Novel drug-induced degradation label

Abstract

The present invention provides novel drug-inducible degradation tags (degradation determinants), as well as fusion proteins, cells and pharmaceutical compositions comprising the drug-inducible degradation tags. Nucleic acid sequences and vectors encoding the new degradation determinants are also provided. Also provided herein are methods of using the novel degradation determinants, fusion proteins, cells, pharmaceutical compositions, nucleic acid sequences, and vectors.

Description

Technical Field

[0001] The present invention provides novel drug-inducible degradation tags (degrons), as well as fusion proteins, cells, and pharmaceutical compositions comprising the drug-inducible degradation tags. Nucleic acid sequences and vectors encoding the novel degrons are also provided. Methods of using the novel degrons, fusion proteins, cells, pharmaceutical compositions, nucleic acid sequences, and vectors are also provided herein. Background Art

[0002] In recent years, chemical biology tools that can induce the degradation of target proteins with drug-like precision have received a lot of attention because they can acutely and reversibly regulate protein levels in cells. These techniques are based on the use of small amino acid tags fused to the protein of interest, which, in the presence of a specific compound, will induce the degradation of that protein. There are currently several available techniques, examples of which include small molecule displacement of cryptic degrons 1 , small molecule-assisted shutdown (SMASh) 2 , degradation by fusing a hydrophobic tag (HaloTag), auxin-inducible degrons (AID1 / 2), HaloPROTAC 3 , and the dTAG system 4,5 . These tools have enabled numerous discoveries at the preclinical stage, but their various limitations (requiring the expression of multiple components, poor PK / PD of the associated compounds, use of non-FDA-approved molecules) have hindered translation into more clinically relevant applications.

[0003] One of the best-described examples of drug-induced protein degradation is the mechanism of action of thalidomide-like derivatives (referred to as immunomodulatory imide drugs IMiD, or more recently Cereblon E3 ligase modulators CELMoD). These degrader drugs bind to a conserved tryptophan cage on the surface of Cereblon (CRBN), which is a substrate receptor of the Cullin4-RING E3 ubiquitin ligase CRL4 CRBN , and induce a change in the substrate specificity of CRL4 CRBN , resulting in the recruitment, ubiquitination, and subsequent proteasomal degradation of new substrates such as Ikaros (IKZF1), Aiolos (IKZF3), casein kinase 1α (CK1a or CSNK1A), G1 to S phase transition protein 1 (GSPT1), and zinc finger protein 91 (ZFP91) 6 .

[0004] The new substrates interact with the degrader-binding surface of CRBN through recognition motifs called degrons, which are characterized by a common β-hairpin loop with a conserved glycine at the apex, which is crucial for the interaction of the degron with the degrader compound. The amino acid sequence adjacent to the sentinel glycine in the β-hairpin loop is variable among the new substrates and is crucial for the specificity of recruitment and degradation of a particular CRBN-binding compound to the new substrate. In the case of the new substrates Ikaros, Aiolos, and ZFP91, the degron motif is located within the C2H2 zinc finger (ZF) domain, and the first half of this domain is characterized by a CxxCG sequence containing a conserved glycine only after the second cysteine of the ZF 7,8 .

[0005] The concept of a molecular switch for regulating chimeric antigen receptor (CAR) expression is widely regarded in the field as an ideal solution to improve the safety and efficacy of CAR-T cells. In proof-of-concept work, the Mackall group at Stanford University demonstrated that in terms of CAR-T efficacy, inducing the degradation of CAR in human T cells has two major advantages over conventional CAR expression 9 . First, the degradation of CAR during T cell manufacturing reduces T cell exhaustion associated with CAR tonic signaling and generates T cell products that enhance T cell survival and lead to increased survival of tumor-bearing animals after administration of the product. Second, if a drug that induces the degradation of CAR on T cells can be administered to tumor-bearing animals in an intermittent dosing regimen, T cells can be transiently rested from antigen-induced activation in the tumor environment. Thus, T cells can recover from exhaustion and have enhanced effector function when CAR is re-expressed. A third potential advantage of the degradation switch is the ability to turn off CAR-T function in the face of toxicity, providing a new additional safety feature

[0006] These data on the functional benefits of transient CAR shutdown in T cells were demonstrated using the FK506-binding protein 12 destabilizing domain, which can be blocked by a chemical probe 9 . To translate this technology into clinical benefit, it is necessary to develop CARs that are reversibly degradable by drugs approved for human use, function at pharmacologically achievable doses, are non-toxic, and can penetrate well into tumor tissue

[0007] Jan and colleagues 10 provided such proof-of-concept data, where they evaluated the molecular glue concept, i.e., that IMiD drugs (thalidomide derivatives) recruit CRL4 CRBNThe E3 ubiquitin ligase binds to a substrate protein known to contain a zinc finger degron motif. A 60 - amino - acid prototype degron based on zinc fingers, a hybrid of ZFP91 and IKZF3, which they call the "super degron", has been identified and shown to cause lenalidomide - dependent reversible degradation of a second - generation (41BB - ζ) chimeric antigen receptor that is tagged with the super degron at the cytoplasmic C - terminus.

[0008] There is a need for an improved degron technology to avoid CAR T - cell toxicity and improve cancer treatment. SUMMARY OF THE INVENTION

[0009] The present inventors developed new drug - inducible degradation tags. Using structural and sequence analysis, they generated 23 different protein tags (potential degrons) based on chimeric structures of IKZF1 (Ikaros) and ZFP91 (zinc finger protein 91). The drug - inducible degradation of each tag was determined, and DCD23 (also referred to herein as iTAG1) was identified as a potential degron candidate due to its relatively small size (60 amino acids) and good IMiD / CELMoD degradation properties ( Figure 1 ). Then, the inventors performed further amino - acid substitutions to further optimize the DCD23 degron sequence. In this process, the inventors identified four core amino - acid substitutions that disrupted the nuclear localization of the mutant degron tag (see iTAG2v1, iTAG2v2, and iTAG2 (note that the latter is also referred to herein as DCD23mut), which will be described in more detail below). Disrupting nuclear localization / accumulation in the nucleus is particularly advantageous when the degron tag is fused to a cell - surface protein such as a chimeric antigen receptor (CAR) because it promotes cell - surface expression of the tagged protein.

[0010] The present invention is based on the surprising discovery that when a new mutant degron tag (such as DCD23mut, also referred to herein as iTAG2) is fused to a CAR, the resulting CAR fusion protein has improved membrane stability compared to CAR labeled with DCD23 in Jurkat cells, 293T cells, and human primary T cells, while still being rapidly degraded in the presence of IMiD / CELMoD such as lenalidomide, pomalidomide, and iberdomide. Advantageously, the expression of DCD23mut-labeled CAR-T is also superior to that reported for the patented degron "super degron" (WO2019089592). In addition, DCD23mut-labeled CAR-T expressed in primary human T cells shows the same level of antigen-dependent reactivity as that observed for unlabeled CAR-T (which is approximately 10-fold superior to DCD23-labeled CAR-T).

[0011] Accordingly, the present invention provides a degron tag comprising the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:66), wherein X1, X2, X3, and X4 are not K, R, or H.

[0012] Suitably, the degron tag may comprise the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:2), wherein:

[0013] X1 is E or a conservative amino acid substitution thereof;

[0014] X2 is A or a conservative amino acid substitution thereof;

[0015] X3 is N or a conservative amino acid substitution thereof; and

[0016] X4 is E or a conservative amino acid substitution thereof.

[0017] Suitably, the tag may comprise the amino acid sequence LQCEICGFTCEQAGNLLNHIELH (SEQ ID NO:5).

[0018] Suitably, the tag may comprise the amino acid sequence LQCEICGFTCEQAGNLLNHIELHSG (SEQ ID NO:12) or LQCEICGFTCEQAGNLLNHIELHTG (SEQ ID NO:13).

[0019] Optionally, the tag may comprise additional N-terminal zinc finger α-helix subdomains and additional C-terminal zinc finger β-hairpin subdomains flanking SEQ ID NO:66, 2, 5, 12 or 13.

[0020] Optionally, the additional C-terminal zinc finger β-hairpin subdomain may comprise the amino acid sequence CHLCNYACR(SEQ ID NO:14)CHLCNYACQ(SEQ ID NO:15), CHLCNYACRRRDAL(SEQ ID NO:69) or CHLCNYACQRRDAL(SEQ ID NO:70).

[0021] Optionally, the additional N-terminal zinc finger α-helix subdomain may comprise the amino acid sequence PNVLMVHX5X6SH(SEQ ID NO:71) or FNVLMVHX5X6SH(SEQ ID NO:72); where X5 and X6 are not R, K or H.

[0022] Optionally, the additional N-terminal zinc finger α-helix subdomain may comprise the amino acid sequence PNVLMVHX5X6SH(SEQ ID NO:16) or FNVLMVHX5X6SH(SEQ ID NO:17); where

[0023] X5 is N or its conservative amino acid substitution; and

[0024] X6 is E or its conservative amino acid substitution.

[0025] Optionally, the additional N-terminal zinc finger α-helix subdomain may comprise the amino acid sequence PNVLMVHNESH(SEQ ID NO:22) or FNVLMVHNESH(SEQ ID NO:23).

[0026] Optionally, the tag may comprise the amino acid sequence:

[0027] PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSG EX8PFKCHLCNYACRRRDAL(SEQ ID NO:73), or

[0028] FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTG EX8PFKCHLCNYACQRRDAL(SEQ ID NO:74);

[0029] where X7 and X8 are not R, K or H.

[0030] Optionally, the tag may comprise the amino acid sequence:

[0031] PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSG EX8PFKCHLCNYACRRRDAL(SEQ ID NO:24), or

[0032] FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTG EX8PFKCHLCNYACQRRDAL(SEQ ID NO:45);

[0033] wherein:

[0034] X7 is I or a conservative amino acid substitution thereof; and

[0035] X8 is I or a conservative amino acid substitution thereof.

[0036] Optionally, the tag may comprise the amino acid sequence:

[0037] PNVLMVHNESHTGEIPLQCEICGFTCEQAGNLLNHIELHSGEIPFKC HLCNYACRRRDAL(SEQ ID NO:43), or

[0038] FNVLMVHNESHTGEIPLQCEICGFTCEQAGNLLNHIELHTGEIPFKC HLCNYACQRRDAL(SEQ ID NO:44).

[0039] Optionally, the tag may have a length of about 23 to about 70 amino acids.

[0040] Optionally, the tag may have a length of about 23 to about 60 amino acids.

[0041] The present invention also provides a fusion protein comprising a protein of interest and at least one degron tag of the present invention.

[0042] Optionally, the degron tag may be located at the C-terminus of the protein of interest.

[0043] Optionally, the protein of interest may be a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor (TCR) fusion construct (TRuC), a T cell antigen conjugate (TAC), a chimeric autoantibody receptor (CAAR), or an antibody-conjugated T cell receptor (ACTR).

[0044] Optionally, the CAR fusion protein may comprise, from the N-terminus to the C-terminus:

[0045] a) An extracellular ligand-binding domain;

[0046] b) A transmembrane domain;

[0047] c) A cytoplasmic domain comprising at least one intracellular signaling domain; and

[0048] d) At least one degron tag of the present invention.

[0049] Optionally, the CAR fusion protein may comprise an extracellular ligand-binding domain that comprises an antibody or an antigen-binding fragment, and the antigen-binding fragment is an scFv that binds B7H3.

[0050] Optionally, the CAR fusion protein may comprise a CD28 transmembrane domain.

[0051] Optionally, the CAR fusion protein may comprise a CD3 signaling domain.

[0052] Optionally, the CAR fusion protein may comprise a CD28 co-stimulatory domain.

[0053] Optionally, the CAR fusion protein may comprise an extracellular ligand-binding domain that comprises an antibody or an antigen-binding fragment (the antigen-binding fragment is an scFv that binds B7H3), a CD8 transmembrane domain, a CD3 signaling domain, a CD28 co-stimulatory domain, and at least one degron tag of the present invention.

[0054] The present invention also provides a non-naturally occurring nucleic acid sequence encoding the degron tag of the present invention or the fusion protein of the present invention.

[0055] The present invention also provides a vector comprising the nucleic acid sequence of the present invention.

[0056] Optionally, the vector may be a viral vector, optionally wherein the viral vector is selected from retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, vaccinia viral vectors, picornaviral vectors, and alphaviral vectors.

[0057] The present invention also provides a cell expressing the nucleic acid sequence of the present invention or the vector of the present invention.

[0058] Optionally, the cell may be an immune effector cell.

[0059] Suitably, the cell can be selected from: T cells, B cells, plasma cells, NK cells, NKT cells, innate lymphoid cells, macrophages, dendritic cells, monocytes, neutrophils, basophils, eosinophils, mast cells, hematopoietic progenitor cells, hematopoietic stem cells, other adult stem cells such as neural, corneal, muscle, skin, small intestine, colon, bone, mesenchymal, embryonic stem cells and induced pluripotent stem cells.

[0060] Suitably, the cell can be a mammalian cell, optionally, wherein the cell is a human cell.

[0061] The present invention also provides a pharmaceutical composition comprising the degrader tag, fusion protein, nucleotide sequence, vector or cell of the present invention, and a pharmaceutically acceptable excipient, carrier, adjuvant and / or diluent.

[0062] The present invention also provides a pharmaceutical composition of the present invention for use as a medicament.

[0063] Suitably, the pharmaceutical composition of the present invention can be used for immunocyte therapy.

[0064] The present invention also provides a method for degrading a protein of interest, comprising:

[0065] Contacting a cell in vitro or in vivo with an effective amount of an immunomodulatory drug (IMiD) or cereblon modulator (CELMoD), wherein the cell expresses a nucleic acid encoding the fusion protein of the present invention.

[0066] The present invention also provides a method for degrading a protein of interest, comprising:

[0067] Administering to a subject an effective amount of an immunomodulatory drug (IMiD) or cereblon modulator (CELMoD), wherein the subject has been previously treated by gene therapy to cause at least some endogenous cells to express a nucleic acid encoding the fusion protein of the present invention.

[0068] Suitably, the IMiD or CELMoD can be thalidomide, pomalidomide, lenalidomide, CC-122, CC-220 or CC-885.

[0069] Throughout the description and claims of this specification, the words "comprising" and "containing" and their variants mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps.

[0070] Unless the context otherwise requires, throughout the description and claims of this specification, the singular includes the plural. In particular, in the case of using an indefinite article, unless the context otherwise requires, the specification should be understood to contemplate both the plural and the singular.

[0071] Features, integers, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention should be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0072] Aspects of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Embodiments of the invention are further described below with reference to the accompanying drawings, in which:

[0074] Figure 1 : Experimental evaluation of DCD tags. (a) Heatmap summary of degradation substrate data for all DCDs tested in a flow cytometry experiment. Normalized values are represented as Log2 fold change relative to the DMSO control. Among the chimeric ZF DCD group (21 - 23), DCD21 produced the strongest degradation, but it was significantly larger in size compared to DCD23 (131 residues and 60 residues respectively). (b) Representation of iTAG1 - DCD23 (60aa). Two Cys and two His in the C2H2 zinc finger motif are highlighted in light gray. Key glycines in the β - hairpin loop degron are highlighted in bold dark gray.

[0075] Figure 2 : Design of iTAG2. Representations of iTAG1 (DCD23) and iTAG2. Two Cys and two His in the C2H2 zinc finger motif are highlighted. Key glycines in the β - hairpin loop degron are highlighted. Mutated amino acids in iTAG2 are also highlighted in bold dark gray and underlined (and described in detail elsewhere herein). The sequence of the super degron is also shown (as described in WO2021188286 and WO2019089592). Differences between the super degron and iTAG are highlighted in italics on the super degron sequence.

[0076] Figure 3 : Experimental validation of iTAG2. (a) HMECs expressing dox - inducible EGFP - iTAG1 / 2 or their corresponding P2A control treated with increasing concentrations of CC - 220 for 4 hours. Loss of the target protein was verified by measuring the EGFP median intensity by flow cytometry. P2A is a ribosomal skipping sequence that can translate EGFP and iTAG separately, which provides evidence for the degradation specificity to the iTAG fusion. (b) Immunofluorescence images of HMECs expressing dox - inducible EGFP - iTAG1 / 2 show abolished nuclear accumulation of iTAG1 using iTAG2.

[0077] Figure 4: iTAG2 mutation. Representation of iTAG2 and its mutations. Two Cys and two His in the C2H2 zinc finger motif are highlighted. Key glycine in the b-hairpin loop of the desolvated stator is highlighted. Mutated amino acids are also highlighted in bold dark gray and underlined (and described in detail elsewhere in this text).

[0078] Figure 5 : Construct for expressing a chimeric antigen receptor tagged. The CAR is expressed by the gammaretroviral construct SFG driven by the EF1α promoter. The RQR8 marker gene expresses the human CD34 epitope, which allows the detection of transduced cells using the anti-CD34 monoclonal antibody clone QBEND10. RQR8 is separated from the CAR by the T2A ribosomal skipping site to allow for the equal translation of the two protein products. The chimeric antigen receptor contains the TE9 anti-B7H3 binder in ScFv format, the CD28 and CD3ζ second-generation signaling domains, and the CD8 hinge sequence and transmembrane sequence. At the C-terminus, iTAG1 (DCD23) or iTAG2 is cloned in-frame with the CD3ζ sequence.

[0079] Figure 6 . Flow cytometry histograms show paired samples for the expression of CAR (upper row) and CD34 (lower row) in transduced 293T (left) and Jurkat cells (right). The left histogram represents the isotype control, while the right histograms represent the expression of CAR or CD34, respectively. The numbers shown indicate the percentage of population staining for the respective markers.

[0080] Figure 7 . Time course of the downregulation of iTAG2- and DCD23-tagged chimeric antigen receptors (TE9-28-Z) after the addition of IMiD drugs to transduced Jurkat cells. CAR expression was determined by direct staining of the TE9 ScFv component and evaluated by flow cytometry.

[0081] Figure 8 . Representative flow cytometry of human primary T cells expressing the CD34 marker gene or TE9-28-Z CAR after 24 hours of culture in the presence or absence of 10 μM lenalidomide. The rectangle depicts the viable population of CAR effectively eliminated by the addition of the drug. The flow plots represent two independent donors.

[0082] Figure 9 . CAR-transduced human primary T cells from two independent donors were cultured with SupT1 cells transduced to express human B7H3 at an effector-to-target ratio of 1:1. Error bars represent the mean and standard deviation of replicate values from two independent donors. UT = untransduced control, DCD2 tag and iTAG2 tag refer to the TE9-28-Z CAR with their respective C-terminal tags.

[0083] Figure 10 . Comparison of the expression of iTAG2-labeled CAR with the non-mutated version after transduction into Jurkat cells.

[0084] Figure 11 . Schematic representation of the TR-FRET assay based on the Aiolos peptide, used to measure the relative affinity of the complexes formed between iTAG1 and iTAG2 for the CRBN / DDB1 complex and various IMiDs.

[0085] Figure 12 . TR-FRET assay results of iTAG1 and iTAG2 obtained in the presence of lenalidomide, pomalidomide, and ibedilomide, including the IC calculated from the TR-FRET curves 50 .

[0086] Figure 13 . By fluorescence microscopy, transiently transfected HMEC cells fused with GFP super degron showed strong nuclear GFP localization. GFP-iTAG1 and GFP-iTAG2 images are shown for comparison and reproduced from Figure 3 b.

[0087] Figure 14 . A - Size-exclusion chromatography (SEC) profiles obtained with samples composed of a mixture of the CRBN / DDB1 complex and iTAG2 in the presence or absence of ibedilomide. B - SDS-PAGE analysis of representative fractions from SEC analysis in the absence of ibedilomide. Peak 1 corresponding to the higher molecular weight species did not contain iTAG2, and iTAG2 was only present in peak 2. C - SDS-PAGE analysis of representative fractions from SEC analysis in the presence of ibedilomide. Peak 1 corresponding to the higher molecular weight species contained iTAG2, confirming the formation of a complex between CRBN / DDB1 and iTAG2 in the presence of ibedilomide.

[0088] Figure 15 . iTAG2 CAR-T is re-upregulated after IMiD washout. (a) γ-retrovirally transduced TE9-only or TE9-iTAG2 CAR-T cells were treated overnight with a series of concentrations of ibedilomide, and then CAR expression was analyzed using flow cytometry. (b) The CAR-T was then washed in PBS to remove the drug and rested for an additional 24 hours in RPMI1640 medium. CAR expression was measured before and after resting. 0.01 uM ibedilomide (shown by the black arrow) was determined to be the optimal IMiD concentration to mediate CAR downregulation after treatment and CAR re-upregulation after drug withdrawal. Representative data from CAR-T of one donor are shown.

[0089] Figure 16 .iTAG2 CAR-T functions in the form of lentivirus. (a) The TE9-iTAG2 anti-B7H3 CAR-T construct was initially expressed using the γ-retrovirus SFG construct (Constructs 1 and 2). Then the TE9-iTAG2 transgene was transferred to the pCCL lentiviral backbone (Construct 3), and finally codon-optimized and simplified by removing the restriction endonuclease cleavage sites used in previous molecular cloning to generate Construct 4. The full expression cassettes of Constructs 3 and 4 are shown in the inset (b). (c) The sensitivities of Constructs 3 and 4 to 0.01 uM lenalidomide treatment overnight were compared (data of technical replicates of three different CAR-T donors are shown). Both Constructs 3 and 4 (C3 and C4) downregulated the CAR response to IMiD, but interestingly, Construct 3 expressed lower levels of RQR8 even in the absence of drug treatment. Therefore, Construct 4 was used as the leading lentiviral vector for additional functional evaluation. (d) Construct 4 TE9-iTAG2 CAR-T cells were co-cultured with SupT1-B7H3 T lymphoma target cells at an E:T ratio of 1:2 overnight (data of six different CAR-T donors are shown). Since complete targeted killing of CAR-T cells was observed, the E:T ratio was increased to 1:10, and approximately 80% of targeted killing was observed overnight. This indicates that TE9-iTAG2 CAR-T cells have high cytotoxicity and are capable of potent and continuous targeted killing. (e, f) To confirm the lack of antigen-nonspecific TE9-iTAG2 CAR-T reactivity, cytokine production was measured by ELISA after culturing overnight at an E:T ratio of 1:10 with SupT1-B7H3 targets or without targets. Cytokines were produced only in the presence of tumor targets (data of three different CAR-T donors are shown). (g) The lack of cytotoxicity of codon-optimized TE9-iTAG2 against B7H3-negative SupT1 targets was confirmed using an overnight luminescence-based assay at an E:T ratio of 1:2 (data of three independent CAR-T donors are shown).

[0090] The patents, scientific and technical literature cited herein establish the knowledge available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications cited herein are incorporated by reference herein to the same extent as if each such content were specifically and individually indicated to be incorporated by reference herein. In case of any inconsistency, the present disclosure shall prevail.

[0091] Aspects of the present invention are further described in detail below. Detailed Description

[0092] The present invention provides new drug - inducible degradation tags (degron tags), as well as fusion proteins, cells, and compositions comprising the drug - inducible degradation tags. Also provided herein are methods of using the new degrons, fusion proteins, cells, and compositions, including methods of modulating protein abundance in a target - specific manner via the degron tag. The present invention can equally target endogenous and exogenous (e.g., therapeutic) proteins. As disclosed herein, a degron tag is a peptide that, when fused to a target protein of interest (POI), converts the POI into a substrate that can be targeted for degradation.

[0093] Degron tags are also referred to in the art as zinc - finger degron domains. The terms "degron tag" and "zinc - finger degron domain" are used interchangeably herein. The degron tags provided herein bind to the complex formed between CRBN and an IMiD; or between CRBN and a cereblon modulator (CELMoD). In other words, the degron tags provided herein bind to the CRBN - IMiD or CRBN - CELMoD complex. Methods for determining the binding of a degron tag to the CRBN - IMiD or CRBN - CELMoD complex are well - known in the art, for example, see Degorce et al., 2009 (PMID 20161833) regarding in vitro HTRF assays and Sievers et al., 2018 (PMID 30385546) regarding using TR - FRET for ZF degron.

[0094] The degron tags provided herein comprise an amino acid sequence that is a mutant form of the minimal degron sequence provided in WO2021 / 188286 (LQCEICGFTC R Q K GNLL R HI K LH (SEQ ID NO:1)) (where the underlined amino acids are the mutants in the present invention). This minimal degron sequence corresponds to a complete C2H2 - type zinc - finger protein domain, consisting of an N - terminal β - hairpin sub - domain containing 2 Cys and a C - terminal α - helix sub - domain containing 2 His, and these 4 residues coordinate with a Zn2+ ion to stabilize the patterned C2H2 zinc - finger protein fold. Thus, the degron tags provided herein comprise an amino acid sequence that is a mutant form of the minimal degron sequence shown to be functional in the prior art.

[0095] The inventors demonstrate herein that although the novel degron tags provided herein contain amino acid sequences that are mutant variants of the minimal degron sequences previously shown to be functional, the mutant variants provided herein retain functionality (in terms of their ability to induce degradation of fusion proteins containing the degron tag), while also providing some surprisingly additional advantageous properties such as increased membrane stability and / or increased cell surface expression of a fusion cell surface protein (such as a CAR).

[0096] The inventors have exemplified the present invention with a novel degron tag that contains four amino acid mutations in a previously identified minimal functional degron sequence. Accordingly, there is provided herein a degron tag that contains the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:66), wherein X1, X2, X3, and X4 are not R, K, or H.

[0097] In one example, there is provided herein a degron tag that contains the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:2), wherein:

[0098] X1 is E or a conservative amino acid substitution thereof;

[0099] X2 is A or a conservative amino acid substitution thereof;

[0100] X3 is N or a conservative amino acid substitution thereof; and

[0101] X4 is E or a conservative amino acid substitution thereof.

[0102] The inventors have shown that by mutating the amino acids X1, X2, X3, and X4 from basic amino acids to acidic, neutral, or aliphatic amino acids, the nuclear localization of the fusion protein tagged with the degron (also referred to as reduced accumulation in the nucleus) can be disrupted. Although the inventors have exemplified the present invention by using a degron wherein X1 is E, X2 is A, X3 is N, and X4 is E, the present invention is equally applicable to other degron variants wherein the basic amino acids at positions X1, X2, X3, and X4 are replaced with appropriate alternative non-basic amino acids. Accordingly, there are provided herein novel degron tags that disrupt the nuclear localization of a fusion protein tagged with a degron, which contain the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:66), wherein X1 is E or any non-basic amino acid substitution of E, X2 is A or any non-basic amino acid substitution of A, X3 is N or any non-basic amino acid substitution of N, and X4 is E or any non-basic amino acid substitution of E.

[0103] In some examples, the non-basic amino acid substitution can be a conservative amino acid substitution of the variant amino acids exemplified herein. As is known to those skilled in the art, "conservative amino acid substitution" refers to an amino acid substitution in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (such as charge, hydrophobicity, and size). Conservative amino acid substitutions result in silent changes and lead to functionally equivalent degrons (in terms of their ability to bind to the CRBN-IMiD complex or the CRBN-CELMOD complex and induce the degradation of a fusion protein containing a degron tag). As long as the endogenous function is retained, intentional amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.

[0104] For example, conservative substitutions can be made according to the following table. Amino acids in the same block in the second column, preferably amino acids in the same row in the third column, can be substituted for each other:

[0105]

[0106] Table 1: Examples of groups of amino acids having similar biochemical properties, such as charge, hydrophobicity, and size (and thus can be used interchangeably at each mutation site provided herein).

[0107] Thus, the degron tag of the present invention can comprise the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:3) wherein:

[0108] X1 is E or D;

[0109] X2 is A, G, P, I, L, or V;

[0110] X3 is N, Q, C, S, T, or M; and

[0111] X4 is E or D.

[0112] In one example, the degron tag of the present invention can comprise the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:4) wherein:

[0113] X1 is E or D;

[0114] X2 is A, G, or P;

[0115] X3 is N or Q; and

[0116] X4 is E or D.

[0117] In one example, X1 is E.

[0118] In one example, X2 is A.

[0119] In one example, X3 is N.

[0120] In one example, X4 is E.

[0121] In one example, the degron tag includes the amino acid sequence LQCEICGFTCEQAGNLLNHIELH (SEQ ID NO:5). SEQ ID NO:5 is the sequence present in iTAG2, iTAGv1, and iTAGv2 described herein (corresponding to the core region of the ZFP91 ZF2β hairpin subdomain and the IKAROS ZF2α helix subdomain sequences of the mutants of these degron tags). It is also the equivalent mutant sequence of the super degron (i.e., the core region of the mutant variants of the ZFP91 ZF2β hairpin subdomain and the AIOLOS ZF2α helix subdomain sequences of the super degron). Thus, a degron containing the amino acid sequence of SEQ ID NO:5 can be a degron based on the iTAG1 or super degron amino acid sequence, in which at least four mutations (X1, X2, X3, and X4 as described above) are introduced.

[0122] Suitably, the presence of the amino acid sequence of SEQ ID NO:66 or any one of SEQ ID NOs:2 to 5 in the degron tag disrupts the nuclear localization of the fusion protein containing the degron tag (e.g., a degron-tagged CAR). In other words, the presence of the amino acid sequence of SEQ ID NO:66 or any one of SEQ ID NOs:2 to 5 in the degron tag reduces the nuclear localization of the fusion protein containing the degron tag compared to a fusion protein containing an equivalent degron tag, wherein the amino acid sequence of SEQ ID NO:66 or any one of SEQ ID NOs:2 to 5 is replaced by the amino acid sequence LQCEICGFTC R Q K GNLL R HI K LH (SEQ ID NO:1). In this context, "reduce" may mean a decrease or reduction of at least 5%, at least 10%, at least 20%, at least 30%, at least 50% or more. It will be clear to those skilled in the art that nuclear localization refers to being present in (localized to) the nucleus. Several methods for determining nuclear localization are known in the art, such as immunofluorescence, as used in the Examples section below.

[0123] The degron tags provided herein may include additional amino acids (e.g., the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 66 or any one of SEQ ID NOs: 2 to 5), provided that the degron tag retains its ability to bind to the CRBN-IMiD complex or the CRBN-CELMoD complex and induce degradation of the fusion protein containing the degron tag. These additional amino acids may correspond to residues in a native zinc finger domain or may be different, provided that the degron tag maintains a zinc finger-like fold and exhibits the necessary binding properties disclosed herein.

[0124] For example, the degron tags provided herein may comprise the sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO: 67) or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO: 68),

[0125] where X1, X2, X3, and X4 are not R, K, or H.

[0126] For example, the degron tags provided herein may comprise the sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO: 6) or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO: 7),

[0127] where:

[0128] X1 is E or a conservative amino acid substitution thereof;

[0129] X2 is A or a conservative amino acid substitution thereof;

[0130] X3 is N or a conservative amino acid substitution thereof; and

[0131] X4 is E or a conservative amino acid substitution thereof.

[0132] In other words, the degron tag may comprise the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO: 8) or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO: 9),

[0133] where:

[0134] X1 is E or D;

[0135] X2 is A, G, P, I, L, or V;

[0136] X3 is N, Q, C, S, T or M; and

[0137] X4 is E or D.

[0138] In one example, the degradation-resolving stator tag of the present invention may comprise the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO:10) or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO:11),

[0139] wherein:

[0140] X1 is E or D;

[0141] X2 is A, G or P;

[0142] X3 is N or Q; and

[0143] X4 is E or D.

[0144] In one example, X1 is E.

[0145] In one example, X2 is A.

[0146] In one example, X3 is N.

[0147] In one example, X4 is E.

[0148] In one example, the degradation-resolving stator tag comprises the amino acid sequence LQCEICGFTCEQAGNLLNHIELHSG (SEQ ID NO:12) or LQCEICGFTCEQAGNLLNHIELHTG (SEQ ID NO:13). In this context, SEQ ID NO:12 is the sequence present in iTAG2, iTAGv1 and iTAGv2 described herein (corresponding to the mutant ZFP91 ZF2β hairpin subdomain and IKAROS ZF2α helix subdomain sequences of these degradation-resolving stator tags). Similarly, SEQ ID NO:13 is the equivalent mutant sequence of the super degradation-resolving stator (i.e., the mutant variant of the ZFP91 ZF2β hairpin subdomain and AIOLOS ZF2α helix subdomain sequences of the super degradation-resolving stator).

[0149] The degron tags provided herein may include additional amino acids flanking the amino acid sequence of any one of SEQ ID NOs: 2 to 13 or 66 to 68 (e.g., additional N-terminal zinc finger α-helical subdomains and / or additional C-terminal zinc finger β-hairpin subdomains). For example, the degron tags of the present invention may contain an additional N-terminal zinc finger α-helical subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence of any one of SEQ ID NOs: 2 to 13 or 66 to 68. Various naturally occurring proteins contain zinc finger regions (also referred to as zinc finger motifs) that contain β-hairpin loops (referred to as zinc finger β-hairpin subdomains in the context of the degron tags described herein) and α-helical regions (referred to as zinc finger α-helical subdomains in the context of the degron tags described herein). In some embodiments, the degron tag may include a first sequence that can be derived from or is at least a part of a first zinc finger region, and a second sequence that can be derived from or is a part of the α-helical region of a second zinc finger region. The first zinc finger region and the second zinc finger region may be the same or different, provided that the resulting degron tag binds to CRBN-IMiD or CRBN-CELMoD. Suitable N-terminal zinc finger α-helical subdomains are well known in the art and include, but are not limited to, the IKAROS ZF1 α-helical subdomain and the AIOLOS ZF1 α-helical subdomain used in the following examples. For example, the ZFP91, ZN276, ZN517, ZN653, ZN654, ZN787, or ZN827 N-terminal zinc finger α-helical subdomains may be used. Suitable C-terminal zinc finger β-hairpin subdomains are also well known in the art and include, but are not limited to, the IKAROS ZF3 β-hairpin subdomain and the AIOLOS ZF1 ZF3 β-hairpin subdomain used in the following examples. For example, the ZFP91, ZN276, ZN517, ZN653, ZN654, ZN787, or ZN827 C-terminal zinc finger β-hairpin subdomains may be used.

[0150] As used herein, the term "flanking" refers to the relative positioning of a feature within the same amino acid sequence. Thus, in this context, it refers to additional amino acids or subdomains at the N-terminus and / or C-terminus (as appropriate) of the core sequence of any one of SEQ ID NOs: 2 to 13 or 66 to 68. It is noted that the flanking sequences do not need to be directly adjacent to the core sequence of SEQ ID NOs: 2 to 13 or 66 to 68; there may be intervening amino acids between the two.

[0151] In one example, the degron contains additional N-terminal zinc finger alpha-helix subdomains and additional C-terminal zinc finger beta-hairpin subdomains flanking the amino acid sequence of SEQ ID NO:5. In other words, the degron can contain a configuration where, in the N-terminal to C-terminal direction: zinc finger alpha-helix subdomain - SEQ ID NO:5 to zinc finger beta-hairpin subdomain (optionally with intervening amino acids between the subdomain and the amino acid sequence of SEQ ID NO:5). In this example, the additional C-terminal zinc finger beta-hairpin subdomain can contain the amino acid sequence CHLCNYACR (SEQ ID NO:14) or CHLCNYACQ (SEQ ID NO:15) (e.g., CHLCNYACRRRDAL (SEQ ID NO:69) or CHLCNYACQRRDAL (SEQ ID NO:70)). In this example, the additional N-terminal zinc finger alpha-helix subdomain can also contain the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:71) or FNVLMVHX5X6SH (SEQ ID NO:72); where X5 and X6 are not R, K, or H. For example, the additional N-terminal zinc finger alpha-helix subdomain can also contain the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:16) or FNVLMVHX5X6SH (SEQ ID NO:17); where X5 is N or its conservative amino acid substitution; and X6 is E or its conservative amino acid substitution. In other words, the additional N-terminal zinc finger alpha-helix subdomain can also contain the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:18) or FNVLMVHX5X6SH (SEQ ID NO:19); where X5 is N, Q, C, S, T, or M; and X6 is E or D. In one example, the additional N-terminal zinc finger alpha-helix subdomain can contain the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:20) or FNVLMVHX5X6SH (SEQ ID NO:21); where X5 is N or Q; and X6 is E or D. In a specific example, the additional N-terminal zinc finger alpha-helix subdomain can contain the amino acid sequence PNVLMVHNESH (SEQ ID NO:22) or FNVLMVHNESH (SEQ ID NO:23). For example, when the degron contains additional N-terminal zinc finger alpha-helix subdomains and additional C-terminal zinc finger beta-hairpin subdomains flanking the amino acid sequence of SEQ ID NO:5, the additional N-terminal zinc finger alpha-helix subdomain can contain the amino acid sequence SEQ ID NO:22 or SEQ ID NO:23; and the additional C-terminal zinc finger beta-hairpin subdomain can contain the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15.

[0152] Thus, in one example, the degradation resolver tag comprises an additional N-terminal zinc finger α-helical subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence of SEQ ID NO:5, wherein the additional N-terminal zinc finger α-helical subdomain comprises the amino acid sequence SEQ ID NO:22, and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence SEQ ID NO:14. This arrangement corresponds to the sequence present in iTAG2.

[0153] In another example, the degradation resolver tag comprises an additional N-terminal zinc finger α-helical subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence of SEQ ID NO:5, wherein the additional N-terminal zinc finger α-helical subdomain comprises the amino acid sequence SEQ ID NO:23, and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence SEQ ID NO:15.

[0154] In one example, the degron stator contains additional N-terminal zinc finger alpha helix subdomains and additional C-terminal zinc finger beta hairpin subdomains flanking the amino acid sequence of SEQ ID NO:12. In other words, the degron stator can contain a configuration that, in the N-terminal to C-terminal direction (from left to right): zinc finger alpha helix substructure - SEQ ID NO:12 to zinc finger beta hairpin subdomain (optionally with intervening amino acids between the subdomain and the amino acid sequence of SEQ ID NO:12). In this example, the additional C-terminal zinc finger beta hairpin subdomain can contain the amino acid sequence CHLCNYACR (SEQ ID NO:14) or CHLCNYACQ (SEQ ID NO:15) (e.g., CHLCNYACRRRDAL (SEQ ID NO:69) or CHLCNYACQRRDAL (SEQ ID NO:70)). In this example, the additional N-terminal zinc finger alpha helix subdomain can also contain the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:71) or FNVLMVHX5X6SH (SEQ ID NO:72); where X5 and X6 are not R, K, or H. For example, the additional N-terminal zinc finger alpha helix subdomain can also contain the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:16) or FNVLMVHX5X6SH (SEQ ID NO:17); where X5 is N or its conservative amino acid substitution; and X6 is E or its conservative amino acid substitution. In other words, the additional N-terminal zinc finger alpha helix subdomain can also contain the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:18) or FNVLMVHX5X6SH (SEQ ID NO:19); where X5 is N, Q, C, S, T, or M; and X6 is E or D. In one example, the additional N-terminal zinc finger alpha helix subdomain can contain PNVLMVHX5X6SH (SEQ ID NO:20) or FNVLMVHX5X6SH (SEQ ID NO:21); where X5 is N or Q; and X6 is E or D. In a particular example, the additional N-terminal zinc finger alpha helix subdomain can contain the amino acid sequence PNVLMVHNESH (SEQ ID NO:22) or FNVLMVHNESH (SEQ ID NO:23). For example, when the degron stator contains additional N-terminal zinc finger alpha helix subdomains and additional C-terminal zinc finger beta hairpin subdomains flanking the amino acid sequence of SEQ ID NO:12, the additional N-terminal zinc finger alpha helix subdomain can contain the amino acid sequence SEQ ID NO:22 or SEQ ID NO:23; and the additional C-terminal zinc finger beta hairpin subdomain can contain the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15.

[0155] Thus, in one example, the degradation resolver tag comprises an additional N-terminal zinc finger α-helical subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence of SEQ ID NO:12, wherein the additional N-terminal zinc finger α-helical subdomain comprises the amino acid sequence PNVLMVHNESH (SEQ ID NO:22), and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence CHLCNYACR (SEQ ID NO:14). This arrangement corresponds to the sequence present in iTAG2.

[0156] In another example, the degradation resolver tag comprises an additional N-terminal zinc finger α-helical subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence of SEQ ID NO:12, wherein the additional N-terminal zinc finger α-helical subdomain comprises the amino acid sequence FNVLMVHNESH (SEQ ID NO:23), and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence CHLCNYACQ (SEQ ID NO:15).

[0157] In one example, the degrading stator comprises additional N-terminal zinc finger α-helix subdomains and additional C-terminal zinc finger β-hairpin subdomains flanking the amino acid sequence of SEQ ID NO:13. In other words, the degrading stator can comprise a configuration that, in the N-terminal to C-terminal direction (from left to right): zinc finger α-helix subdomain - SEQ ID NO:13 to zinc finger β-hairpin subdomain (optionally with intervening amino acids between the subdomain and the amino acid sequence of SEQ ID NO:13). In this example, the additional C-terminal zinc finger β-hairpin subdomain can comprise the amino acid sequence CHLCNYACR (SEQ ID NO:14) or CHLCNYACQ (SEQ ID NO:15) (e.g., CHLCNYACRRRDAL (SEQ ID NO:69) or CHLCNYACQRRDAL (SEQ ID NO:70)). In this example, the additional N-terminal zinc finger α-helix subdomain can also comprise the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:71) or FNVLMVHX5X6SH (SEQ ID NO:72); where X5 and X6 are not R, K, or H. In this example, the additional N-terminal zinc finger α-helix subdomain can also comprise the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:16) or FNVLMVHX5X6SH (SEQ ID NO:17); where X5 is N or its conservative amino acid substitution; and X6 is E or its conservative amino acid substitution. In other words, the additional N-terminal zinc finger α-helix subdomain can also comprise the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:18) or FNVLMVHX5X6SH (SEQ ID NO:19); where X5 is N, Q, C, S, T, or M; and X6 is E or D. In one example, the additional N-terminal zinc finger α-helix subdomain can comprise the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:20) or FNVLMVHX5X6SH (SEQ ID NO:21); where X5 is N or Q; and X6 is E or D. In a particular example, the additional N-terminal zinc finger α-helix subdomain can comprise the amino acid sequence PNVLMVHNESH (SEQ ID NO:22) or FNVLMVHNESH (SEQ ID NO:23). For example, when the degrading stator comprises additional N-terminal zinc finger α-helix subdomains and additional C-terminal zinc finger β-hairpin subdomains flanking the amino acid sequence of SEQ ID NO:13, the additional N-terminal zinc finger α-helix subdomain can comprise the amino acid sequence SEQ ID NO:22 or SEQ ID NO:23; and the additional C-terminal zinc finger β-hairpin subdomain can comprise the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15.

[0158] Thus, in one example, the degradation resolver tag comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence of SEQ ID NO:13, wherein the additional N-terminal zinc finger α-helix subdomain comprises the amino acid sequence PNVLMVHNESH (SEQ ID NO:22), and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence CHLCNYACR (SEQ ID NO:14).

[0159] In another example, the degradation resolver tag comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence of SEQ ID NO:13, wherein the additional N-terminal zinc finger α-helix subdomain comprises the amino acid sequence FNVLMVHNESH (SEQ ID NO:23), and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence CHLCNYACQ (SEQ ID NO:15).

[0160] In one example, the degradation resolver tag comprises the amino acid sequence:

[0161] PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSG EX8PFKCHLCNYACRRRDAL (SEQ ID NO:73), wherein:

[0162] X1, X2, X3, X4, X5, X6, X7, and X8 are not R, K or H.

[0163] In one example, the degradation resolver tag comprises the amino acid sequence:

[0164] PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSG EX8PFKCHLCNYACRRRDAL (SEQ ID NO:24), wherein:

[0165] X1 is E or a conservative amino acid substitution thereof;

[0166] X2 is A or a conservative amino acid substitution thereof;

[0167] X3 is N or a conservative amino acid substitution thereof;

[0168] X4 is E or a conservative amino acid substitution thereof.

[0169] X5 is N or a conservative amino acid substitution thereof;

[0170] X6 is E or a conservative amino acid substitution thereof;

[0171] X7 is I or a conservative amino acid substitution thereof; and

[0172] X8 is I or a conservative amino acid substitution thereof.

[0173] In other words, the degron tag can comprise the amino acid sequence PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO:25), wherein:

[0174] X1 is E or D;

[0175] X2 is A, G, P, I, L or V;

[0176] X3 is N, Q, C, S, T or M;

[0177] X4 is E or D;

[0178] X5 is N, Q, C, S, T or M;

[0179] X6 is E or D;

[0180] X7 is I, L, V, G, A or P; and

[0181] X8 is I, L, V, G, A or P.

[0182] In one example, the degron tag comprises the amino acid sequence PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO:26), wherein:

[0183] X1 is E or D;

[0184] X2 is A, G or P;

[0185] X3 is N or Q;

[0186] X4 is E or D;

[0187] X5 is N or Q;

[0188] X6 is E or D;

[0189] X7 is I, L or V; and

[0190] X8 is I, L or V.

[0191] In one example, X1 is E.

[0192] In one example, X2 is A.

[0193] In one example, X3 is N.

[0194] In one example, X4 is E.

[0195] In one example, X5 is N.

[0196] In one example, X6 is E.

[0197] In one example, X7 is I.

[0198] In one example, X8 is I.

[0199] In one example, the degron tag contains the amino acid sequence SEQ ID NO:27. (See Table 2 below).

[0200] In one example, the degron tag contains the amino acid sequence SEQ ID NO:29 (See Table 2 below).

[0201] In one example, the degron tag contains the amino acid sequence SEQ ID NO:31 (See Table 2 below).

[0202] In one example, the degron tag contains the amino acid sequence SEQ ID NO:33 (See Table 2 below).

[0203] In one example, the degron tag contains the amino acid sequence SEQ ID NO:35 (See Table 2 below).

[0204] In one example, the degron tag contains the amino acid sequence SEQ ID NO:37 (See Table 2 below).

[0205] In one example, the degron tag contains the amino acid sequence SEQ ID NO:39 (See Table 2 below).

[0206] In one example, the degron tag contains the amino acid sequence SEQ ID NO:41 (See Table 2 below).

[0207] In one example, the degron tag contains the amino acid sequence SEQ ID NO:43 (See Table 2 below).

[0208] In one example, the degron tag contains the amino acid sequence:

[0209] FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTG EX8PFKCHLCNYACQRRDAL (SEQ ID NO:74), wherein: X1, X2, X3, X4, X5, X6, X7, and X8 are not R, K, or H.

[0210] In one example, the degradation resolver tag comprises the amino acid sequence:

[0211] FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTG EX8PFKCHLCNYACQRRDAL (SEQ ID NO:45), wherein:

[0212] X1 is E or a conservative amino acid substitution thereof;

[0213] X2 is A or a conservative amino acid substitution thereof;

[0214] X3 is N or a conservative amino acid substitution thereof;

[0215] X4 is E or a conservative amino acid substitution thereof.

[0216] X5 is N or a conservative amino acid substitution thereof;

[0217] X6 is E or a conservative amino acid substitution thereof;

[0218] X7 is I or a conservative amino acid substitution thereof; and

[0219] X8 is I or a conservative amino acid substitution thereof.

[0220] In other words, the degradation resolver tag can comprise the amino acid sequence FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO:46), wherein:

[0221] X1 is E or D;

[0222] X2 is A, G, P, I, L, or V;

[0223] X3 is N, Q, C, S, T, or M;

[0224] X4 is E or D;

[0225] X5 is N, Q, C, S, T, or M;

[0226] X6 is E or D;

[0227] X7 is I, L, V, G, A, or P; and

[0228] X8 is I, L, V, G, A or P.

[0229] In one example, the degradation-resolving stator tag comprises the amino acid sequence FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO:47), wherein:

[0230] X1 is E or D;

[0231] X2 is A, G or P;

[0232] X3 is N or Q;

[0233] X4 is E or D;

[0234] X5 is N or Q;

[0235] X6 is E or D;

[0236] X7 is I, L or V; and

[0237] X8 is I, L or V.

[0238] In one example, X1 is E.

[0239] In one example, X2 is A.

[0240] In one example, X3 is N.

[0241] In one example, X4 is E.

[0242] In one example, X5 is N.

[0243] In one example, X6 is E.

[0244] In one example, X7 is I.

[0245] In one example, X8 is I.

[0246] In one example, the degradation-resolving stator tag comprises the amino acid sequence SEQ ID NO:28 (see Table 2 below).

[0247] In one example, the degradation-resolving stator tag comprises the amino acid sequence SEQ ID NO:30 (see Table 2 below).

[0248] In one example, the degradation-resolving stator tag comprises the amino acid sequence SEQ ID NO:32 (see Table 2 below).

[0249] In one example, the degron tag contains the amino acid sequence SEQ ID NO:34 (see Table 2 below).

[0250] In one example, the degron tag contains the amino acid sequence SEQ ID NO:36 (see Table 2 below).

[0251] In one example, the degron tag contains the amino acid sequence SEQ ID NO:38 (see Table 2 below).

[0252] In one example, the degron tag contains the amino acid sequence SEQ ID NO:40 (see Table 2 below).

[0253] In one example, the degron tag contains the amino acid sequence SEQ ID NO:42 (see Table 2 below).

[0254] In one example, the degron tag contains the amino acid sequence SEQ ID NO:44 (see Table 2 below).

[0255] The following table lists the specific amino acid sequences that may be contained in the degron tags of the present invention, where the specific mutations identified herein are introduced into the corresponding iTAG1 and super degron sequences described herein. As can be seen from the table, four specific mutations are considered in the minimal degron sequence, and optional (one, two, three, or four) additional mutations are also considered in the flanking sequences. In each case, the mutations are underlined for ease of review, and the minimal degron sequence is italicized and bolded.

[0256]

[0257]

[0258]

[0259] Table 2. Examples of specific new degron sequences contemplated herein

[0260] The degron tags provided herein are generally described using the core amino acid sequence of any one of SEQ ID NOs: 2 to 13 or 66 to 68, in combination with LQCEICGFTC R Q K GNLL R HI KCompared to the known minimal functional degron of LH, it includes four amino acid substitutions (where the underlined amino acids indicate the mutated amino acids in the present invention). Although the degron tags provided herein are generally described as having all four amino acid substitutions, it will be clear to those skilled in the art that degron tags having one or more, two or more, three or more of these mutations may also be contemplated herein.

[0261] Accordingly, provided herein is a degron tag comprising the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:75), wherein one, two or three of the following amino acid substitutions are made:

[0262] At position X1, R is replaced with any amino acid other than R, K or H; and / or

[0263] At position X2, K is replaced with any amino acid other than R, K or H; and / or

[0264] At position X3, R is replaced with any amino acid other than R, K or H; and / or

[0265] At position X4, K is replaced with any amino acid other than R, K or H.

[0266] In one example, provided herein is a degron tag comprising the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:48), wherein one, two or three of the following amino acid substitutions are made:

[0267] At position X1, R is replaced with E or a conservative amino acid substitution thereof; and / or

[0268] At position X2, K is replaced with A or a conservative amino acid substitution thereof; and / or

[0269] At position X3, R is replaced with N or a conservative amino acid substitution thereof; and / or

[0270] At position X4, K is replaced with E or a conservative amino acid substitution thereof.

[0271] It will be clear to those skilled in the art that at any position where no amino acid substitution is made, the original amino acid is retained (i.e., R at position X1, K at position X2, R at position X3, K at position X4, etc.).

[0272] Thus, the degron stator tags provided herein may include the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:49), where one, two, or three of the following amino acid substitutions are made:

[0273] At position X1, R is replaced with E or D; and / or

[0274] At position X2, K is replaced with A, G, P, I, L, or V; and / or

[0275] At position X3, R is replaced with N, Q, C, S, T, or M; and / or

[0276] At position X4, K is replaced with E or D.

[0277] In one example, the degron stator tags provided herein may include the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:50), where one, two, or three of the following amino acid substitutions are made:

[0278] At position X1, R is replaced with E or D; and / or

[0279] At position X2, K is replaced with A, G, or P; and / or

[0280] At position X3, R is replaced with N or Q; and / or

[0281] At position X4, K is replaced with E or D.

[0282] In one example, the degron stator tags provided herein may thus include the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:51), where one, two, or three of the following amino acid substitutions are made:

[0283] At position X1, R is replaced with E; and / or

[0284] At position X2, K is replaced with A; and / or

[0285] At position X3, R is replaced with N; and / or

[0286] At position X4, K is replaced with E.

[0287] Thus, in one example, the degron stator tags provided herein may thus include the amino acid sequence LQCEICGFTC E QKGNLLRHIKLH (SEQ ID NO:52), LQCEICGFTCRQ AGNLLRHIKLH (SEQ ID NO:53), LQCEICGFTCRQKGNLL N HIKLH (SEQ ID NO:54) or LQCEICGFTCRQKGNLLRHI E LH (SEQ ID NO:55) (all of which have one of the substitutions set forth above). Examples are also contemplated herein in which the degradation determinant tag will have two or three of said substitutions, and those skilled in the art will readily recognize these examples. Examples of such tags include: LQCEICGFTC E Q A GNLLRHIKLH (SEQ ID NO:56); LQCEICGFTC E QKGNLL N HIKLH (SEQ ID NO:57); LQCEICGFTC E QKGNLLNHI E LH (SEQ ID NO:58); LQCEICGFTCRQ A GNLL N HIKLH (SEQ ID NO:59); LQCEICGFTCRQ A GNLLRHI E LH (SEQ ID NO:60); LQCEICGFTCRQKGNLL N HI E LH (SEQ ID NO:61); LQCEICGFTC E Q A GNLL N HIKLH (SEQ ID NO:62); LQCEICGFTC E Q A GNLLRHI E LH (SEQ ID NO:63); LQCEICGFTC E QKGNLL N HI E LH (SEQ ID NO:64); or LQCEICGFTCRQ A GNLL N HI E LH (SEQ ID NO:65). In each of these examples, the mutant amino acid(s) relative to SEQ ID NO:1 are underlined.

[0288] The degron tags of the present invention are peptides, typically having from about 23 to about 70 amino acids, typically from about 23 to about 60 amino acids. In some examples, the degron tags of the present invention are peptides typically having from about 23 to about 30 amino acids. The terms "peptide", "polypeptide" and "protein" are used herein in accordance with their generally recognized meanings in the art.

[0289] The present disclosure also provides fusion proteins comprising a protein of interest (POI) and at least one degron tag of the present invention. When linked to a therapeutic protein of interest (POI), the degron tag can serve as a "safety switch" as it can be used to target the degradation of the POI in the absence of the need for POI expression. The ability to generate POI-degron tag fusions and administer an IMiD or CELMoD to degrade a specific endogenous protein of interest can be used to treat conditions in which intracellular protein expression above a specific threshold level results in a disease state. Other applications of the technology include 1) targeting the degradation of proteins where the pathology is a function of gain-of-function mutation(s), 2) targeting the degradation of proteins where the pathology is a function of amplification or increased expression, 3) targeting the degradation of proteins that manifest as monogenic diseases, 4) targeting the degradation of proteins where the genetic susceptibility manifests over a longer period of time and typically after alternative biological compensatory mechanisms are no longer sufficient, such as, but not limited to, hypercholesterolemia and proteinopathies. In addition, POI-degron tag fusions can be used to evaluate the function of endogenous proteins or to validate endogenous proteins as targets for the treatment of disease states. Thus, the degron tags of the present invention can be used to generate stably expressed endogenous protein-degron tag fusion proteins or exogenous protein-degron tag fusion proteins. Endogenous proteins originate within an organism, tissue or cell and are expressed by the same organism, tissue or cell, while exogenous proteins originate outside an organism, tissue or cell and are introduced into the organism, tissue or cell.

[0290] As described elsewhere herein, the fusion proteins provided herein can comprise a degron of the present invention fused to a protein of interest. The protein of interest can be an immune surface receptor and / or a "chimeric immunomodulatory receptor". The protein of interest can be selected from chimeric antigen receptors (CARs), T cell receptors (TCRs), T cell receptor (TCR) fusion constructs (TRuCs), T cell antigen conjugates (TACs), chimeric autoantibody receptors (CAARs) or antibody-conjugated T cell receptors (ACTRs).

[0291] When the POI is a chimeric antigen receptor (CAR) protein, the degron of the present invention is particularly useful. Accordingly, CAR-degron tag fusion proteins are specifically provided herein. Genetically modified T cells expressing a chimeric antigen receptor (CAR-T therapy) have proven to be therapeutically efficacious against a variety of cancers, including lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, and neuroblastoma. Two autologous CAR-T cell therapies (Kymriah TM and Yescarta TM ) have received approval from the US Food and Drug Administration. Generally, both are CD19-specific CAR-T cell therapies that lyse CD19-positive targets (normal and malignant B-lineage cells). However, CAR-T therapy is not without significant side effects. Although most adverse events of CAR-T are tolerable and acceptable, in many cases, the administration of CAR-T cells can lead to severe systemic inflammatory responses, including cytokine release syndrome (CRS) and tumor lysis syndrome. The significant clinical activity of CAR-T cell therapy indicates the need to implement safety strategies to rapidly reverse or abort the T cell response in patients experiencing CRS or related adverse events.

[0292] Accordingly, the present invention encompasses fusion proteins comprising a CAR and at least one degron tag. The CARs are further characterized in that they comprise, in this order starting from the N-terminal side, an extracellular ligand-binding domain capable of binding an antigen, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises at least one signaling domain. The degron tag(s) can be located at the N-terminus or between the extracellular binding domain and the transmembrane domain, provided that there is no interruption to antigen binding or insertion into the membrane. Similarly, the degron tag(s) can be located at the C-terminus, between the transmembrane domain and the intracellular domain, or between signaling domains when there is more than one signaling domain, provided that there is no interruption to intracellular signaling or insertion into the membrane. The degron tag is preferably located at the C-terminus. In other words, the degron is preferably at the 3' end of the fusion protein such that it is fused to the 3' end of the C-terminal intracellular signaling domain in the cytoplasmic domain of the CAR.

[0293] In one embodiment, the fusion protein comprises a CAR that is tisagenlecleucel (Kymriah TM) and a degron tag as described herein. Tisagenlecleucel is generally a genetically modified antigen - specific autologous T cell targeting CD19. The extracellular domain of the CAR is a murine anti - CD19 single - chain antibody fragment (scFv) from the murine monoclonal FMC63 hybridoma. The intracellular domain of the CAR is a T cell signaling domain derived from human CD3z and a co - stimulatory domain derived from human 4 - 1BB (CD137). The transmembrane domain and the spacer located between the scFv domain and the transmembrane domain are derived from human CD8a. Kymriah TM (Tisagenlecleucel) is approved for the treatment of patients 25 years of age and younger with refractory or relapsed B - cell precursor acute lymphoblastic leukemia (ALL), and for the treatment of adult patients with relapsed / refractory (R / R) diffuse large B - cell lymphoma (DLBCL), which is the most common non - Hodgkin lymphoma, as well as high - grade B - cell lymphoma and DLBCL arising from follicular lymphoma. The degron tag can be any of the degron tags disclosed herein.

[0294] In one embodiment, the fusion protein comprises a CAR, which is axicabtagene ciloleucel (Yescarta TM ) and a degron tag. Axicabtagene ciloleucel is a genetically modified antigen - specific autologous T cell targeting CD19. The extracellular domain of the CAR is a murine anti - CD19 single - chain antibody fragment (scFv). The intracellular domain of the CAR is two signaling domains, one derived from human CD3z and the other derived from CD28. Yescarta TM (Axicabtagene ciloleucel) has been approved for the treatment of adult patients with R / R large B - cell lymphoma, including DLBCL not otherwise specified, primary mediastinal large B - cell lymphoma, high - grade B - cell lymphoma, and DLBCL arising from follicular lymphoma. The degron tag can be any of the degron tags disclosed herein.

[0295] In one embodiment, the antigen - binding portion of the CAR is designed to treat a specific cancer. For example, a CAR designed to target CD19 can be used to treat cancers and disorders, including precursor B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B - cell lymphoma, and salvage after allogeneic bone marrow transplantation.

[0296] Further features of CAR proteins, nucleic acids encoding CAR proteins, immune effector cells expressing CAR, and methods of treating diseases using cells expressing CAR are disclosed in U.S. Patent Application Publication 2018 / 0169109A1, which is incorporated herein by reference.

[0297] As described elsewhere herein, the fusion proteins provided herein can comprise a degron of the invention fused to a protein of interest. The protein of interest can be a T cell receptor (TCR). Several different types of TCRs can be used in the context of the present invention. For example, the TCR can be a chimeric T cell receptor, an artificial T cell receptor, or a synthetic T cell receptor. Additionally, the TCR can be an antibody-coupled T cell receptor (ACTR), a T cell receptor fusion construct (TRuC), or a T cell antigen coupler (TAC).

[0298] In one example, the POI can be a T cell receptor fusion construct (TRuC). A T cell receptor fusion construct (TRuC) is a receptor protein that includes an antibody-based binding domain fused to a T cell receptor (TCR) subunit and is designed to efficiently recognize a target cell surface antigen. The TRuC includes a specific ligand antibody fused to the extracellular N-terminus of a number of TCR subunits (e.g., TCRα, TCRβ, CD3ε, CD3γ, and CD3δ). The TRuC provides target specificity and HLA-independent target cell elimination ability. The TRuC can integrate into the native TCR complex on the surface of cells such as T cells. Different from CAR, the TRuC becomes a functional component of the TCR complex. TRuC-T cells have shown strong anti-tumor activity in both liquid tumor and solid tumor xenograft models.

[0299] In another example, the POI can be an antibody-coupled T cell receptor (ACTR). An ACTR is a non-naturally occurring molecule that can be expressed on the surface of a host cell and comprises an extracellular domain capable of binding to a target molecule containing an Fc portion (e.g., the CD16A extracellular domain) and one or more cytoplasmic signaling domains for triggering effector functions of an immune cell expressing the ACTR polypeptide, wherein at least two domains in the ACTR polypeptide can be derived from different molecules. The ACTR polypeptide can comprise a CD16A extracellular domain capable of binding to a target molecule containing an Fc portion, a transmembrane domain, one or more co-stimulatory signaling domains, and a CD3ζ cytoplasmic signaling domain. At least one of the co-stimulatory signaling domains can be a CD28 co-stimulatory domain. The ACTR polypeptide can be devoid of any hinge domain of non-CD16A receptors, or can comprise more than one co-stimulatory signaling domain if the transmembrane domain is a CD8 transmembrane domain.

[0300] In another example, the POI can be a T cell antigen conjugate (TAC). A T cell antigen conjugate (TAC) is a platform that combines the endogenous TCR with MHC-independent mechanisms to induce a more effective target cell response and reduce toxicity. The TAC chimeric protein is conjugated to the TCR to recognize the antigen through binding to the CD3 domain, thereby forming a TCR / CD3 complex.

[0301] Alternatively, the antigen-binding cell surface protein can be a chimeric autoantibody receptor (CAAR). A CAAR is a modified form of CAR that is used to recognize antibody-secreting cells, such as autoreactive B cells. A CAAR includes a specific antigen, a transmembrane domain, and an intracellular signaling domain with or without a co-stimulatory domain. The CAAR recognizes and binds to the target autoantibody expressed on autoreactive cells through the specific antigen, and subsequently destroys it.

[0302] Also provided are nucleic acid sequences and nucleic acid molecules encoding the degrons or fusion proteins described herein. The nucleic acid sequences and nucleic acid molecules can be non-naturally occurring nucleic acid sequences encoding the degron tags or fusion proteins described herein.

[0303] Also provided are vectors comprising nucleic acid sequences encoding the novel degrons or fusion proteins described herein. A "vector" is a composition of matter containing a nucleic acid that can be used to deliver the nucleic acid into the interior of a cell. Many vectors are known in the art, including linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes or lipid nanoparticles. Thus, a vector can be a non-viral vector (e.g., a plasmid, a polylysine compound, and a liposome or lipid nanoparticle) or a viral vector. Representative examples of viral vectors include: retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, vaccinia viral vectors, picornaviral vectors, and alphaviral vectors. An example of a retroviral vector is a lentiviral vector.

[0304] The vector can be delivered by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intrathecal, intratracheal, subcutaneous, or intracranial infusion) or local application. Alternatively, the vector can be delivered ex vivo to cells, such as cells transplanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, or tissue biopsies) or universal donor hematopoietic stem cells, and then the cells are typically re-implanted into the patient after selecting the cells that have incorporated the vector.

[0305] In certain embodiments, the nucleic acid encoding the degron stator tag can be genomically inserted in-frame with a gene encoding a protein involved in a disorder. Representative examples of specific genes involved in disorders where the degron stator tag insertion can be targeted include alpha-1 antitrypsin (A1AT), apolipoprotein B (apoB), angiopoietin-like protein 3 (ANGPTL3), proprotein convertase subtilisin / kexin type 9 (PCSK9), apolipoprotein C3 (APOC3), catenin (CTNNB1), low density lipoprotein receptor (LDLR), C-reactive protein (CRP), apolipoprotein a (Apo(a)), factor VII, factor XI, antithrombin III (SERPINC1), phosphatidylinositol glycan class A (PIG-A), C5, alpha-1 antitrypsin (SERPINA1), hepcidin regulation (TMPRSS6), (delta-aminolevulinate synthase 1 (ALAS-1), acyl-CoA:diacylglycerol acyltransferase (DGAT), miR-122, miR-21, miR-155, miR-34a, prekallikrein (KLKB1), connective tissue growth factor (CCN2), intercellular adhesion molecule 1 (ICAM-1), glucagon receptor (GCGR), glucocorticoid receptor (GCCR), protein tyrosine phosphatase (PTP-1B), C-Raf kinase (RAF1), fibroblast growth factor receptor 4 (FGFR4), vascular cell adhesion molecule-1 (VCAM-1), very late antigen-4 (VLA-4), transthyretin (TTR), survival motor neuron 2 (SMN2), growth hormone receptor (GHR), dystrophia myotonica protein kinase (DMPK), cellular nucleic acid binding protein (CNBP or ZNF9), clusterin (CLU), eukaryotic translation initiation factor 4E (eIF-4E), MDM2, MDM4, heat shock protein 27 (HSP 27), signal transducer and activator of transcription 3 protein (STAT3), vascular endothelial growth factor (VEGF), kinesin spindle protein (KIF11), hepatitis B genome, androgen receptor (AR), atonal homolog 1 (ATOH1), vascular endothelial growth factor receptor 1 (FLT1), retinoschisin 1 (RS1), retinal pigment epithelium-specific 65 kDa protein (RPE65), Rab escort protein 1 (CHM), and sodium channel, voltage-gated, type X, alpha subunit (PN3 or SCN10A). Additional proteins of interest that may be targeted by degron stator tag insertion include proteins associated with gain-of-function mutations, e.g., oncogenic proteins.

[0306] In certain embodiments, the protein of interest is apoB-100, ANGPTL3, PCSK9, APOC3, CRP, ApoA, Factor XI, Factor VII, Antithrombin III, phosphatidylinositol glycan class A (PIG-A), C5 component of complement, alpha-1-antitrypsin (A1AT), TMPRSS6, ALAS-1, DGAT-2, KLB1, CCN2, ICAM, glucagon receptor, glucocorticoid receptor, PTP-1B, FGFR4, VCAM-1, VLA-4, GCCR, TTR, SMN1, GHR, DMPK, or sodium channel subtype NaV1.8.

[0307] In one embodiment, the degron tag is integrated in-frame into a gene encoding an endogenous protein associated with a proteinopathy at the 5' or 3' genome. In one embodiment, the degron tag is integrated in-frame at the 5' or 3' genome into a gene encoding an endogenous protein associated with proteinopathies such as Alzheimer's disease (amyloid-beta peptide (Aβ); Tau protein), cerebral amyloid angiopathy (amyloid-beta peptide (Aβ)), glaucoma retinal ganglion cell degeneration (amyloid-beta peptide (Aβ)), prion disease (prion protein), Parkinson's disease and other synucleinopathies (alpha-synuclein), Tauopathies (microtubule-associated protein tau (Tau protein)), frontotemporal lobar degeneration (FTLD) (Ubi+, Tau) (TDP-43), FTLD-FUS (fused in sarcoma (FUS) protein), amyotrophic lateral sclerosis (ALS) (superoxide dismutase, TDP-43, FUS), Huntington's disease and other triplet repeat diseases (proteins with tandem glutamine expansions), familial British dementia (ABri), familial Danish dementia (Adan), hereditary cerebral hemorrhage with amyloidosis (Icelandic type) (HCHWA-I) (cystatin C), CADASIL (Notch3), Alexander disease (glial fibrillary acidic protein (GFAP)), Seipinopathies (Seipin), familial amyloid neuropathy, senile systemic amyloidosis (transthyretin), Seipinopathies (Serpins), AL (light chain) amyloidosis (primary systemic amyloidosis) (monoclonal immunoglobulin light chain), AH (heavy chain) amyloidosis (immunoglobulin heavy chain), AA (secondary) amyloidosis (amyloid A), type 2 diabetes (islet amyloid polypeptide (IAPP;in genes of endogenous proteins associated with disorders such as amylin, medial aortic amyloidosis (Medin (galectin)), ApoAI amyloidosis (apolipoprotein AI), ApoAII amyloidosis (apolipoprotein AII), ApoAIV amyloidosis (apolipoprotein AIV), familial amyloidosis Finnish type (FAF) (gelsolin), lysozyme amyloidosis (lysozyme), fibrinogen amyloidosis (fibrinogen), dialysis amyloidosis (β-2 microglobulin), inclusion body myositis / myopathy (amyloid beta peptide (Ab)), cataract (crystallin), retinitis pigmentosa with rhodopsin mutation (rhodopsin), medullary thyroid carcinoma (calcitonin), cardiac amyloidosis (atrial natriuretic factor), pituitary prolactinoma (prolactin), hereditary lattice corneal dystrophy (corneal epitheliopathy), lichen amyloidosis of skin (keratin), Mallory bodies (keratin intermediate filament protein), corneal lactoferrin amyloidosis (lactoferrin), alveolar proteinosis (surfactant protein C (SP-C)), odontogenic (pindborg) tumor amyloid (odontogenic ameloblast-associated protein), seminal vesicle amyloid (seminal vesicle protein I), cystic fibrosis (cystic fibrosis transmembrane conductance regulator (CFTR) protein), sickle cell disease (hemoglobin), and critical illness myopathy (CIM) (high proteolytic state of myosin ubiquitination);

[0308] In-frame insertion of a nucleic acid sequence encoding a degron tag can be performed or achieved by any known and effective genome editing process. In one aspect, the present invention utilizes the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a knock-in endogenous protein degron tag fusion protein, which is produced from an endogenous locus and is readily degraded in a reversible and dose-responsive manner upon administration of an IMiD or CELMoD. In certain embodiments, the CRISPR-Cas9 system is used to insert an expression cassette of a degron tag present in a homologous recombination (HR) “donor” sequence, wherein the degron tag nucleic acid sequence is inserted into the genomic locus of the protein of interest as a “donor” sequence during homologous recombination after Cas nuclease cleavage within the CRISPR. The HR targeting vector contains homology arms at the 5′ and 3′ ends of the expression cassette that are homologous to the genomic DNA surrounding the target gene locus of interest. By fusing the nucleic acid sequence encoding the degron tag in-frame with the target gene of interest, the resulting fusion protein contains a degron tag that is targeted by the CRBN-IMiD complex or the CRBN-CELMOD complex.

[0309] In certain embodiments, a nucleic acid or vector encoding a degron tag or fusion protein described herein can be introduced into a cell, whereby the encoded degron tag or fusion protein is expressed by the cell. Accordingly, cells expressing the nucleic acid sequences described herein or the vectors described herein are also provided. Cells expressing the degron or fusion protein of the present invention are also provided.

[0310] The cell can be any suitable cell. In certain embodiments, the cell is an immune effector cell. For example, the cell can be selected from: T cells, B cells, plasma cells, NK cells, NKT cells, innate lymphoid cells, macrophages, dendritic cells, monocytes, neutrophils, basophils, eosinophils, mast cells, hematopoietic progenitor cells, hematopoietic stem cells, other adult stem cells such as neural, corneal, muscle, skin, small intestine, colon, bone, mesenchymal, embryonic stem cells, and induced pluripotent stem cells. The cells can be derived from a mammal, for example, they can be human cells, or cells derived from a non-human mammal such as a monkey, mouse, rat, pig, horse, or dog.

[0311] For example, cells collected, isolated, purified, or induced from body fluids, tissues, or organs such as blood (peripheral blood, cord blood, etc.) or bone marrow can be used. Peripheral blood mononuclear cells (PBMCs), immune cells (dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells, or hematopoietic cells (neutrophils, basophils)), cord blood mononuclear cells, fibroblasts, preadipocytes, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines, or neural stem cells can be used. In the present invention, T cells, precursor cells of T cells (hematopoietic stem cells, lymphocyte precursor cells, etc.), or cell populations containing them are preferably used. Representative examples of T cells include CD8-positive T cells, CD4-positive T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. Cell populations containing T cells and precursor cells of T cells include PBMCs. The above cells can be collected from a living body, obtained by amplification culture of cells collected from a living body, or established as a cell line. When it is necessary to transplant cells expressing a degron or fusion protein into a living body, it is preferred to introduce the nucleic acid into cells collected from the living body itself or its syngeneic living body. Accordingly, the immune effector cells can be autologous or allogeneic.

[0312] Immune effector cells expressing the degron or fusion protein of the present invention can be engineered by introducing a nucleic acid encoding the degron or integrin protein into the cells. In one embodiment, this step is performed ex vivo. For example, cells can be transformed ex vivo with a vector carrying the nucleic acid of the present invention to generate cells expressing the degron or fusion protein of the present invention.

[0313] Immune effector cells expressing a fusion protein containing a POI and a degron tag can be used as therapeutic agents for diseases. The therapeutic agent can be a cell expressing the POI as an active ingredient, and can also include a suitable excipient. The diseases targeted by administering cells expressing the POI are not limited as long as the disease shows sensitivity to the transformed immune effector cells. Representative examples of diseases treatable with immune effector cells expressing a nucleic acid encoding a fusion protein containing a POI and a degron tag include cancer (neuroblastoma, blood cancer (leukemia), solid tumors, etc.), inflammatory diseases / autoimmune diseases (asthma, eczema), hepatitis, and infectious diseases, for example, those caused by viruses such as influenza and HIV, bacteria or fungi, such as tuberculosis, MRSA, VRE, and deep mycosis. The transformed immune effector cells can bind to an antigen presented by a target cell, and it is desired to reduce or eliminate the target cell to treat the above diseases, that is, tumor antigens, viral antigens, bacterial antigens, etc. are administered for the treatment of these diseases.

[0314] The immune effector cells can be administered intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, intravenously, intratumorally, or into the afferent lymphatic vessels, parenterally, for example, by injection or infusion, however the route of administration is not limited. For example, the cells can be directly injected into the tumor, lymph node, or site of infection.

[0315] The degron tags provided herein bind to the CRBN-IMiD complex or the CRBN-CELMoD complex. When the degrons provided herein are included in a fusion protein (e.g., a degron-POI fusion protein, such as a degron-CAR fusion protein), they can bind to the CRBN-IMiD complex or the CRBN-CELMoD complex and induce degradation of the fusion protein in the cell. Thus, the degrons can be advantageously used as a "safety switch" by inducing degradation of the POI in cases where expression of the POI is not desired.

[0316] Notably, some IMiDs, such as pomalidomide, are effective in crossing the blood-brain barrier. Thus, the regulation of iTAG2 is highly relevant to brain tumors in adults and children that are amenable to treatment with CAR-T cells, such as high-grade gliomas, medulloblastomas, and meningiomas.

[0317] IMiD (immunomodulatory drug) and CELMoD (cereblon modulator) compounds are known in the art, examples of which include thalidomide, pomalidomide, lenalidomide, CC-122, CC-220, and CC-885, or pharmaceutically acceptable salts thereof (e.g., HCl salts). The IMiD compound, thalidomide (sold under the name ), lenalidomide (sold under the name ), and pomalidomide (sold under the name The names of which (sales) have all been approved by the US Food and Drug Administration for the treatment of multiple myeloma (among other diseases). Currently available in capsule form containing 50 mg, 100 mg, 150 mg or 200 mg of thalidomide. Currently available in capsule form containing 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg or 25 mg of lenalidomide. Currently available in capsule form containing 1 mg, 2 mg, 3 mg or 4 mg of pomalidomide. The CELMoD compounds CC-122, CC-220 and CC-885 are currently under review by the FDA.

[0318] In certain examples, the degradation of the degron tags or fusion proteins provided herein is lenalidomide-dependent. In another example, the degradation of the degron tags or fusion proteins provided herein is pomalidomide-dependent. In additional examples, the degradation of the degron tags or fusion proteins provided herein is iboridomide-dependent. As is known to those skilled in the art, iboridomide is also known as CC-220, and these terms may be used interchangeably herein. Similarly, the terms "avadomide" and "CC-122" may be used interchangeably herein.

[0319] IMiD and CELMoD compounds can be in the form of free acids or free bases or pharmaceutically acceptable salts. As used herein, in the context of salts, the term "pharmaceutically acceptable" refers to salts of the compounds that do not eliminate the biological activity or properties of the compounds and are relatively non-toxic, i.e., the salt form of the compounds can be administered to a subject without causing undesirable biological effects (such as dizziness or stomach discomfort) and without interacting in a harmful manner with any other components of the composition containing it. The term "pharmaceutically acceptable salts" refers to products obtained by reacting the compounds of the present invention with suitable acids or bases. Examples of pharmaceutically acceptable salts of IMiD and CELMoD compounds include those derived from suitable inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Al, Zn, and Mn salts. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of the amino group formed with inorganic acids, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, mesylate, esylate, benzenesulfonate, 4-methylbenzenesulfonate, or p-toluenesulfonate, etc. Certain compounds of the present invention can form pharmaceutically acceptable salts with various organic bases, such as lysine, arginine, guanidine, diethanolamine, or metformin.

[0320] IMiD and CELMoD compounds can have at least one chiral center and thus can be in the form of stereoisomers. As used herein, the stereoisomers include all isomers of a single compound that differ only in the orientation of their atoms in space. The term stereoisomers includes enantiomers (including (R-) or (S-) configurations of the compound), mixtures of enantiomers of the compound (physical mixtures of enantiomers, and racemates or racemic mixtures), geometric (cis / trans or E / Z, R / S) isomers of the compound, and isomers of the compound having more than one chiral center and not being mirror images of each other (diastereoisomers). The chiral centers of the compounds can epimerize in vivo; thus, for these compounds, administering the compound in its (R-) form is considered equivalent to administering the compound in its (S-) form. Therefore, IMiD and CELMoD compounds can be used in the form of individual isomers and substantially free of other isomers, or in the form of mixtures of various isomers, such as racemic mixtures of stereoisomers.

[0321] In some embodiments, an IMiD or CELMoD compound is an isotopic derivative in that it has at least one desired atomic isotope substitution in an amount above the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound comprises deuterium or multiple deuterium atoms. Substitution with a heavier isotope such as deuterium (i.e., 2 H) may provide certain therapeutic advantages due to greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and may thus be advantageous in certain instances.

[0322] In addition, IMiD and CELMoD compounds include the use of N-oxides, crystal forms (also known as polymorphs), active metabolites of compounds having the same type of activity, tautomers, and non-solvated forms of the compounds and solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. Solvated forms of the conjugates presented herein are also considered to be disclosed herein.

[0323] Also provided herein is a pharmaceutical composition comprising a degrader tag, fusion protein, nucleotide sequence, vector, or cell of the invention, and a pharmaceutically acceptable excipient, carrier, adjuvant, and / or diluent. The pharmaceutical composition can generally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, supplementary immune enhancers such as adjuvants and cytokines, and optionally other therapeutic agents or compounds.

[0324] As used herein, "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual in conjunction with the selected compound without eliciting any undesirable biological effects and without interacting in a harmful manner with any other components of the pharmaceutical composition in which it is contained.

[0325] An excipient is a natural or synthetic substance formulated with an active ingredient (e.g., a compound of the present invention), including for the purpose of increasing the weight of the composition or conferring an enhanced therapeutic effect on the active ingredient in the final dosage form, such as promoting drug absorption or solubility. Excipients can also be used during the manufacturing process to assist in handling the relevant active substance, such as by promoting powder flowability or non-stickiness, and also contribute to in vitro stability, such as preventing denaturation during the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. Thus, a person of ordinary skill in the art can easily identify suitable excipients. For example, suitable pharmaceutically acceptable excipients include water, saline, aqueous glucose solution, glycerol, ethanol, etc. An adjuvant is a pharmacological and / or immunological reagent that modifies the action of other reagents in a composition. Pharmaceutically acceptable adjuvants are well known in the art. Thus, a person of ordinary skill in the art can easily identify suitable adjuvants. A diluent is a diluent. Pharmaceutically acceptable diluents are well known in the art. Thus, a person of ordinary skill in the art can easily identify suitable diluents. At the doses and concentrations employed, the carrier is non-toxic to the recipient and compatible with the other components of the formulation. The term "carrier" denotes a natural or synthetic organic or inorganic component with which the active ingredient is combined to facilitate application. Pharmaceutically acceptable carriers are well known in the art. Thus, a person of ordinary skill in the art can easily identify suitable carriers.

[0326] The pharmaceutical compositions of the present invention can be used as drugs. As detailed above, they may be particularly suitable for immunocytotherapy, where the degron tags provided herein can be used as a safety switch to regulate the expression of the POI.

[0327] Accordingly, a method for degrading a protein of interest (POI) is also provided, comprising: contacting a cell with an effective amount of an immunomodulatory drug (IMiD) or cereblon modulator (CELMoD) or a pharmaceutically acceptable salt or stereoisomer thereof, either in vitro or in vivo, wherein the cell expresses a nucleic acid encoding a fusion protein of the present invention (i.e., a fusion protein comprising the POI and a degron tag of the present invention). These methods can be carried out in vivo or in vitro. The POI may be exogenous or endogenous. The cell can be allogeneic or autologous. The method can be carried out in a subject. The subject can be a human.

[0328] A method for degrading a protein of interest is also provided, comprising:

[0329] Administering to a subject an effective amount of an immunomodulatory drug (IMiD) or cereblon modulator (CELMoD) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the subject has been previously treated by gene therapy to cause at least some endogenous cells to express a nucleic acid encoding the fusion protein of the present invention (i.e., a fusion protein comprising a POI and a degron tag of the present invention). Gene therapy is a medical method for treating or preventing diseases by introducing genes into cells to correct underlying genetic problems. Gene therapy includes introducing the nucleic acid or vector of the present invention into a subject, wherein the nucleic acid or vector encodes the degron tag or fusion protein described herein (i.e., a fusion protein comprising a POI and a degron tag of the present invention). The nucleic acid or vector can be introduced into cells ex vivo and then the cells can be introduced into the subject, or the nucleic acid or vector can be introduced into the subject's cells in vivo. In some embodiments, the subject has been administered immune effector cells, such as autologous T cells (CAR-T cells), which have been genetically modified to express a chimeric antigen receptor protein (CAR)-degron tag fusion protein and are experiencing an adverse immune reaction (e.g., cytokine release syndrome or neurotoxicity) due to the treatment. In some other embodiments, gene therapy includes gene knock-in, administration of viral vectors, or clustered regularly interspaced short palindromic repeats (CRISPR)-mediated knock-in.

[0330] Another aspect of the present invention relates to a method for reducing gene overexpression in a subject, comprising introducing a nucleic acid sequence encoding a degron tag into one or more relevant cells of the subject, the degron tag being genomically in-frame integrated with the nucleic acid sequence of an endogenous protein associated with a disease caused by overexpression of the endogenous protein; and administering to the subject an effective amount of an IMiD or CELMoD. In some embodiments, the endogenous protein is associated with a disease caused by a gain-of-function mutation, amplification, or increased expression, a monogenic disease, a proteinopathy, or a combination thereof.

[0331] Another aspect of the present invention relates to a method for assessing the function of an endogenous protein or validating an endogenous protein as a target for treating a disease state, comprising introducing a nucleic acid sequence encoding a degron tag into one or more relevant cells, the degron tag being genomically in-frame integrated with the nucleic acid sequence of an endogenous protein suspected of being associated with a disease; and contacting the cells with an effective amount of an IMiD or CELMoD. These methods can be performed in vivo (e.g., in an animal model) or in vitro (e.g., in cell culture).

[0332] Any of the methods of the present invention may require contacting cells or administering an IMiD or CELMoD to a subject, which is thalidomide, pomalidomide, lenalidomide, CC-122, CC-220, or CC-885.

[0333] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2nd Edition, John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide one of ordinary skill in the art with a general dictionary of many of the terms used in the present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined below will be described more fully by reference to the entire specification. Further, as used herein, unless the context clearly dictates otherwise, the singular terms "a," "an," and "the" include plural references. Unless otherwise indicated, nucleic acids are written left to right in a 5' to 3' direction; amino acid sequences are written left to right in an amino to carboxy direction. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described, as these may vary according to the context used by one of ordinary skill in the art.

[0334] The following non-limiting examples demonstrate various aspects of the present invention.

[0335] Example

[0336] Thalidomide-like derivatives (referred to as immunomodulatory drugs IMiDs, or more recently as Cereblon E3 ligase modulators CELMoDs) are degrader drugs that bind to a conserved tryptophan cage on the surface of Cereblon (CRBN), and CRL4CRL4CRBN is the substrate receptor of the Cullin4-RING E3 ubiquitin ligase CRL4 CRBN and induce an alteration in CRL4 CRBN substrate specificity, resulting in the recruitment, ubiquitination, and subsequent proteasomal degradation of new substrates such as Ikaros (IKZF1), Aiolos (IKZF3), casein kinase 1α (CK1a or CSNK1A), G1 to S phase transition protein 1 (GSPT1), and zinc finger protein 91 (ZFP91) (Table 1) 7,11-16 .

[0337]

[0338] Table 1: Summary of IMiD / CELMoD Substrate Specificity: From Thoma and colleagues 8 , Ebert / Fisher and colleagues 7,17,18 , Chamberlain and colleagues 16 ' data

[0339] The new substrates interact with the degrader-binding surface of CRBN via an identification motif called a degron, which is characterized by a common β-hairpin loop with a conserved glycine at the apex, which is crucial for the interaction of the degron with the degrader compound. The amino acid sequence adjacent to the sentinel glycine in the β-hairpin loop is variable among the new substrates and is crucial for the specificity of recruitment and degradation of new substrates by specific CRBN binders. In the case of the new substrates Ikaros, Aiolos, and ZFP91, the degron motif is located within the C2H2 zinc finger (ZF) domain, and the first half of this domain is characterized only by a CxxCG sequence containing a conserved glycine after the second cysteine of the ZF 7,8.

[0340] Generation of DCD-EGFP fusion construct

[0341] To address the limitations of currently available protein regulation tools, the inventors developed a new and robust system for targeted protein degradation by exploiting the fusion of IMiD / CeLMOD recognition sequences with the protein of interest. By systematically evaluating the substrates of 23 degron-containing domains (DCDs) from various ZF and non-ZF proteins with a panel of IMiD / CeLMOD small molecules (see Figure 1 ), they identified a DCD tag (DCD23) based on the chimeric sequences of Ikaros and ZFP91, which induces acute target degradation in vitro and in vivo ( Figure 1 ). This 60-amino acid sequence is referred to herein as iTAG1 (DCD23).

[0342] Based on the structural analysis of the DCD23 sequence, mutations were introduced to evaluate whether improved degron sequences could be identified. Many different constructs were tested (e.g., iTAG2v1, iTAG2v2, iTAG2), and the best construct was selected, which is referred to herein as DCD23mut or iTAG2 ( Figure 2 ).

[0343] The inventors found that when fused to an EGFP construct ( Figure 3 .a), iTAG2 induces degradation similar to iTAG1 (DCD23) and eliminates nuclear accumulation ( Figure 3 .b). Figure 4 Variants of iTAG2 that are also envisioned herein are shown.

[0344] Generation of iTAG2 chimeric antigen receptor (CAR) fusion construct

[0345] Then, the inventors cloned iTAG2 and iTAG1 (DCD23) into the C-terminus of a second-generation anti-B7H3 chimeric antigen receptor containing the intracellular domains of CD28 and CD3ζ, respectively. The tagged CAR was co-expressed with a CD34 marker gene from a γ-retroviral vector via a 2A sequence ( Figure 5 ). The inventors sought to determine whether the two tagged CARs were successfully degraded after cells expressing the tagged CARs were exposed to IMiD drugs.

[0346] Preliminary experiments evaluated the expression and degradation of human Jurat T cell leukemia cells or human 293T cells stably transduced with untagged, iTAG1 (DCD23)-tagged, and iTAG2-tagged anti-B7H3 CAR-T cells. Here, the relative expression of the CAR and the CD34 marker gene was determined by direct staining and flow cytometry assessment. In both target cell lines, although the iTAG2-CAR and the untagged CAR had similar CD34 and CAR expression levels, for the iTAG1 (DCD23)-tagged CAR, the CAR expression was lower than that of CD34, indicating relatively poor cell surface expression ( Figure 6 ). These data indicate that the iTAG2 sequence results in higher cell surface expression of the chimeric antigen receptor compared to the iTAG1 (DCD23) sequence.

[0347] The inventors next evaluated the downregulation of the iTAG2-tagged CAR in transduced Jurkat cells after addition of a series of concentrations of IMiD drugs (lenalidomide, pomalidomide, and elotuzumab). Both the iTAG1 (DCD23)-tagged and the iTAG2-tagged CARs showed a similar downregulation pattern after addition of the drugs, with a maximum reduction in surface expression detectable 4 hours after drug treatment. Elotuzumab showed the highest degradation potency, with almost maximum downregulation observed at a drug concentration as low as 0.01 μM ( Figure 7 ).

[0348] After demonstrating expression and drug-induced degradation in the Jurkat cell line, the inventors subsequently evaluated these parameters in primary human T cells. Human PBMCs from two independent donors were transduced with the TE9-28Z anti-B7H3 CAR in untagged DCD23 and iTAG2 formats and expanded for 7 days after addition of 10 μM elotuzumab for 24 hours, and then CAR expression was evaluated. Interestingly, in primary T cells at 24 hours, elotuzumab treatment resulted in almost complete loss of expression of the viable CAR-expressing population, although weak expression was still observed ( Figure 8 ).

[0349] To evaluate whether the more robust CAR expression of the iTAG2-tagged CAR translated into greater effector function compared to the DCD23-tagged CAR, the corresponding CAR-T populations from two independent donors were co-cultured overnight with the human SupT1 leukemia target cells syngeneic to the target antigen B7H3. Wild-type SupT1 has no detectable B7H3 expression. After culturing the T cells overnight with the target, the culture supernatant was harvested and the inflammatory cytokines interferon γ and interleukin-2( Figure 9 ) were evaluated by ELISA. The data showed that the DCD23 fusion protein had approximately 5-fold reduced effector function compared to the iTAG2 fusion protein in terms of interferon γ secretion and approximately 30-fold reduced IL-2 secretion( Figure 9 ). The effector function of the iTGA2-tagged CAR was not significantly different from that of the untagged CAR.

[0350] The inventors compared the surface expression of both the 41BB and CD28 versions of the TE9 CAR and compared the surface expression of the iTAG2 fusion protein with both DCD23 and a previously published super-degrader (referred to herein as the super-degrader stator). In transduced Jurkat cells, the DCD23- and super-degrader-tagged CARs had similar weak surface expression, while in this experiment, the iTAG2-tagged CAR had the same expression as the wild-type (untagged CAR). Contrary to the published reports, the super-degrader only worked in the context of the 41BB CAR and the inventors found no evidence of differential surface expression between the two co-stimulation arrangements( Figure 10 ). Thus, the data presented herein show that the tested DCD sequences (iTAG2, DCD23, and super-degrader) work in both the 41BB and CD28 versions of the second-generation CAR.

[0351] TR-FRET assay based on Aiolos peptide for measuring the relative affinity of the complex formed between iTAG1 and iTAG2 with CRBN / DDB1 (DNA damage-binding protein 1) and various IMiDs

[0352] Figure 11 A schematic of a time-resolved Förster resonance energy transfer (TR-FRET) assay based on the Aiolos peptide is shown. The inventors used this assay to evaluate the ability of iTAG1 and iTAG2 to displace a fluorescently labeled Aiolos peptide probe from complexes formed between CRBN, DDB1, and various IMiDs (lenalidomide, pomalidomide, and elotuzumab), enabling measurement of the relative affinity of iTAG1 and iTAG2 for these complexes. The results are as Figure 12 shown, including the IC 50 calculated from the TR-FRET curves. Overall, the IC 50 obtained for iTAG2 was higher than the IC obtained for iTAG150 , with a difference of approximately 10-fold. The relative affinities obtained for the 3 compounds were the same as the trends for iTAG1 and iTAG2, obtaining the tightest binding to lenalidomide, followed by pomalidomide and thalidomide.

[0353] Super-degron-tagged GFP mainly has nuclear localization

[0354] GFP tagged with iTAG2 had a cytoplasmic localization, in contrast to iTAG1, which had a predominantly nuclear localization. To determine the localization of a "super degron" that has a certain degree of homology to iTAG1, the inventors cloned the super degron into an expression vector to replace iTAG1 as the degron fused to GFP. After transient transfection in HMEC (human mammary epithelial cells), the super degron tag was shown to result in strong nuclear localization of GFP, as Figure 13 shown.

[0355] Size-exclusion chromatography confirms the formation of a complex between CRBN / DDB1 and iTAG2 in the presence of lenalidomide

[0356] Biochemical evidence for IMiD-dependent formation of a direct interaction between iTAG2 and the CRBN / DDB1 complex was generated using size exclusion chromatography (SEC). Purified CRBN / DDB1 protein complex and iTAG2 protein were mixed together and SEC was performed in the presence or absence of lenalidomide, as Figure 14 shown. In the presence of lenalidomide, SEC peak 1 corresponding to the higher molecular weight species shifted to a lower elution time and there was a slight increase in the optical density at 280 nm, which is consistent with the formation of a larger complex in the presence of lenalidomide ( Figure 14 A). In the absence of lenalidomide, SDS-PAGE analysis of representative fractions from the SEC analysis confirmed that SEC peak 1 contained only DDB1 and CRBN proteins, while iTAG2 was only present in SEC peak 2 ( Figure 14 B). In the presence of lenalidomide, SEC peak 1 contained all three proteins, demonstrating the formation of a complex between DDB1, CRBN, and iTAG2 in the presence of lenalidomide ( Figure 14 C).

[0357] iTAG2-labeled human CAR-T cells rapidly re-express after cell washing and IMiD drug withdrawal

[0358] After clearing the degrader drug, the re-expression of the degraded chimeric antigen receptor is important for CAR-T cells to be able to switch between an active and a resting state, thus allowing additional therapeutic manipulations and fine-tuning. To determine reversibility, TE9-28Z-iTAG2 CAR-T cells from PBMCs were treated with lenalidomide concentrations to reduce CAR-T surface expression, then washed twice, and then cultured for 24 hours in fresh medium lacking lenalidomide to demonstrate the re-expression of cell surface CAR, as Figure 15 shown in A and B.

[0359] Expression and function of iTAG2-labeled anti-B7H3 CAR in lentiviral vector

[0360] To evaluate the general function of the TE9-28Z-iTAG2 anti-B7H3 CAR-T construct when expressed in different viral vectors driven by alternative promoters, the construct was cloned into the third-generation lentiviral vector pCLL2 backbone, and viral supernatants were generated by transient transfection of 293T cells, and the viruses were pseudotyped with VSV-G. Two versions of the TE9-28Z-iTAG2 sequence were evaluated in this format, with and without codon optimization and clone scar removal. The amino acid sequence of iTAG2 was identical between the original (construct 3) and codon-optimized (construct 4) versions ( Figure 16 a and b). To test the degradation after codon optimization of the CAR and iTAG2 sequences, CAR-T cells generated from three independent donors using GMP-like conditions transduced with the pCCL2 lentiviral vector were expanded to 9 days after stimulation and then treated overnight with 100 nM lenalidomide before direct staining of both CAR and the co-expressed RQR8 marker gene by flow cytometry. The log reduction of CAR expression, expressed as geometric mean fluorescence intensity (gMFI), was highly consistent. It was found that codon-optimized iTAG2 in the lentiviral backbone showed the same sensitivity to lenalidomide-induced degradation as non-codon-optimized iTAG2 ( Figure 16 c). Codon-optimized lentivirus-expressed TE9-28z-iTAG2 CAR-T cells showed high cytotoxicity levels against antigen-positive target cells and inflammatory cytokine secretion ( Figure 16 d-f). Cytotoxicity of TE9-iTAG2 lacking codon optimization against B7H3-negative SupT1 targets was confirmed using an overnight luminescence-based assay at an E:T ratio of 1:2 ( Figure 16 G shows data from three independent CAR-T donors).

[0361] Methods:

[0362] Design of iTAG2

[0363] The inventors generated three mutant versions of iTAG1:

[0364] -iTAG2v1, in which only the central ZFP91 / Ikaros chimeric zinc finger is mutated:

[0365] iTAG2v1

[0366]

[0367] -iTAG2v2, in which one more residue is mutated in the linker between ZF1 and ZF2 of Ikaros:

[0368] iTAG2v2

[0369]

[0370] -iTAG2, in which several residues present in the construct are mutated:

[0371] iTAG2

[0372]

[0373] The sequences were commercially synthesized (GeneArt, ThermoFischer Scientific) and inserted into the pLVX-TetOne-Puro lentiviral vector.

[0374] Evaluation of the EGFP-iTAG(1 / 2) construct

[0375] Viral transduction and cell line generation

[0376] To produce viral particles, HEK293T cells were plated 24 hours before transfection and transfected with pLVX-TetOne-Puro lentiviral vector psPAX2 (packaging plasmid) pMD.2G using Lipofectamine TM 3000 (ThermoFisher). The viral-containing supernatant was harvested 48 hours after transfection and filtered through a 0.45 μm filter (Sartorius Stedim) for storage in cryotubes (ThermoFischer Scientific) at -80 °C.

[0377] For cell transduction, HMEC cells were infected at an MOI of 3 (viral particle titer was determined using the Lenti-XTM GoStixTM kit (Clontech)), and polybrene (4 μg / ml) was used to enhance the transduction efficiency. The cells were cultured for at least two generations and then induced with 1 μg / ml doxycycline (DOX) for 24 hours for construct expression. Fluorescence-activated cell sorting (FACS) was used to select the top 30% cell population with the highest EGFP expression.

[0378] Testing degradation using flow cytometry

[0379] Construct expression was induced by treating cells with 1 μg / ml of DOX for 24 hours. Cells were treated with IMiD / CELMoD at the indicated concentrations and time points for each experimental setup. Cells were collected in sterile 5 ml polystyrene round-bottom tubes on ice. DAPI (4′,6-diamidino-2-phenylindole; ThermoFisher Scientific) was added to the cell suspension at a ratio of 1:1000 as a marker of cell viability. EGFP signals were measured on a BD LSR II flow cytometer (BD Biosciences). Results were further processed using FlowJo 10 software (FlowJo).

[0380] Testing nuclear localization using immunofluorescence

[0381] HMEC cells were seeded at 1000 cells / well in glass-bottom 96-well plates (Perkin Elmer) and treated with 1 μg / ml of DOX for 24 hours to induce the construct. Cells were fixed with 4% paraformaldehyde in PBS solution for 15 minutes at 37 °C and permeabilized with 0.05% Triton-X-100 detergent for 10 minutes at room temperature. Prior to starting cell imaging, cells were incubated with 1 μg / ml of DAPI (ThermoFisher Scientific) for one minute to stain the cell nuclei. Cells were visualized on a Zeiss Axio Vert.A1 FL-LED inverted epifluorescence microscope (Zeiss) and images were captured using ZENBlue software (Zeiss).

[0382] Evaluation of the CAR-iTAG(1 / 2) construct

[0383] Construct generation and cloning

[0384] The degrader tags iTAG1 (DCD23) and iTAG2 were synthesized as gene blocks (IDT) and cloned into the MMLV-derived γ-retroviral vector SFG using standard restriction site ligation. The vector drives the transcription of a single transcript (where the RQR8 epitope and the CAR construct (anti-B7H3 scFv (TE9), CD8 transmembrane domain, CD28 co-stimulation, and CD3 signaling domain)) and is separated by a T2A ribosomal skipping site. The degrader tags were inserted directly after the CD3 signaling domain without a spacer sequence.

[0385] Virus production

[0386] When approximately 80% fusion is achieved using the standard protocol, Phoenix ampho packaging cells are grown and transfected with viral constructs containing the CAR+ / - tag using GeneJuice transfection reagent. Viral supernatants are harvested at 48 hours and 72 hours post-transfection and pooled prior to snap-freezing.

[0387] Transduction of primary cells and cell lines:

[0388] Jurkat and 293T cell lines are grown in DMEM containing 10% fetal bovine serum and transduced with constructs containing the CAR+ / - degrader tag. Transduction efficiency is analyzed by flow cytometry 72 hours post-transduction, staining for the CD34 epitope encoded by the RQR8 gene.

[0389] PBMC-derived T cells from blood cones of 2 healthy donors are cultured after CD56 depletion (day 0) using Miltenyi CD56 depletion beads and magnetic negative selection using an LD column according to the manufacturer's instructions. After depletion, PBMCs are stimulated with CD3+CD28 antibody beads (day 1) and activated with IL-2 at 100 iU / ml (day 3). On day 4, cells are transduced with CAR+ / - degrader tag virus using undiluted PBMC viral supernatant and diluent to generate a multiplicity of infection of 1 - 5 for the cell lines. Transduction efficiency is evaluated 4 days post-transduction by flow cytometry staining for RQR8 and CAR expression.

[0390] IMiD-induced CAR degradation and CAR function experiments:

[0391] Degradation assays are performed in transduced Jurkat cell lines by adding pomalidomide, lenalidomide, or elotuzumab or DMSO at concentrations of 10 μM, 1 μM, 0.1 μM, 0.01 μM for 2 hours, 4 hours, or 24 hours, and CAR degradation is evaluated by flow cytometry.. CAR expression in PBMC-derived T cells after degradation with 10 uM, 1 uM, 0.1 uM, 0.01 uM, 0.001 uM, or 0 uM elotuzumab is evaluated by flow cytometry 24 hours later. To evaluate CAR function + / - degrader tag, transduced T cells are cultured with SUPT1+ / -B7H3 at a 1:1 ratio for 24 hours. Supernatants are harvested and IL-2 and IFN levels are measured by ELISA.

[0392] Flow cytometry analysis:

[0393] Using an LSR2 cell counter, the CAR surface expression in primary cells and cell lines for transduction efficiency and degradation experiments was quantified using his-tagged B7H3 protein binding to an anti-his-tagged fluorescent antibody (J095G45, BioLegend) and a directly fluorescently labeled anti-CD34 antibody (QBEnd10, R&D). Analysis was performed using Flo-Jo software.

[0394] Aiolos peptide-based TR-FRET assay:

[0395] The methods for the production and purification of the proteins used in the TR-FRET assay have been described in our previous publication on iTAG discovery (Bouguenina, Nicolaou and Le Bihan et al., 2023. iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells. iScience, Volume 26, Issue 7 https: / / doi.org / 10.1016 / j.isci.2023.107059) 19 。

[0396] All TR-FRET assays were performed in black 384-well Proxi plates + (Perkin-Elmer, USA) in a buffer containing 20 mM HEPES (pH 8.0), 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween 20 and 0.05% BSA, with a final assay volume of 10 μL. The Echo E550 (Beckman Coulter, USA) acoustic liquid dispenser was used to create a final concentration range of 82 μM to 2.05 nM for iTAG1 and 27 μM to 0.68 nM for iTAG2. The wild-type full-length CRBN / DDB1 complex at a final concentration of 5 nM, 750 nM sulfo-Cy5 fluorescent Aiolos-based peptide probe (Cambridge Research Biochemicals, UK), 750 nM Zn(OAc)2 and 0.5 nM MAb anti-6HIS-terbium cryptate gold (Cisbio, France) were added using a Tempest liquid handler (Formulatrix, USA). Finally, IMiD was added using the Echo until a final concentration of 10 μM. The plates were sealed, centrifuged at 200 g for 1 minute and stored overnight at 4 °C. The TR-FRET signal was read using a PHERAstar FSX microplate reader (BMG Labtech, Germany). The final signal was measured as the following ratio:

[0397] TR-FRET signal = Channel 1 / Channel 2

[0398] Channel 1 at 665 nm represents positive FRET, and Channel 2 at 620 nm represents terbium emission when no FRET occurs.

[0399] Size-exclusion chromatography:

[0400] A method for preparing and purifying proteins for reconstituting the complex formed between CRBN / DDB1, lenalidomide, and iTAG2 by size-exclusion chromatography (SEC) has been described in our previous publication on iTAG discovery (Bouguenina, Nicolaou and Le Bihan et al., 2023. iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells. iScience, Volume 26, Issue 7, https: / / doi.org / 10.1016 / j.isci.2023.107059). 19 。

[0401] SEC was performed using a Superose 6 5 / 150GL column (Cytiva Life Sciences) installed on an Agilent 1260 Infinity II LC system HPLC system. Two 60 μL samples were prepared by mixing purified CRBN / DDB1 and iTAG2 at 15 μM and 50 μM, respectively, in a buffer consisting of 20 mM HEPES pH 7.0, 200 mM NaCl, and 1 mM TCEP. One of the samples included 100 μM lenalidomide, and both samples were incubated on ice for 1 hour and then centrifuged at 21000 g for 10 minutes before injection to remove potential aggregates. Before injecting 50 μL of each sample, the Superose 6 column was pre-equilibrated in the same buffer (20 mM HEPES pH 7.0, 200 mM NaCl, and 1 mM TCEP) at a flow rate of 0.25 mL / min. 100 μL fractions were collected, and samples across the elution peak were analyzed by SDS-PAGE on precast NuPAGE 4-12% Bis-Tris gels (Invitrogen), while SeeBlue Plus2 prestained protein standards (Invitrogen) were loaded in the first lane of each gel.

[0402] The reader's attention is directed to all papers and documents that were filed simultaneously with or before this specification in relation to this application, and these papers and documents may be publicly available for inspection together with this specification, and the contents of all these papers and documents are incorporated herein by reference.

[0403] All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations where at least some of these features and / or steps are mutually exclusive.

[0404] Unless otherwise expressly stated, each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by an alternative feature for the same, equivalent or similar purpose. Accordingly, unless otherwise expressly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0405] The present invention is not limited to the details of any of the foregoing embodiments. The present invention extends to any new feature or any combination of new features among the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any new method or process or any combination of new methods or processes among the steps of any method or process so disclosed.

[0406] References

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[0421] 15. Gandhi, A.K. et al. Immunomodulatory agents lenelidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors lkaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4CRBN. Br. J. Haematol. 164, 811 - 821 (2014).

[0422] 16. Matyskiela, M.E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252 - 257 (2016).

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[0425] 19. Bouguenina, Nicolaou and Le Bihan et al. iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells. iScience, Volume 26, Issue 7 (2023).

Claims

1. A degron tag, comprising the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:66), where X1, X2, X3, and X4 are not K, R, or H.

2. The degron tag according to claim 1, comprising the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO:2), where: X1 is E or its conservative amino acid substitution; X2 is A or its conservative amino acid substitution; X3 is N or its conservative amino acid substitution; and X4 is E or its conservative amino acid substitution.

3. The degron tag according to claim 2, wherein the tag comprises the amino acid sequence LQCEICGFTCEQAGNLLNHIELH (SEQ ID NO:5).

4. The degron tag according to claim 3, wherein the tag comprises the amino acid sequence LQCEICGFTCEQAGNLLNHIELHSG (SEQ ID NO:12) or LQCEICGFTCEQAGNLLNHIELHTG (SEQ ID NO:13).

5. The degron tag according to any one of the preceding claims, wherein the tag comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain flanking the amino acid sequence in any one of SEQ ID NO:66, 2, 5, 12, or 13.

6. The degron tag according to claim 5, wherein the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence CHLCNYACR (SEQ ID NO:14), CHLCNYACQ (SEQ ID NO:15), CHLCNYACRRRDAL (SEQ ID NO:69), or CHLCNYACQRRDAL (SEQ ID NO:70).

7. The degron tag according to claim 5 or 6, wherein the additional N-terminal zinc finger α-helix subdomain comprises the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:71) or FNVLMVHX5X6SH (SEQ ID NO:72); where X5 and X6 are not R, K, or H.

8. The degron tag according to claim 7, wherein the additional N-terminal zinc finger α-helix subdomain comprises the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO:16) or FNVLMVHX5X6SH (SEQ ID NO:17); where X5 is N or its conservative amino acid substitution; and X6 is E or its conservative amino acid substitution.

9. The degron tag according to claim 8, wherein the additional N-terminal zinc finger α-helix subdomain comprises the amino acid sequence PNVLMVHNESH (SEQ ID NO:22) or FNVLMVHNESH (SEQ ID NO:23).

10. The degron tag according to claim 8 or 9, wherein the tag comprises the amino acid sequence: PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO:73), or FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO:74); wherein X7 and X8 are not R, K or H.

11. The degron tag according to claim 10, wherein the tag comprises the amino acid sequence: PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO:24), or FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO:45); wherein: X7 is I or a conservative amino acid substitution thereof; and X8 is I or a conservative amino acid substitution thereof.

12. The degron tag according to claim 11, wherein the tag comprises the amino acid sequence: PNVLMVHNESHTGEIPLQCEICGFTCEQAGNLLNHIELHSGEIPFKCHLCNYACRRRDAL (SEQ ID NO:43), or FNVLMVHNESHTGEIPLQCEICGFTCEQAGNLLNHIELHTGEIPFKCHLCNYACQRRDAL (SEQ ID NO:44).

13. The degron tag according to any one of the preceding claims, wherein the tag has a length of about 23 to about 70 amino acids.

14. The degron tag according to claim 10, wherein the tag has a length of about 23 to about 60 amino acids.

15. A fusion protein comprising a protein of interest and at least one degron tag according to any one of claims 1 to 14.

16. The fusion protein according to claim 15, wherein the degron tag is located at the C-terminus of the protein of interest.

17. The fusion protein according to claim 15 or 16, wherein the protein of interest is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor (TCR) fusion construct (TRuC), a T cell antigen conjugate (TAC), a chimeric autoantibody receptor (CAAR), or an antibody-conjugated T cell receptor (ACTR).

18. The fusion protein according to claim 17, wherein the CAR fusion protein comprises, from the N-terminus to the C-terminus: a) an extracellular ligand-binding domain; b) a transmembrane domain; c) a cytoplasmic domain comprising at least one intracellular signaling domain; and d) at least one degron tag according to any one of claims 1 to 14.

19. The fusion protein according to claim 18, wherein the extracellular ligand-binding domain comprises an antibody or an antigen-binding fragment, the antigen-binding fragment being a scFv that binds B7H3, the transmembrane domain being a CD8 transmembrane domain, and the intracellular signaling domain being a CD3 signaling domain, and the CAR fusion protein further comprises a CD28 co-stimulatory domain.

20. A non-naturally occurring nucleic acid sequence encoding the degron tag according to any one of claims 1 to 14 or the fusion protein according to any one of claims 15 to 19.

21. A vector comprising the nucleic acid sequence according to claim 20.

22. The vector according to claim 21, wherein the vector is a viral vector, optionally wherein the viral vector is selected from the group consisting of: retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, vaccinia viral vectors, picornaviral vectors, and alphaviral vectors.

23. A cell that expresses the nucleic acid sequence according to claim 20 or the vector according to claim 21 or 22.

24. The cell according to claim 23, wherein the cell is an immune effector cell.

25. The cell according to claim 23 or 24, wherein the cell is selected from the group consisting of: T cells; B cells; plasma cells; NK cells; NKT cells; innate lymphoid cells; macrophages; dendritic cells; monocytes; neutrophils; basophils; eosinophils; mast cells; hematopoietic progenitor cells; hematopoietic stem cells; other adult stem cells, such as neural, corneal, muscle, skin, small intestine, colon, bone, mesenchymal; embryonic stem cells; and induced pluripotent stem cells.

26. The cell according to any one of claims 23 to 25, wherein the cell is a mammalian cell, optionally wherein the cell is a human cell.

27. A pharmaceutical composition comprising the degron tag, fusion protein, nucleotide sequence, vector, or cell according to any one of the preceding claims, and a pharmaceutically acceptable excipient, carrier, adjuvant, and / or diluent.

28. The pharmaceutical composition according to claim 27, for use as a medicament.

29. The pharmaceutical composition according to claim 27, for use in immunocyte therapy.

30. A method of degrading a protein of interest, comprising: contacting a cell, in vitro or in vivo, with an effective amount of an immunomodulatory drug (IMiD) or a cereblon modulator (CELMoD), wherein the cell expresses a nucleic acid encoding the fusion protein according to any one of claims 15 to 19.

31. A method of degrading a protein of interest, comprising: Administering to a subject an effective amount of an immunomodulatory drug (IMiD) or a cereblon modulator (CELMoD), wherein the subject has been previously treated by gene therapy, causing at least some endogenous cells to express a nucleic acid encoding the fusion protein of any one of claims 15 to 19.

32. The method according to claim 30 or 31, wherein the IMiD or CELMoD is thalidomide, pomalidomide, lenalidomide, CC-122, CC-220 or CC-885.

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