • an IMiD molecule e.g., thalidomide analogue
  • 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 relative to the wild-type Cys 2 -His 2 zinc finger domain from which the beta-hairpins and/or the alpha helix subdomains are derived.
  • the enhanced or increased sensitivity to one or more IMiD molecules allows for a reduction in the amount of IMiD molecule administered to induce degradation by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more.
  • the amount of small molecule, e.g., IMiD molecule, administered is reduced by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 110, 120, 130, 140, 150 or more.
  • a hybrid zinc finger degron containing the ZFP91 ZF4 beta hairpin and IKZF1 ZF2 alpha helix was found to show increase sensitivity towards IMiD induced degradation as compared to non-hybrid ZFP91 ZF4 and IKZF1 ZF2 zinc finger degrons.
  • We previously created a ZFP91 ZF4 beta hairpin- IKZF1 ZF2 alpha helix- IKZF1 ZF3 degron (abbreviated as single hybrid, dual zinc finger prior art degron, Figure 1) and demonstrated that CRASH-IT switch designs that contain this single hybrid, dual zinc finger prior art degron domain can be regulated by IMiD molecules (W02021080427A1).
  • FLYRD18 Sigma-Aldrich
  • NKIRTIL006 Keratborg et al. Oncoimmunology. 2012 Jul 1 ;1 (4):409-418
  • Jurkat cells were cultured in IMDM (ThermoFisher), supplemented with 8 % FCS (ThermoFisher) and penicillin-streptomycin (100 lll/ml penicillin, 100 pg/ml streptomycin, Sigma-Aldrich).
  • FLYRD18 and NKIRTIL006 cells were passaged every 2-3 days with trypsin-EDTA (ThermoFisher). All cell lines were tested for mycoplasma using PCR based screening and found negative.
  • Retroviral particles were produced in FLYRD18 packaging cells.
  • 700,000 FLYRD18 packaging cells were plated per 10 cm dish one day prior to transfection. The next day, cell culture medium was refreshed with IMDM supplemented with 8% FCS without antibiotics.
  • 25 ⁇ l of X-tremeGENE 9 (Roche) was mixed with 800 ⁇ l Opti-MEM (ThermoFisher) and incubated for 5 minutes. Subsequently, the Optimem-X-tremeGENE 9 mixture was added on top of 10 ⁇ g retroviral plasmid DNA dissolved in water and incubated for 15 minutes, and the resulting transfection mixture was added dropwise onto the packaging cells.
  • Retrovirus containing supernatant was harvested 48 hours after transfection and immediately used or snap-frozen in liquid nitrogen.
  • the FKBP12F36V encoding sequence in this vector was replaced by gene synthesis products encoding double hybrid degron ( Figure 3) or double hybrid degron variants containing the G14N/K21A, G14M/N15R, L17I/K21 L, E4R/Q12L, Q12R/K1T or Q12R/K13V dual substitutions, or Q12R/K13V/G14N/K21A quadruple substitutions (SED ID NO: 135; SEQ ID NO: 136) using the Gibson assembly method.
  • Jurkat cells expressing the SynFinger library were treated with 50 nM lenalidomide (a suboptimal dose that activates only -50% of cells expressing a CRASH-IT switch containing the parental double hybrid degron in Jurkat cells, as compared to a maximally effective 1000 nM lenalidomide dose) or were mock treated for 24 hours, and subsequently activated with anti-CD3/anti-CD28 antibodies in the presence or absence of 50 nM lenalidomide for 5 hours. Afterwards, cells were stained with IR dye (1 :400) and anti-CD69-PE (1 :200). Cells were sorted for live (IR dye negative), EGFP high end CD69 high (top 5%) or CD69 low (bottom 5%) expression using Aria Fusion cytometers.
  • 50 nM lenalidomide a suboptimal dose that activates only -50% of cells expressing a CRASH-IT switch containing the parental double hybrid degron in Jurkat cells, as compared to a maximally effective
  • Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions, using 5x10 ⁇ 6 cells per column. DNA was eluted in 22 ⁇ l RNAse free water. Deep sequencing adapters and indexes were added to SynFinger encoding DNA sequences using PCR amplification. Briefly, 3 ⁇ g genomic DNA (20 ul), indexed forward and reverse primers (10 ⁇ M, 2.5 ⁇ l each) and 25 ⁇ l NEBNext® High-Fidelity 2X PCR Master Mix were added to the PCR reaction.
  • CD69-HI and CD60-LO read counts from top 100 SynFingers were compared with read counts from the parental double hybrid degron using a two-sided 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.
  • the results for SEQ ID NO: 1 - 100 are shown in Table 6 and 9.
  • the top 100 SynFingers identified with this method were all dual amino acid substitution (Table 6). Importantly, certain amino acid substitutions such as Q12R, Q12K, N15R and L22R (present in 11 %, 8%, 9% and 6% of top 100 SynFingers, respectively) were observed frequently among the top 100 SynFinger containing dual substitution mutations ( Figure 4). Notably, these mutations were also ranked among the top hits when analyzed as single amino acid substitutions.
  • T cells expressing G14N/K21A, G14M/N15R, L17I/K21 L, E4R/Q12L, Q12R/K1T and Q12R/K13V dual substitution containing SynFingers showed improved cytokine production at very low (5 nM) lenalidomide concentration levels as compared to T cells expressing the double hybrid degron containing CRASH-IT switch that formed the starting point of the genetic screen ( Figure 5).
  • CRASH-IT switch variants containing parental ZF, or SynFinger degrons with dual substitutions (Q12R/K13V or G14N/K21A) or quadruple substitutions (Q12R/K13V/G14N/K21A; SEQ ID NO: 135 and SEQ ID NO: 136).
  • Primary human T cells were transduced to express the CDK4 TOR, together with indicated CRASH-IT variants.
  • the synthetic zinc finger (SynFinger) sequences we identified in this study may be used in a range of applications beyond their use in the CRASH-IT switch platform.
  • important regulators of cell function such as receptors that determine the antigen specificity of immune cells, but also regulators controlling antigen sensitivity, cytokine signaling, or cell differentiation may be regulated by creating fusion proteins with such novel SynFinger sequences.
  • the ability to induce protein degradation at reduced IMiD levels opens new possibilities to implement this protein degradation technology in clinical settings both in cellular therapies and beyond.

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