Novel drug-inducible degradation tag

Abstract

The present invention provides novel drug-inducible degradation tags (degrons), as well as fusion proteins, cells, and pharmaceutical compositions containing them. Nucleic acid sequences and vectors encoding the novel degrons are also provided. Methods for using the novel degrons, fusion proteins, cells, pharmaceutical compositions, nucleic acid sequences, and vectors are also provided herein.

Description

[Technical Field]

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

[0002] background Chemical biology tools that induce targeted protein degradation with drug-like precision have attracted considerable attention in recent years due to their ability to acutely and reversibly modulate protein levels within cells. These techniques are based on the use of small amino acid tags fused to proteins of interest that induce degradation in the presence of specific compounds. Several techniques are currently available, examples of which include cryptic degrons. 1 Small molecule substitution, small molecule assisted shutoff (SMASh) 2 , Degradation of fused hydrophobic tag (HaloTag), Auxin-inducible degron (AID1 / 2), HaloPROTAC 3 , dTAG system 4,5 Although these tools have enabled many discoveries at the preclinical level, their various limitations (the need to express multiple components, poor PK / PD of related compounds, and the use of non-FDA-approved molecules) have hindered their translation to more clinically relevant applications.

[0003] One of the best-known examples of drug-induced protein degradation is the mechanism of action of thalidomide-like derivatives (immunomodulatory imide drugs, IMiDs, or more recently, cereblon E3 ligase modulators, CELMoDs). These degrader drugs inhibit the Cullin4-RING E3 ubiquitin ligase CRL4. CRBN It binds to the conserved tryptophan cage on the surface of cereblon (CRBN), the substrate receptor for CRL4. CRBNIt induces changes in the substrate specificity of Ikaros (IKZF1), Aiolos (IKZF3), casein kinase 1 alpha (CK1a or CSNK1A), G1 to S phase transition protein 1 (GSPT1), and zinc finger protein 91 (ZFP91). 6 This leads to the recruitment, ubiquitination and subsequent proteasomal degradation of neo-substrates such as ATP.

[0004] Neosubstrates interact with the degron-binding surface of CRBN through a recognition motif known as a degron. This recognition motif is characterized by a common β-hairpin loop with a conserved glycine at its apex and is crucial for the interaction of the degron with degron-binding compounds. The amino acid sequence adjacent to the sentinel glycine in the β-hairpin loop varies among neosubstrates and is crucial for the specificity of neosubstrate recruitment and degradation by specific CRBN-binding compounds. In the case of the neosubstrates Ikaros, Aiolos, and ZFP91, the degron motif is located within the C2H2 zinc finger (ZF) domain, and the first half of the domain is the ZF domain. 7,8 It has a CxxCG sequence with a conserved glycine immediately following the second cysteine.

[0005] The concept of molecular switches to control chimeric antigen receptor (CAR) expression is well recognized in the field as a desirable solution for enhancing the safety and efficacy of CAR-T cells. In a proof-of-concept study, McCall's research group at Stanford University demonstrated that inducing CAR degradation in human T cells significantly improved the efficacy of CAR-T. 9We demonstrated two major advantages over conventional CAR expression in this regard. First, CAR degradation during T cell manufacturing reduces T cell exhaustion associated with CAR tonic signaling, resulting in a T cell product that improves T cell survival and leads to improved survival in tumor-bearing animals after administration. Second, if a drug that induces CAR degradation on T cells can be administered to tumor-bearing animals on an intermittent dosing schedule, it can temporarily rest T cells from antigen-induced activation in the tumor environment. As a result, T cells can recover from exhaustion, and re-expression of the CAR enhances effector function. A third potential advantage of the degradation switch is its ability to turn off CAR-T function in the face of toxicity, providing a novel additional safety feature.

[0006] These data on the functional benefits of transiently switching off CAR in T cells are supported by chemical probes 9 This has been demonstrated using the FK506-binding protein 12 destabilizing domain, which can be blocked by IL-1. Clinical application of this technology requires the development of CARs that are reversibly degraded in response to drugs approved for human use, function at pharmacologically achievable doses, are nontoxic, and have good tumor tissue penetration.

[0007] Such proof-of-concept data suggest that IMiD drugs (thalidomide derivatives) inhibit CRL4 CRBN Jan and colleagues evaluated the molecular glue concept of linking E3 ubiquitin ligases with known substrate proteins containing zinc finger degron motifs. 10 provided by The authors identified a prototype zinc finger-based 60-amino acid degron, a hybrid of ZFP91 and IKZF3, which they termed a "Super-Degron," and showed that it reversibly degraded a second-generation (41BB-zeta) chimeric antigen receptor tagged with the cytoplasmic C-terminus of the superdegron in a lenalidomide-dependent manner.

[0008] Improved degron technology is needed to avoid the toxicity of CAR T cells and improve cancer treatment. Summary of the Invention

[0009] Summary of the Invention We developed a novel drug-inducible degradation tag. Based on a chimeric structure of IKZF1 (Ikaros) and ZFP91 (zinc finger protein 91), we performed structural and sequence analyses to generate 23 different protein tags (degron candidates). Drug-inducible degradation of each tag was determined, and DCD23 (also referred to herein as iTAG1) was identified as a degron candidate due to its relatively small size (60 amino acids) and favorable IMiD / CELMoD degradation profile (Figure 1). We then performed additional amino acid substitutions to further optimize the DCD23 degron sequence. During this process, we identified four core amino acid substitutions that inhibited the nuclear localization of mutant degron tags (iTAG2v1, iTAG2v2, and iTAG2 (the latter also referred to herein as DCD23mut)). It is particularly advantageous to fuse the degron tag to a cell surface protein, such as a chimeric receptor antigen (CAR), to promote cell surface expression of the tagged protein, thereby preventing its localization / accumulation in the cell nucleus.

[0010] The present invention is based on the surprising discovery that fusing a novel mutant degron tag (such as DCD23mut, also referred to herein as iTAG2) to a CAR results in improved membrane stability of the resulting CAR fusion protein in Jurkat cells, 293T cells, and primary human T cells compared to DCD23-tagged CARs, while being rapidly degraded in the presence of IMiDs / CELMoDs such as lenalidomide, pomalidomide, and iverdomide. Advantageously, expression of DCD23mut-tagged CAR-Ts is superior to that reported for the patented degron "superdegron" (WO2019089592). Furthermore, DCD23mut-tagged CAR-Ts expressed in primary human T cells exhibited antigen-dependent reactivity at levels similar to that observed with untagged CAR-Ts (approximately 10-fold superior to DCD23-tagged CAR-Ts).

[0011] Thus, the present invention provides a degron tag comprising the amino acid sequence of 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 of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 2), wherein: X1 is E or a conservative amino acid substitution thereof; X2 is A or a conservative amino acid substitution thereof; X3 is N or a conservative amino acid substitution thereof, and X4 is E or a conservative amino acid substitution thereof.

[0013] Preferably, the degron tag may comprise the amino acid sequence LQCEICGFTCEQAGNLLNHIELH (SEQ ID NO: 5).

[0014] Preferably, the degron tag may comprise the amino acid sequence LQCEICGFTCEQAGNLLNHIELHSG (SEQ ID NO: 12) or LQCEICGFTCEQAGNLLNHIELHTG (SEQ ID NO: 13).

[0015] Suitably, the degron tag may comprise 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 any one of SEQ ID NOs: 66, 2, 5, 12 or 13.

[0016] Suitably, 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).

[0017] Suitably, the additional N-terminal zinc finger α-helical 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.

[0018] Suitably, the additional N-terminal zinc finger α-helical subdomain may comprise the amino acid sequence PNVLMVHX5X6SH (SEQ ID NO: 16) or FNVLMVHX5X6SH (SEQ ID NO: 17); X5 is N or a conservative amino acid substitution thereof; and X6 is E or a conservative amino acid substitution thereof.

[0019] Suitably, the additional N-terminal zinc finger α-helical subdomain may comprise the amino acid sequence PNVLMVHNESH (SEQ ID NO: 22) or FNVLMVHNESH (SEQ ID NO: 23).

[0020] Preferably, the degron tag has 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.

[0021] Preferably, the degron tag has the amino acid sequence: PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO: 24), or FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO: 45) where: X7 is I or a conservative amino acid substitution thereof; and X8 is I or a conservative amino acid substitution thereof.

[0022] Preferably, the degron tag has the amino acid sequence: PNVLMVHNESHTGEIPLQCEICGFTCEQAGNLLNHIELHSGEIPFKCHLCNYACRRRDAL (SEQ ID NO: 43), or FNVLMVHNESHTGEIPLQCEICGFTCEQAGNLLNHIELHTGEIPFKCHLCNYACQRRDAL (SEQ ID NO: 44) may include:

[0023] Preferably, the degron tag has a length of about 23 to about 70 amino acids.

[0024] Preferably, the degron tag has a length of about 23 to about 60 amino acids.

[0025] The present invention also provides a fusion protein comprising a protein of interest and at least one degron tag of the present invention.

[0026] Preferably, the degron tag may be located at the C-terminus of the protein of interest.

[0027] Suitably, 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 coupler (TAC), a chimeric autoantibody receptor (CAAR), or an antibody-bound T cell receptor (ACTR).

[0028] Suitably, the CAR fusion protein comprises, from N-terminus to C-terminus: a) extracellular ligand-binding domain; b) transmembrane domain; c) a cytoplasmic domain containing at least one intracellular signaling domain; and d) at least one degron tag of the present invention may include:

[0029] Suitably, the CAR fusion protein may comprise an extracellular ligand-binding domain comprising an antibody or antigen-binding fragment that is an scFv that binds to B7H3.

[0030] Suitably, the CAR fusion protein may comprise the CD28 transmembrane domain.

[0031] Suitably, the CAR fusion protein may comprise a CD3 signalling domain.

[0032] Suitably, the CAR fusion protein may comprise a CD28 costimulatory domain.

[0033] Suitably, the CAR fusion protein may comprise an extracellular ligand-binding domain comprising an antibody or antigen-binding fragment that is an scFv that binds to B7H3, a CD8 transmembrane domain, a CD3 signaling domain, a CD28 costimulatory domain, and at least one degron tag of the invention.

[0034] The present invention also provides non-naturally occurring nucleic acid sequences encoding the degron tags of the present invention or the fusion proteins of the present invention.

[0035] The present invention also provides a vector comprising a nucleic acid sequence of the present invention.

[0036] Preferably, the vector is a viral vector, and optionally the viral vector is selected from the group consisting of a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, a vaccinia viral vector, a picornavirus vector, and an alphavirus vector.

[0037] The present invention also provides cells expressing the nucleic acid sequences of the invention or the vectors of the invention.

[0038] Preferably, the cell is an immune effector cell.

[0039] Preferably, the cells are selected from the group consisting of T cells, B cells, plasma cells, NK cells, NKT cells, natural lymphocytes, 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.

[0040] Preferably the cell is a mammalian cell, and optionally the cell is a human cell.

[0041] The present invention also provides pharmaceutical compositions comprising a degron tag, fusion protein, nucleotide sequence, vector, or cell of the present invention and a pharmaceutically acceptable excipient, carrier, adjuvant, and / or diluent.

[0042] The present invention also provides a pharmaceutical composition of the present invention for use as a medicament.

[0043] Preferably, the pharmaceutical composition of the present invention can be used in immune cell therapy.

[0044] The present invention also provides a method for degrading a protein of interest, comprising the steps of: Contacting cells in vitro or in vivo with an effective amount of an immunomodulatory drug (IMiD) or cereblon modulator (CELMoD), wherein the cells express a nucleic acid encoding a fusion protein of the invention.

[0045] The present invention also provides a method for degrading a protein of interest, comprising the steps of: Administering an effective amount of an immunomodulatory drug (IMiD) or cereblon modulator (CELMoD) to a subject, wherein the subject has previously been treated with gene therapy to cause at least some endogenous cells to express a nucleic acid encoding a fusion protein of the invention.

[0046] Preferably, the IMiD or CELMoD is thalidomide, pomalidomide, lenalidomide, CC-122, CC-220 or CC-885.

[0047] Throughout the description and claims of this specification, the terms "comprise" and "contain" and variations thereof mean "including, but not limited to," and are not intended to (and do not) exclude other moieties, additives, components, integers, or steps.

[0048] Throughout the description and claims of this specification, the singular encompasses the plural unless the context clearly dictates otherwise. In particular, where the indefinite article is used, the specification is understood to contemplate the plural as well as the singular, unless the context clearly dictates otherwise.

[0049] It is understood that any feature, integer, property, compound, chemical moiety or group described in connection with a particular aspect, embodiment or example of the invention is also applicable to other aspects, embodiments or examples described herein, except where inconsistent therewith.

[0050] Various aspects of the invention are described in further detail below. [Brief explanation of the drawings]

[0051] BRIEF DESCRIPTION OF THE DRAWINGS Aspects of the present invention will now be further described with reference to the accompanying drawings. [Figure 1]Figure 1: Experimental evaluation of DCD tags. (a) Heatmap summary of degradation matrix data for all DCDs tested in flow cytometry experiments. Normalized values ​​are expressed as Log2 fold change relative to the DMSO control. In the group of chimeric ZF DCDs (21-23), DCD21 provided the most potent degradation but was significantly larger in size than DCD23 (131 residues vs. 60 residues, respectively). (b) Representation of iTAG1-DCD23 (60 aa). The two Cys and two His residues in the C2H2 zinc finger motif are highlighted in light gray. The critical glycine in the β-hairpin loop degron is highlighted in bold, dark gray. [Figure 2] Figure 2: iTAG2 design. Representation of iTAG1 (DCD23) and iTAG2. The two Cys and two His residues in the C2H2 zinc finger motif are highlighted. The critical glycine in the degron of the β-hairpin loop is highlighted. The mutated amino acids in iTAG2 are also highlighted in bold, dark gray, and underlined (as detailed elsewhere herein). The sequence of the superdegron (described in WO2021188286 and WO2019089592) is also shown. Differences between the superdegron and iTAG are highlighted in italics on the superdegron sequence. [Figure 3] Figure 3: Experimental validation of iTAG2. (a) HMECs expressing dox-inducible EGFP-iTAG1 / 2, or their corresponding P2A controls, were treated with CC-220 for 4 hours. Target protein loss was verified by measuring EGFP mean intensity by flow cytometry. P2A is a ribosomal skip sequence that allows separate translation of EGFP and iTAG, providing evidence that degradation is specific to the iTAG fusion. (b) Immunofluorescence images of HMECs expressing dox-inducible EGFP-iTAG1 / 2, showing nuclear accumulation of iTAG1, which is abolished with iTAG2. [Figure 4]Figure 4: iTAG2 Variations. iTAG2 and its variations. The two Cys and two His residues in the C2H2 zinc finger motif are highlighted. The critical glycine in the degron of the β-hairpin loop is highlighted. The mutated amino acids are highlighted in bold, dark gray, and underlined (as detailed elsewhere herein). [Figure 5] Figure 5: Constructs used for expression of tagged chimeric antigen receptors. CAR was expressed from the gammaretroviral construct SFG driven by the EF1α promoter. The RQR8 marker gene expresses the human CD34 epitope, allowing detection of transduced cells using the anti-CD34 monoclonal antibody clone QBEND10. RQR8 is cleaved from CAR by a T2A ribosomal skip site, allowing equal translation of the two protein products. The chimeric antigen receptor contains a TE9 anti-B7H3 conjugate in ScFv format, CD28 and CD3zeta second-generation signaling domains, a CD8 hinge sequence, and a transmembrane sequence. At the C-terminus, iTAG1 (DCD23) or iTAG2 was cloned in-frame with the CD3zeta sequence. [Figure 6] Figure 6: Flow cytometry histograms showing paired samples of CAR (top) and CD34 (bottom) expression in transduced 293T cells (left) and Jurkat cells (right). The histogram on the left represents the isotype control, while the histogram on the right shows CAR or CD34 expression, respectively. The numbers indicate the percentage of the population stained with each marker. [Figure 7] Figure 7. Time course of downregulation of iTAG2- and DCD23-tagged chimeric antigen receptor (TE9-28-Z) following addition of IMiD drugs to transfected Jurkat cells. CAR expression was determined by direct staining of the TE9 ScFv component and assessed by flow cytometry. [Figure 8]Figure 8: Representative flow cytometry of human primary T cells directly stained for CD34 marker gene or TE9-28-Z CAR expression after 24 hours of culture in the presence or absence of 10 μM iberdomide. Rectangles indicate CAR-positive populations that are effectively eliminated by drug addition. Flow plots are representative of two independent donors. [Figure 9] Figure 9: CAR-transduced human primary T cells from two independent donors were cultured with SupT1 cells transduced to express human B7H3 at a 1:1 effector-to-target ratio. Error bars represent the mean and standard deviation of duplicate values ​​from two independent donors. UT = untransduced control; DCD2 tag and iTAG2 tag represent the TE9-28-Z CAR with their respective C-terminal tags. [Figure 10] Figure 10: Comparison of expression of iTAG2-tagged CAR with the non-mutated version after transfection into Jurkat cells. [Figure 11] Figure 11: Schematic of the Aiolos peptide-based TR-FRET assay used to measure the relative affinities of iTAG1 and iTAG2 for complexes formed between the CRBN / DDB1 complex and various IMiDs. [Figure 12] Figure 12: TR-FRET assay results of iTAG1 and iTAG2 in the presence of lenalidomide, pomalidomide, and iverdomide, including IC50 values ​​calculated from the TR-FRET curves. [Figure 13] Figure 13: HMEC cells transiently transfected with GFP-superdegron fusions showed strong nuclear GFP localization by fluorescence microscopy. Images of GFP-iTAG1 and GFP-iTAG2 are shown for comparison and adapted from Figure 3b. [Figure 14]Figure 14: A. Size-exclusion chromatography (SEC) profiles obtained in the presence or absence of iberdomide for a sample consisting of a mixture of the CRBN / DDB1 complex and iTAG2. B. SDS-PAGE analysis of representative fractions from SEC analysis in the absence of iberdomide. iTAG2 was not present in peak 1, corresponding to the high-molecular-weight species, but was present only in peak 2. C. SDS-PAGE analysis of representative fractions from SEC analysis in the presence of iberdomide. iTAG2 was present in peak 1, corresponding to the high-molecular-weight species, confirming that CRBN / DDB1 and iTAG2 formed a complex in the presence of iberdomide. [Figure 15] Figure 15: iTAG2 CAR-T cells are re-upregulated after IMiD washout. (a) Gamma-retrovirally transduced TE9-only or TE9-iTAG2 CAR-T cells were treated overnight with various concentrations of iberdomide and analyzed for CAR expression by flow cytometry. (b) CAR-T cells were then washed with PBS to remove the drug and incubated in RPMI 1640 medium for an additional 24 hours. CAR expression was measured before and after incubation. 0.01 μM iberdomide (indicated by the black arrow) was identified as the optimal IMiD concentration mediating CAR downregulation with treatment and CAR re-upregulation with drug withdrawal. Representative data from CAR-T cells from one donor. [Figure 16]Figure 16: iTAG2 CAR-T functions in a lentiviral format. (a) The TE9-iTAG2 anti-B7H3 CAR-T construct was originally expressed using a gamma-retroviral SFG construct (constructs 1 and 2). The TE9-iTAG2 transgene was then introduced into a pCCL lentiviral backbone (construct 3), and finally codon-optimized and streamlined by removing previously used molecular cloning restriction sites to generate construct 4. The complete expression cassettes for constructs 3 and 4 are shown in panel (b). (c) Constructs 3 and 4 were compared for their sensitivity to overnight 0.01 μM iberdomide treatment (data shown for technical replicates of three different CAR-T donors). Both constructs 3 and 4 (C3 and C4) downregulated the CAR in response to IMiDs; however, interestingly, construct 3 expressed RQR8 at lower levels even in the absence of drug treatment. Therefore, Construct 4 was adopted as the lead lentiviral vector for further functional evaluation. (d) Construct 4 TE9-iTAG2 CAR-T cells were co-cultured overnight with SupT1-B7H3 T lymphoma target cells at an E:T ratio of 1:2 (data from six different CAR-T donors are shown). Complete target killing by CAR-T cells was observed, so the E:T ratio was increased to 1:10, and approximately 80% target killing was observed overnight. This demonstrated that TE9-iTAG2 CAR-T cells have high cytotoxicity and potent continuous target killing capacity. (e, f) To confirm the lack of antigen-nonspecific TE9-iTAG2 CAR-T reactivity, cytokine production was measured by ELISA after overnight culture with SupT1-B7H3 targets at an E:T ratio of 1:10 or without targets. Cytokines were produced only in the presence of tumor targets (data from three different CAR-T donors are shown). (g) Lack of codon-optimized TE9-iTAG2 cytotoxicity against the B7H3-negative SupT1 target was confirmed using an overnight luminescence-based assay at an E:T ratio of 1:2 (data shown for three independent CAR-T donors). DETAILED DESCRIPTION OF THE INVENTION

[0052] The patent, scientific, and technical literature referred to in this specification demonstrates the knowledge available to those skilled in the art at the time of filing. The entire disclosures of issued patents, published and pending patent applications, and other publications cited in this specification are incorporated herein by reference to the same extent as if each were specifically and individually indicated to be incorporated by reference. In the event of a conflict, the disclosures in this specification will control.

[0053] Various aspects of the invention are described in further detail below.

[0054] Detailed Description The present invention provides novel drug-inducible degradation tags (degron tags), as well as fusion proteins, cells, and compositions comprising them. Methods for using the novel degrons, fusion proteins, cells, and compositions are also provided herein, including methods for target-specifically regulating protein abundance via degron tags. The present invention can target endogenous and exogenous (e.g., therapeutic) proteins alike. As described 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.

[0055] Degron tags are also referred to in the art as zinc finger degradation domains. The terms "degron tag" and "zinc finger degradation domain" are used interchangeably herein. The degron tags provided herein bind to complexes formed between CRBN and IMiD; or CRBN and cereblon modulator (CELMoD). In other words, the degron tags provided herein bind to CRBN-IMiD or CRBN-CELMoD complexes. Methods for determining the binding of degron tags to CRBN-IMiD or CRBN-CELMoD complexes are well known in the art; see, for example, Degorce et al., 2009 (PMID 20161833) for an in vitro HTRF assay and Sievers et al., 2018 (PMID 30385546) for the use of TR-FRET on ZF degrons.

[0056] The degron tag provided herein is a minimal degron sequence (LQCEICGFTC) provided in WO2021 / 188286. R Q K GNLL R HI K LH (SEQ ID NO: 1) (where the underlined amino acids indicate the amino acids that are mutated in the present invention). This minimal degron sequence corresponds to a complete C2H2-type zinc finger protein domain, including an N-terminal β-hairpin subdomain containing two Cys and a C-terminal α-helical subdomain containing two His, and these four residues form a single Zn 2+ It coordinates ions and stabilizes the folding of typical C2H2-type zinc finger proteins. Thus, the degron tags provided herein comprise amino acid sequences that are variants of minimal degron sequences shown to be functional in the prior art.

[0057] The inventors have shown herein that, although the novel degron tags provided herein comprise amino acid sequences that are variants of minimal degron sequences previously shown to be functional, the variants provided herein retain functionality (in terms of their ability to induce degradation of fusion proteins containing the degron tag) while also providing several surprising additional advantageous properties (such as increased membrane stability and / or increased cell surface expression of fused cell surface proteins such as CAR).

[0058] The present inventors have exemplified the present invention using a novel degron tag that contains four amino acid mutations of a previously identified minimal functional degron sequence. Thus, a degron tag is provided herein that contains the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 66), where X1, X2, X3, and X4 are not R, K, or H.

[0059] In one example, it comprises the amino acid sequence of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 2), wherein: X1 is E or a conservative amino acid substitution thereof; X2 is A or a conservative amino acid substitution thereof; X3 is N or a conservative amino acid substitution thereof; and X4 is E or a conservative amino acid substitution thereof Degron tags are provided herein.

[0060] The present inventors have shown that it is possible to disrupt nuclear localization (also known as reducing accumulation in the cell nucleus) of degron-tagged fusion proteins by mutating amino acids X1, X2, X3, and X4 from basic amino acids to acidic, neutral, or aliphatic amino acids. Although the present inventors have exemplified the invention using a degron in which X1 is E, X2 is A, X3 is N, and X4 is E, the invention is equally applicable to other degron mutants in which the basic amino acids at positions X1, X2, X3, and X4 are replaced with appropriate alternative non-basic amino acids. Thus, a novel degron tag that inhibits nuclear localization of a degron-tagged fusion protein is provided herein and comprises LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 66), wherein X1 is either E or a non-basic amino acid substitution of E, X2 is either A or a non-basic amino acid substitution of A, X3 is either N or a non-basic amino acid substitution of N, and X4 is either E or a non-basic amino acid substitution of E.

[0061] In some instances, non-basic amino acid substitutions may be conservative amino acid substitutions of the mutant amino acids exemplified herein. As is well known to those skilled in the art, a "conservative amino acid substitution" refers to an amino acid substitution that changes a given amino acid in a protein to a different amino acid with similar biochemical properties, such as charge, hydrophobicity, or size. Conservative amino acid substitutions result in a silent change, resulting in a functionally equivalent degron (in terms of its ability to bind to the CRBN-IMiD complex or CRBN-CELMOD complex and induce degradation of a fusion protein containing a degron tag). Deliberate amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, as long as the intrinsic function is preserved. 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 with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, tyrosine, etc.

[0062] Conservative substitutions are possible, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: [Table 1]

[0063] Thus, a degron tag of the present invention has the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 3), wherein: X1 is E or D; X2 is A, G, P, I, L or V; X3 is N, Q, C, S, T, or M; and X4 is E or D It may comprise an amino acid sequence.

[0064] In one example, a degron tag of the invention has the amino acid sequence of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 4), wherein: X1 is E or D; X2 is A, G or P; X3 is N or Q; and X4 is E or D It may comprise an amino acid sequence.

[0065] In one example, X1 is E. In one example, X2 is A. In one example, X3 is N. In one example, X4 is E.

[0066] In one example, the degron tag contains 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 regions of the mutant ZFP91 ZF2 β hairpin subdomain and IKAROS ZF2 α helix subdomain sequences of these degron tags). It is also a mutant sequence equivalent to a superdegron (i.e., the core regions of the mutant ZFP91 ZF2 β hairpin subdomain and AIOLOS ZF2 α helix subdomain sequences of the superdegron). Thus, a degron containing the amino acid sequence of SEQ ID NO: 5 may be based on the iTAG1 or superdegron amino acid sequence with at least four mutations (X1, X2, X3, and X4, as described above) introduced.

[0067] Preferably, 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 nuclear localization of the fusion protein comprising the degron tag (e.g., a degron-tagged CAR). SEQ ID NO: 66 or any one of SEQ ID NOs: 2 to 5 is the amino acid sequence LQCEICGFTC R Q K GNLL R HI K LH (SEQ ID NO: 1). As used herein, "reduce" can mean a decrease or reduction of at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, or more. As will be appreciated by those skilled in the art, nuclear localization means being present (located) in the nucleus of a cell. Several methods for determining nuclear localization are known in the art, such as immunofluorescence, which is used in the Examples section below.

[0068] The degron tags provided herein can contain additional amino acids (e.g., at the N-terminus and / or C-terminus of SEQ ID NO: 66, or the amino acid sequence set forth in any one of SEQ ID NOs: 2-5), provided that the degron tag retains the ability to bind to a CRBN-IMiD complex or a CRBN-CELMoD complex and induce degradation of the fusion protein containing the degron tag. These additional amino acids can correspond to or differ from residues in a naturally occurring zinc finger domain, provided that the degron tag maintains a zinc finger-like fold and exhibits the required binding properties as disclosed herein.

[0069] For example, a degron tag provided herein is the sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO: 67), or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO: 68), wherein: X1, X2, X3 and X4 are not R, K or H It may contain sequences.

[0070] For example, a degron tag provided herein is the sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO: 6), or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO: 7), wherein: X1 is E or a conservative amino acid substitution thereof; X2 is A or a conservative amino acid substitution thereof; X3 is N or a conservative amino acid substitution thereof; and X4 is E or a conservative amino acid substitution thereof It may contain sequences.

[0071] In other words, the degron tag is the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO: 8), or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO: 9), wherein: X1 is E or D; X2 is A, G, P, I, L or V; X3 is N, Q, C, S, T or M; and X4 is E or D It may contain sequences.

[0072] In one example, a degron tag of the invention has the amino acid sequence LQCEICGFTCX1QX2GNLLX3HIX4LHSG (SEQ ID NO: 10), or LQCEICGFTCX1QX2GNLLX3HIX4LHTG (SEQ ID NO: 11), wherein: X1 is E or D; X2 is A, G or P; X3 is N or Q; and X4 is E or D It may contain sequences.

[0073] In one example, X1 is E. In one example, X2 is A. In one example, X3 is N. In one example, X4 is E.

[0074] In one example, the degron tag comprises the amino acid sequence LQCEICGFTCEQAGNLLNHIELHSG (SEQ ID NO: 12) or LQCEICGFTCEQAGNLLNHIELHTG (SEQ ID NO: 13). Herein, SEQ ID NO: 12 is the sequence present in iTAG2, iTAGv1, and iTAGv2 described herein (corresponding to the mutant ZFP91 ZF2 β hairpin subdomain and IKAROS ZF2 α helix subdomain sequences of these degron tags). Similarly, SEQ ID NO: 13 is the equivalent mutant sequence of a superdegron (i.e., mutants of the ZFP91 ZF2 β hairpin subdomain and AIOLOS ZF2 α helix subdomain sequences of the superdegron).

[0075] The degron tags provided herein can comprise additional amino acids (e.g., an additional N-terminal zinc finger α-helix subdomain and / or an additional C-terminal zinc finger β-hairpin subdomain) adjacent to the amino acid sequence of any one of SEQ ID NOs: 2-13, or 66-68. For example, a degron tag of the present invention can comprise an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain adjacent to the amino acid sequence of any one of SEQ ID NOs: 2-13, or 66-68. Various naturally occurring proteins contain zinc finger regions (also known as zinc finger motifs) that include a beta hairpin loop (referred to as a zinc finger β-hairpin subdomain in the context of the degron tags described herein) and an alpha helix region (referred to as a zinc finger α-helix subdomain in the context of the degron tags described herein). In some embodiments, a degron tag can include a first sequence derived from or at least a portion of a first zinc finger region, and a second sequence derived from or a portion of the α-helical region of a second zinc finger region. The first and second zinc finger regions can 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 N-terminal zinc finger α-helical subdomains of ZFP91, ZN276, ZN517, ZN653, ZN654, ZN787, or ZN827 can 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 Examples below.For example, ZFP91, ZN276, ZN517, ZN653, ZN654, ZN787 or ZN827 C-terminal zinc finger beta hairpin subdomains can be used.

[0076] As used herein, the term "adjacent" refers to the relative position of features within the same amino acid sequence. Accordingly, the term refers herein 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: 2-13, or 66-68. It is noted that the adjacent sequences need not be directly adjacent to the core sequence of SEQ ID NOS: 2-13, or 66-68, and may have intervening amino acids.

[0077] In one example, a degron 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: 5. In other words, a degron may comprise, from N- to C-terminal, the following structure: zinc finger α-helix subdomain-SEQ ID NO: 5-zinc finger β-hairpin subdomain (with intervening amino acids between the subdomain and the amino acid sequence of SEQ ID NO: 5, if necessary). In this example, the additional C-terminal zinc finger β-hairpin subdomain may 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. For 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 a conservative amino acid substitution thereof; and X6 is E or a conservative amino acid substitution thereof. 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 alpha-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 alpha-helix subdomain can comprise the amino acid sequence PNVLMVHNESH (SEQ ID NO: 22) or FNVLMVHNESH (SEQ ID NO: 23).For example, when a degron comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain adjacent to the amino acid sequence of SEQ ID NO:5, the additional N-terminal zinc finger α-helix subdomain can comprise the amino acid sequence of 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 of SEQ ID NO:14 or SEQ ID NO:15.

[0078] Thus, in one example, a degron 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: 5, where the additional N-terminal zinc finger α-helix subdomain comprises the amino acid sequence of SEQ ID NO: 22 and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence of SEQ ID NO: 14. This arrangement corresponds to the sequences present in iTAG2.

[0079] In another example, the degron tag comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain adjacent to the amino acid sequence of SEQ ID NO:5, where the additional N-terminal zinc finger α-helix subdomain comprises the amino acid sequence of SEQ ID NO:23 and the additional C-terminal zinc finger β-hairpin subdomain comprises the amino acid sequence of SEQ ID NO:15.

[0080] In one example, a degron 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: 12. In other words, a degron can comprise the following structure from N- to C-terminus (left to right): zinc finger α-helix subdomain-SEQ ID NO: 12-zinc finger β-hairpin subdomain (with intervening amino acids between the subdomain and the amino acid sequence of SEQ ID NO: 12, if necessary). 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. For 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 a conservative amino acid substitution thereof; and X6 is E or a conservative amino acid substitution thereof. 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 a degron comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain adjacent to the amino acid sequence of SEQ ID NO:12, the additional N-terminal zinc finger α-helix subdomain can comprise the amino acid sequence of 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 of SEQ ID NO:14 or SEQ ID NO:15.

[0081] Thus, in one example, a degron 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: 12, where 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). This arrangement corresponds to the sequences present in iTAG2.

[0082] In another example, the degron tag comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain adjacent to the amino acid sequence of SEQ ID NO: 12, where 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).

[0083] In one example, a degron 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. In other words, a degron can comprise the following structure from N- to C-terminus (left to right): zinc finger α-helix subdomain-SEQ ID NO: 13-zinc finger β-hairpin subdomain (with intervening amino acids between the subdomain and the amino acid sequence of SEQ ID NO: 13, if necessary). 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 a conservative amino acid substitution thereof; and X6 is E or a conservative amino acid substitution thereof. 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 a degron comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain adjacent to the amino acid sequence of SEQ ID NO:13, the additional N-terminal zinc finger α-helix subdomain can comprise the amino acid sequence of 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 of SEQ ID NO:14 or SEQ ID NO:15.

[0084] Thus, in one example, a degron 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, where 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).

[0085] In another example, the degron tag comprises an additional N-terminal zinc finger α-helix subdomain and an additional C-terminal zinc finger β-hairpin subdomain adjacent to the amino acid sequence of SEQ ID NO: 13, where 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).

[0086] In one example, the degron tag has the amino acid sequence: PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (sequence number 73), wherein X1, X2, X3, X4, X5, X6, X7 and X8 are not R, K or H.

[0087] In one example, the degron tag has the amino acid sequence: PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO: 24), wherein X1 is E or a conservative amino acid substitution thereof; X2 is A or a conservative amino acid substitution thereof; X3 is N or a conservative amino acid substitution thereof; X4 is E or a conservative amino acid substitution thereof; X5 is N or a conservative amino acid substitution thereof; X6 is E or a conservative amino acid substitution thereof; X7 is I or a conservative amino acid substitution thereof; and X8 is I or a conservative amino acid substitution thereof.

[0088] In other words, the degron tag can comprise the amino acid sequence PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO: 25), wherein: X1 is E or D; X2 is A, G, P, I, L or V; X3 is N, Q, C, S, T or M; X4 is E or D; X5 is N, Q, C, S, T or M; X6 is E or D; X7 is I, L, V, G, A, or P; and X8 is I, L, V, G, A or P.

[0089] In one example, the degron tag comprises the amino acid sequence PNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHSGEX8PFKCHLCNYACRRRDAL (SEQ ID NO: 26), wherein: X1 is E or D; X2 is A, G or P; X3 is N or Q; X4 is E or D; X5 is N or Q; X6 is E or D; X7 is I, L, or V; and X8 is I, L or V.

[0090] In one example, X1 is E. In one example, X2 is A. In one example, X3 is N. In one example, X4 is E. In one example, X5 is N. In one example, X6 is E. In one example, X7 is I. In one example, X8 is I.

[0091] In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 27 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 29 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 31 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 33 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 35 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 37 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 39 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 41 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 43 (see Table 2 below).

[0092] In one example, the degron tag has the amino acid sequence: FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (sequence number 74), wherein X1, X2, X3, X4, X5, X6, X7 and X8 are not R, K or H.

[0093] In one example, the degron tag has the amino acid sequence: FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO: 45), wherein X1 is E or a conservative amino acid substitution thereof; X2 is A or a conservative amino acid substitution thereof; X3 is N or a conservative amino acid substitution thereof; X4 is E or a conservative amino acid substitution thereof; X5 is N or a conservative amino acid substitution thereof; X6 is E or a conservative amino acid substitution thereof; X7 is I or a conservative amino acid substitution thereof; and X8 is I or a conservative amino acid substitution thereof.

[0094] In other words, the degron tag may comprise the amino acid sequence FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO: 46), wherein: X1 is E or D; X2 is A, G, P, I, L or V; X3 is N, Q, C, S, T or M; X4 is E or D; X5 is N, Q, C, S, T or M; X6 is E or D; X7 is I, L, V, G, A, or P; and X8 is I, L, V, G, A or P.

[0095] In one example, the degron tag comprises the amino acid sequence FNVLMVHX5X6SHTGEX7PLQCEICGFTCX1QX2GNLLX3HIX4LHTGEX8PFKCHLCNYACQRRDAL (SEQ ID NO: 47), wherein: X1 is E or D; X2 is A, G or P; X3 is N or Q; X4 is E or D; X5 is N or Q; X6 is E or D; X7 is I, L, or V; and X8 is I, L or V.

[0096] In one example, X1 is E. In one example, X2 is A. In one example, X3 is N. In one example, X4 is E. In one example, X5 is N. In one example, X6 is E. In one example, X7 is I. In one example, X8 is I.

[0097] In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 28 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 30 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 32 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 34 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 36 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 38 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 40 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 42 (see Table 2 below). In one example, the degron tag comprises the amino acid sequence of SEQ ID NO: 44 (see Table 2 below).

[0098] Specific amino acid sequences that may be included within the degron tags of the present invention are shown in the table below, with the specific mutations identified therein being introduced into the corresponding iTAG1 and superdegron sequences described herein. As can be seen from the table, four specific mutations are contemplated within the minimal degron sequence, with additional mutations (one, two, three, or four) contemplated in the flanking sequences, if desired. In each case, the mutations are underlined and the minimal degron sequence is italicized and bolded for ease of identification.

[0099] [Table 2-1] [Table 2-2] [Table 2-3]

[0100] The degron tags provided herein generally have the structure: LQCEICGFTC R Q K GNLL R HI K The degron tags are described using a core amino acid sequence of any one of SEQ ID NOS: 2-13 or 66-68, which contains four amino acid substitutions compared to the known minimal functional degron sequence of LH (where the underlines indicate the amino acids that are mutated in the present invention). While the degron tags provided herein are generally described with all four amino acid substitutions, it will be apparent to one of skill in the art that degron tags with one or more, two or more, or three or more of these mutations are also contemplated herein.

[0101] Thus, provided herein is a degron tag comprising the amino acid sequence of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 75), in which one, two, or three of the following amino acid substitutions are substituted: At position X1, R is substituted with any amino acid other than R, K or H; and / or At position X2, K is substituted with any amino acid other than R, K or H; and / or At position X3, R is substituted with any amino acid other than R, K or H; and / or At position X4, K is substituted with an amino acid other than R, K or H.

[0102] In one example, provided herein is a degron tag comprising the amino acid sequence of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 48) in which one, two, or three of the following amino acid substitutions are substituted: At position X1, R is replaced with E or a conservative amino acid substitution thereof; and / or At position X2, K is replaced with A or a conservative amino acid substitution thereof; and / or At position X3, R is substituted with N or a conservative amino acid substitution thereof; and / or At position X4, K is replaced with E or a conservative amino acid substitution thereof.

[0103] As will be apparent to one of skill in the art, at positions where no amino acid is substituted, 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.).

[0104] Thus, a degron tag provided herein can comprise the amino acid sequence of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 49), in which one, two, or three of the following amino acid substitutions are substituted: At position X1, R is substituted with E or D; and / or At position X2, K is substituted with A, G, P, I, L or V; and / or At position X3, R is substituted with N, Q, C, S, T or M; and / or In position X4, K is substituted with E or D.

[0105] In one example, a degron tag provided herein may comprise the amino acid sequence of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 50), in which one, two, or three of the following amino acid substitutions are substituted: At position X1, R is substituted with E or D; and / or At position X2, K is replaced with A, G or P; and / or At position X3, R is substituted with N or Q; and / or In position X4, K is substituted with E or D.

[0106] Thus, in one example, a degron tag provided herein can comprise the amino acid sequence of LQCEICGFTCX1QX2GNLLX3HIX4LH (SEQ ID NO: 51), in which one, two, or three of the following amino acid substitutions are substituted: At position X1, R is substituted with E; and / or In position X2, K is substituted with A; and / or At position X3, R is substituted with N; and / or In position X4, K is substituted with E.

[0107] Thus, in one example, the degron tag provided herein is LQCEICGFTC E QKGNLLRHIKLH (SEQ ID NO: 52), LQCEICGFTCRQ A GNLLRHIKLH (SEQ ID NO: 53), LQCEICGFTCRQKGNLL N HIKLH (SEQ ID NO: 54) or LQCEICGFTCRQKGNLLRHIE LH (SEQ ID NO: 55), all of which have one of the substitutions described above. Examples of degron tags with two or three substitutions are also contemplated herein and may be readily identifiable by one of skill in the art. 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 mutated amino acids relative to SEQ ID NO: 1 are underlined.

[0108] Degron tags of the present invention are generally peptides having about 23 to about 70 amino acids, typically about 23 to about 60 amino acids. In some examples, degron tags of the present invention are generally peptides having about 23 to about 30 amino acids. The terms "peptide," "polypeptide," and "protein" are used herein consistent with their art-recognized meanings.

[0109] Fusion proteins comprising a protein of interest (POI) and at least one degron tag of the present invention are also provided herein. When linked to a therapeutic protein of interest (POI), the degron tag can be used as a "safety switch" to target the degradation of the POI in situations where POI expression is undesirable. The ability to degrade specific endogenous proteins of interest by creating a POI-degron tag fusion and administering an IMiD or CELMoD can be used to treat disorders in which protein expression above a certain threshold level in cells leads to a disease state. Other applications of this technology include, but are not limited to, 1) targeted degradation of proteins whose pathology is a function of gain-of-function mutations; 2) targeted degradation of proteins whose pathology is a function of amplification or increased expression; 3) targeted degradation of proteins that are symptoms of monogenic diseases; and 4) targeted degradation of proteins in which genetic predispositions manifest over a longer period of time, often after alternative biological compensatory mechanisms are no longer appropriate, such as hypercholesterolemia and proteopathies. Furthermore, POI-degron tag fusions can be used to evaluate the function of endogenous proteins or to validate endogenous proteins as therapeutic targets for disease states. Thus, the degron tags of the present invention can be utilized to produce stably expressed endogenous or exogenous protein-degron tag fusion proteins. An endogenous protein originates within an organism, tissue, or cell and is expressed by the same organism, tissue, or cell, whereas an exogenous protein originates from outside the organism, tissue, or cell and is introduced into that organism, tissue, or cell.

[0110] 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 the group consisting of a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor (TCR) fusion construct (TRuC), a T cell antigen coupler (TAC), a chimeric autoantibody receptor (CAAR), or an antibody-bound T cell receptor (ACTR).

[0111] The degrons of the present invention are particularly useful when the POI is a chimeric antigen receptor (CAR) protein. Accordingly, CAR-degron tag fusion proteins are specifically provided herein. Genetically modified T cells expressing chimeric antigen receptors (CAR-T therapy) have been shown to be effective in treating many cancers, including lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, and neuroblastoma. Two autologous CAR-T cell therapies (Kymriah et al., 2014) have been shown to be effective in treating cancers including lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, and neuroblastoma. (商標) and Yescarta (商標) ) have been approved by the FDA. What they have in common is that 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 serious side effects. While most CAR-T-related adverse events are tolerable, there have been a number of cases in which administration of CAR-T cells has induced severe systemic inflammatory reactions, such as cytokine release syndrome (CRS) and tumor lysis syndrome. The dramatic clinical activity of CAR-T cell therapy highlights the need for safety strategies to rapidly reverse or halt T-cell responses in patients with CRS or related adverse events.

[0112] Thus, the present invention includes a fusion protein comprising a CAR and at least one degron tag. The CAR further comprises an extracellular ligand-binding domain capable of binding to an antigen, a transmembrane domain, and an intracellular domain, in that order from the N-terminus, with the intracellular domain comprising at least one signaling domain. The degron tag can be positioned at the N-terminus or between the extracellular binding domain and the transmembrane domain, as long as it does not inhibit antigen binding or membrane insertion. Similarly, the degron tag can be positioned at the C-terminus, between the transmembrane domain and the intracellular domain, or between multiple signaling domains if multiple domains are present. The degron tag is preferably located at the C-terminus. In other words, the degron is preferably located at the 3'-end of the fusion protein, fused to the 3'-end of the intracellular signaling domain C-terminal to the cytoplasmic domain of the CAR.

[0113] In one aspect, the fusion protein is a fusion protein comprising tisagenlecleucel (Kymriah), as described herein. (商標) ) and a degron tag, CAR. Tisagenlecleucel is a genetically engineered antigen-specific autologous T cell targeting CD19. The extracellular domain of CAR is a mouse anti-CD19 single-chain antibody fragment (scFv) derived from the mouse monoclonal FMC63 hybridoma. The intracellular domain of CAR is a T cell signaling domain derived from human CD3z and a costimulatory domain derived from human 4-1BB (CD137). The transmembrane domain and spacer between the scFv domain and the transmembrane domain are derived from human CD8a. Kymriah (商標) (tisagenlecleucel) is approved for the treatment of patients up to 25 years of age with refractory or relapsed (R / R) B-cell precursor acute lymphoblastic leukemia (ALL), and adults with R / R diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin's lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma. The degron tag can be any of the degron tags disclosed herein.

[0114] In one embodiment, the fusion protein is axicabtageneciloleucel (Yescarta(商標) It contains a CAR (carrier antigen) and a degron tag. Axicabtageneciloleucel is a genetically engineered antigen-specific autologous T cell that targets CD19. The extracellular domain of the CAR is a murine anti-CD19 single-chain antibody fragment (scFv). The intracellular domain of the CAR contains two signaling domains, one derived from human CD3z and the other from human CD28. Yescarta (商標) (Axicabtagene Siloleucel) is approved for the treatment of adults 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.

[0115] 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 diseases such as pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, and salvage therapy after allogeneic bone marrow transplantation.

[0116] Further features of CAR proteins, nucleic acids encoding CAR proteins, immune effector cells expressing CARs, and methods of using CAR-expressing cells for the treatment of disease are disclosed in U.S. Patent Application Publication No. 2018 / 0169109 A1, which is incorporated herein by reference.

[0117] As described elsewhere herein, the fusion proteins provided herein can include a degron of the present invention fused to a protein of interest. The protein of interest can be a T cell receptor (TCR). For example, the TCR can be a chimeric T cell receptor, an artificial T cell receptor, or a synthetic T cell receptor. Furthermore, 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).

[0118] In one example, the POI is a T cell receptor fusion construct (TRuC). A T cell receptor fusion construct (TRuC) is a receptor protein containing an antibody-based binding domain fused to a T cell receptor (TCR) subunit designed to effectively recognize target cell surface antigens. TRuC contains a specific ligand antibody fused to the extracellular N-terminus of multiple TCR subunits (e.g., TCRα, TCRβ, CD3ε, CD3γ, and CD3δ). TRuC provides target specificity and HLA-independent target cell elimination. TRuC can be incorporated into the native TCR complex on the surface of cells such as T cells. Unlike CARs, TRuC is a functional component of the TCR complex. TRuC-T cells have demonstrated potent antitumor activity in liquid and solid tumor xenograft models.

[0119] In another example, the POI is an antibody-binding T cell receptor (ACTR). ACTR is a non-naturally occurring molecule that can be expressed on the surface of a host cell and includes an extracellular domain (e.g., a CD16A extracellular domain) capable of binding to a target molecule containing an Fc portion and one or more cytoplasmic signaling domains for inducing effector functions of immune cells expressing the ACTR polypeptide, and at least two domains of the ACTR polypeptide may be derived from different molecules. The ACTR polypeptide may include a CD16A extracellular domain capable of binding to a target molecule containing an Fc portion, a transmembrane domain, one or more costimulatory signaling domains, and a CD3ζ cytoplasmic signaling domain. At least one of the costimulatory signaling domains may be a CD28 costimulatory domain. The ACTR polypeptide may not include a hinge domain of a non-CD16A receptor, or may include two or more costimulatory signaling domains if the transmembrane domain is a CD8 transmembrane domain.

[0120] In another example, the POI is a T cell antigen coupler (TAC). T cell antigen couplers (TACs) are a platform that utilizes endogenous TCRs in an MHC-independent manner to induce more efficient target cell responses and reduce toxicity. TAC chimeric proteins bind to TCRs that recognize antigens via CD3 domain binding, forming a TCR / CD3 complex.

[0121] Alternatively, the antigen-binding cell surface protein may be a chimeric autoantibody receptor (CAAR). CAARs are an improved version of CARs that identify antibody-secreting cells, such as autoreactive B cells. CAARs contain a specific antigen, a transmembrane domain, and an intracellular signaling domain (with or without a costimulatory domain). CAARs recognize and bind to target autoantibodies expressed on autoreactive cells via the specific antigen, and then destroy the autoantibodies.

[0122] Nucleic acid sequences and molecules encoding the degrons or fusion proteins described herein are also provided. The nucleic acid sequences and molecules may be non-naturally occurring nucleic acid sequences encoding the degron tags or fusion proteins described herein.

[0123] Also provided are vectors containing nucleic acid sequences encoding the novel degrons or fusion proteins described herein. A "vector" refers to a composition of matter that contains a nucleic acid and can be used to deliver the nucleic acid into a cell. Numerous vectors are known in the art, including linear polynucleotides, polynucleotides conjugated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This 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, vectors may be non-viral vectors (e.g., plasmids, polylysine compounds, liposomes or lipid nanoparticles) or viral vectors. Representative examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors, herpes simplex virus vectors, vaccinia virus vectors, picornavirus vectors, and alphavirus vectors. An example of a retroviral vector is a lentiviral vector.

[0124] Vectors can be delivered in vivo by administration to an individual subject, typically systemically (e.g., intravenous, intraperitoneal, intramuscular, intrathecal, intratracheal, subcutaneous, or intracranial injection) or by local application. Alternatively, vectors can be introduced into ex vivo cells, such as cells removed from an individual patient (e.g., lymphocytes, bone marrow aspirate, or tissue biopsy) or hematopoietic stem cells from a universal donor, which can then be reimplanted into the patient, typically after selection of cells that have taken up the vector.

[0125] In certain embodiments, the nucleic acid encoding the degron tag can be inserted into the genome in frame with the gene encoding the protein involved in the disorder.Representative examples of specific genes involved in disorders that can be targeted for degron tag insertion 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, and antithrombin III (SERPI). NC1), phosphatidylinositol glycan class A (PIG-A), C5, alpha-1 antitrypsin (SERPINA1), hepcidin regulation (TMPRSS6), delta-aminolevulinic acid synthase 1 (ALAS-1), acyl-CaA: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) R), protein tyrosine phosphatase (PTP-1B), c-Raf kinase (RAF1), fibroblast growth factor receptor 4 (FGFR4), vascular adhesion molecule-1 (VCAM-1), very late antigen-4 (VLA-4), transthyretin (TTR), survival of motor neuron 2 (SMN2), growth hormone receptor (GHR), myotonic dystrophy 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 kinase C (HSCT) These include heat shock protein 27 (HSP27), 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), retinopathy 1 (RS1), retinal pigment epithelium-specific 65 kDa protein (RPE65), Rab escort protein 1 (CHM), sodium channel, voltage-gated, type X, alpha subunit (PN3 or SCN10A).Additional proteins of interest that can be targeted by insertion of a degron tag include proteins associated with gain-of-function mutations, such as proteins that cause cancer.

[0126] In particular 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), the 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 isoform NaV1.8.

[0127] In one embodiment, the degron tag is genomically inserted in-frame, either 5' or 3', to a gene encoding an endogenous protein associated with a proteopathy. In one embodiment, the degron tag is a tag that identifies a protein that is specifically targeted to Alzheimer's disease (amyloid peptide (Ab); tau protein), cerebral amyloid angiopathy (amyloid b peptide (Ab)), retinal ganglion cell degeneration in glaucoma (amyloid b peptide (Ab)), prion disease (prion protein), Parkinson's disease and other synucleinopathies (α-synuclein), tauopathies (microtubule-associated protein tau (tau protein)), frontotemporal lobar degeneration (FTLD) (Ubi+, tau) (TDP-43), FTLD-FUS (fused sarcoma (FUS) protein), amyotrophic lateral sclerosis (ALS) (superoxide dismutase, TDP-43, FUS), Huntington's disease and other triplet repeat disorders (tandem glutamine expansion). Protein), Familial British Dementia (ABri), Familial Danish Dementia (Adan), Hereditary Cerebral Hemorrhage with Amyloidosis (Iceland) (HCHWA-I) (Cystatin C), CADASIL (Notch3), Alexander Disease (Glial Fibrillary Acidic Protein (GFAP)), Seipinopathy (Seipin), Familial Amyloidotic Neuropathy, Senile Systemic Amyloidosis (Transthyretin), Serpinopathy (Serpin), AL (Light Chain) Amyloidosis (Primary Systemic Amyloidosis) (Monoclonal Immunoglobulin Light Chain), AH (Heavy Chain) Amyloidosis (Immunoglobulin Heavy Chain), AA (Secondary) Amyloidosis (Amyloid A Protein), Type II Diabetes (Islet Amyloid Polypeptide (IAPP;Amylin), medial aortic amyloidosis (medin (lactadherin)), ApoAI amyloidosis (apolipoprotein AI), ApoAII amyloidosis (apolipoprotein AII), ApoAIV amyloidosis (apolipoprotein AIV), Finnish-type familial amyloidosis (FAF) (gelsolin), lysozyme amyloidosis (lysozyme), fibrinogen amyloidosis (fibrinogen), dialysis amyloidosis (beta2 microglobulin), inclusion body myositis / myopathy (amyloid b peptide (Ab)), cataracts (crystallin), retinitis pigmentosa with rhodopsin mutations (rhodopsin), medullary thyroid carcinoma (calcitonin), atrial amyloidosis (atrial natriuretic factor), pituitary prolactin They are inserted in-frame, either 5' or 3', into genes encoding endogenous proteins associated with disorders such as rhabdomyosarcoma (prolactin), hereditary lattice corneal dystrophy (keratoepithelin), cutaneous lichen amyloidosis (keratin), Mallory bodies (keratin intermediate filament protein), corneal lactoferrin amyloidosis (lactoferrin), pulmonary alveolar proteinosis (surfactant protein C (SP-C)), odontogenic (Pindborg) tumor amyloid (odontogenic ameloblast-associated protein), seminal vesicle amyloid (semenogelin I), cystic fibrosis (cystic fibrosis transmembrane conductance regulator (CFTR) protein), sickle cell disease (hemoglobin), and myasthenia gravis (CIM) (highly protected degradation state of myosin ubiquitination);

[0128] In-frame insertion of a nucleic acid sequence encoding a degron tag can be performed or achieved by known effective genome editing processes. In one aspect, the present invention utilizes the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to produce a knock-in endogenous protein-degron tag fusion protein that is produced from the endogenous locus and easily degraded in a reversible and dose-responsive manner depending on the administration of an IMiD or CELMoD. In a specific embodiment, the CRISPR-Cas9 system is employed to insert an expression cassette of a degron tag present in a homologous recombination (HR) "donor" sequence, where the degron tag nucleic acid sequence serves as the "donor" sequence inserted into the genomic locus of the protein of interest during homologous recombination after CRISPR-Cas endonucleation. 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 targeting locus of interest. By fusing a nucleic acid sequence encoding a degron tag in frame with a 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.

[0129] In certain embodiments, a nucleic acid or vector encoding a degron tag or fusion protein described herein can be introduced into a cell, thereby expressing the encoded degron tag or fusion protein by the cell. Thus, cells expressing the nucleic acid sequences or vectors described herein are also provided. Cells expressing the degron or fusion protein of the present invention are also provided herein.

[0130] The cells may be any suitable cells. In certain embodiments, the cells are immune effector cells. For example, the cells may be selected from the group consisting of T cells, B cells, plasma cells, NK cells, NKT cells, innate lymphocytes, 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 may be derived from mammals, such as human cells, or may be derived from non-human mammals, such as monkeys, mice, rats, pigs, horses, and dogs.

[0131] For example, cells collected, isolated, purified, or derived from body fluids, tissues, or organs such as blood (peripheral blood, umbilical 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)), umbilical cord blood mononuclear cells, fibroblasts, preadipocytes, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines, neural stem cells, etc. can be used. In the present invention, it is preferable to use T cells, T cell precursors (hematopoietic stem cells, lymphocyte precursor cells, etc.), or cell populations containing them. Representative examples of T cells include CD8+ T cells, CD4+ T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. Cell populations containing T cells and T cell precursors include PBMCs. The above cells can be collected from a living body, obtained by expanding and culturing cells collected from a living body, or established cell lines. When degron- or fusion protein-expressing cells are transplanted into an organism, it is preferable to introduce the nucleic acid into cells taken from the organism itself or from an organism of the same species. Thus, the immune effector cells can be autologous or allogeneic.

[0132] Immune effector cells expressing a degron or fusion protein of the invention can be engineered by introducing a nucleic acid encoding the degron or fusion protein into a cell. In one aspect, this step is performed ex vivo. For example, cells can be transformed ex vivo with a vector carrying a nucleic acid of the invention to produce cells expressing a degron or fusion protein of the invention.

[0133] Immune effector cells expressing a fusion protein containing a POI and a degron tag can be used as a therapeutic agent for diseases. The therapeutic agent is a cell expressing a POI as an active ingredient and may further contain appropriate excipients. The disease to which the POI-expressing cells are administered is not limited, as long as the disease is susceptible 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, such as infections caused by viruses such as influenza and HIV, bacteria, and fungi, including tuberculosis, MRSA, VRE, and deep-seated fungi. The transformed immune effector cells can bind to antigens presented by target cells whose reduction or elimination is desired for the treatment of the above diseases; i.e., tumor antigens, viral antigens, bacterial antigens, etc., are administered for the treatment of these diseases.

[0134] The immune effector cells can be administered intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, intravenously, intratumorally, or into afferent lymphatic vessels, or parenterally, e.g., by injection or infusion, but the route of administration is not particularly limited. The cells can be, for example, injected directly into a tumor, lymph node, or site of infection.

[0135] The degron tags provided herein bind to the CRBN-IMiD complex or the CRBN-CELMOD complex. When the degrons provided herein are contained within a fusion protein (e.g., a degron-POI fusion protein, a degron-CAR fusion protein, etc.), they can bind to the CRBN-IMiD complex or the CRBN-CELMOD complex and induce degradation of the fusion protein within the cell. Thus, degrons can be advantageously used as a "safety switch" by inducing degradation of the POI in situations where expression of the POI is undesirable.

[0136] Some IMiDs, such as pomolidin, efficiently cross the blood-brain barrier, making iTAG2 modulation particularly suitable for adult and pediatric brain tumors amenable to CAR-T cell treatment, such as high-grade gliomas, medulloblastomas, and meningiomas.

[0137] IMiD (immunomodulatory drug) and CELMoD (cereblon modulatory drug) 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., hydrochloride salts). IMiD compound thalidomide (THALOMID) (登録商標) ), lenalidomide (REVLIMID (登録商標) (sold under the name POMALYST) and pomalidomide (POMALYST) (登録商標) THALOMID and THALOMID-1 are both approved by the FDA for the treatment of multiple myeloma (among other diseases). (登録商標) REVLIMID is currently available as capsules containing 50 mg, 100 mg, 150 mg, or 200 mg of thalidomide. (登録商標) POMALYST is currently available as capsules containing 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg of lenalidomide. (登録商標)is currently available as capsules containing 1 mg, 2 mg, 3 mg, or 4 mg of pomalidomide. CELMoD compounds CC-122, CC-220, and CC-885 are currently under FDA review.

[0138] In certain examples, the degradation of the degron tag or fusion protein provided herein is lenalidomide-dependent. In another example, the degradation of the degron tag or fusion protein provided herein is pomalidomide-dependent. In a further example, the degradation of the degron tag or fusion protein provided herein is iberdomide-dependent. As known to those skilled in the art, iberdomide is also known as CC-220, and these terms are used interchangeably herein. Similarly, the terms "avadomide" and "CC-122" are used interchangeably herein.

[0139] The IMiD and CELMoD compounds may be in the form of a free acid or free base, or a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" in the context of a salt refers to a salt of a compound that does not destroy the biological activity or properties of the compound and is relatively non-toxic; i.e., the compound in salt form can be administered to a subject without causing undesirable biological effects (such as dizziness or upset stomach) or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The term "pharmaceutically acceptable salt" refers to the product obtained by reacting a compound of the present invention with an appropriate acid or base. Examples of pharmaceutically acceptable salts of IMiD and CELMoD compounds include salts derived from appropriate 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 amino salts formed with inorganic bases such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate, or p-toluenesulfonate. Certain compounds of the present invention can form pharmaceutically acceptable salts with various organic bases such as lysine, arginine, guanidine, diethanolamine, or metformin.

[0140] IMiD compounds and CELMoD compounds have at least one chiral center and therefore may be in the form of stereoisomers, which as used herein include all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes enantiomers (enantiomers containing the (R-) or (S-) configuration of a compound), mixtures of enantiomers of a compound (physical mixtures of enantiomers, and racemates or racemic mixtures), geometric isomers of a compound (cis / trans or E / Z, R / S), and isomers of a compound with two or more chiral centers that are not mirror images of each other (diastereoisomers). The chiral centers of a compound may undergo epimerization in vivo. Therefore, for these compounds, administration of the (R-) form of a compound is considered equivalent to administration of the (S-) form of a compound. Thus, the IMiD and CELMoD compounds can be used in the form of individual isomers, substantially free of other isomers, or in the form of mixtures of various isomers, for example racemic mixtures of stereoisomers.

[0141] In some embodiments, the IMiD or CELMoD compound is an isotopic derivative in that it has at least one desired isotopic substitution of an atom at or above the natural abundance of that isotope, i.e., is enriched. In one embodiment, the compound contains deuterium or multiple deuterium atoms. A heavy isotope such as deuterium, i.e., 2 Substitution with H may confer certain therapeutic advantages resulting from greater metabolic stability, for example increased half-life in vivo, or reduced dosage requirements, and may therefore be advantageous in some circumstances.

[0142] Additionally, IMiD and CELMoD compounds encompass the use of N-oxides, crystalline forms (also known as polymorphs), active metabolites of the compounds having the same type of activity, tautomers, and undissolved forms of the compounds as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. Solvates of the conjugates provided herein are also considered to be disclosed herein.

[0143] Also provided herein are pharmaceutical compositions comprising the degron tags, fusion proteins, nucleotide sequences, vectors, or cells of the present invention and pharmaceutically acceptable excipients, carriers, adjuvants, and / or diluents. Pharmaceutical compositions may conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and supplemental immune-enhancing agents such as cytokines, and any other therapeutic agents or compounds.

[0144] As used herein, "pharmaceutically acceptable" means a substance that is biologically or otherwise undesirable, i.e., a substance that may be administered to an individual together with a selected compound without causing undesirable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is included.

[0145] An excipient is a natural or synthetic substance formulated with an active ingredient (e.g., a compound of the present invention) to bulk the composition or to impart a therapeutic effect to the active ingredient in the final dosage form, such as by promoting drug absorption or solubility. Excipients may also be useful in the manufacturing process to aid in handling of the active ingredient, such as promoting powder flowability or non-stick properties, as well as in vitro stability, such as preventing degradation during expected storage periods. Pharmaceutically acceptable excipients are well known in the art. Therefore, suitable excipients can be readily identified by those skilled in the art. Examples of suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like. Adjuvants are pharmacological and / or immunological agents that modify the effects of other agents in the composition. Pharmaceutically acceptable adjuvants are well known in the art. Therefore, suitable adjuvants can be readily identified by those skilled in the art. A diluent is a diluent. Pharmaceutically acceptable diluents are well known in the art. Therefore, suitable diluents can be easily identified by those skilled in the art. Carriers are non-toxic to recipients at the dosages and concentrations used, and are compatible with other ingredients of the formulation. The term "carrier" refers to a natural or synthetic organic or inorganic component that is combined with active ingredients to facilitate application. Pharmaceutically acceptable carriers are well known in the art. Therefore, suitable carriers can be easily identified by those skilled in the art.

[0146] The pharmaceutical composition of the present invention can be used as a medicine. The degron tag provided herein functions as a safety switch to regulate the expression of the POI.

[0147] Thus, a method for degrading a protein of interest (POI) is also provided, the method comprising contacting cells in vitro or in vivo with an effective amount of an immunomodulatory drug (IMiD) or cereblon-modulating drug (CELMoD), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the cells express a nucleic acid encoding a fusion protein of the present invention (i.e., a fusion protein comprising a POI of the present invention and a degron tag). The method can be performed in vivo or in vitro. The POI can be exogenous or endogenous. The cells can be allogeneic or autologous cells. The method can be performed in vivo on a subject. The subject can be a human.

[0148] Also provided is a method for degrading a protein of interest, the method comprising: The present invention includes administering an effective amount of an immunomodulatory drug (IMiD) or cereblon-modulating drug (CELMoD), or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject who has previously been treated with gene therapy to cause at least some endogenous cells to express a nucleic acid encoding a 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 approach that corrects an underlying genetic problem and treats or prevents disease by introducing a gene into cells. Gene therapy of the present invention involves introducing a nucleic acid or vector of the present invention into a subject, wherein the nucleic acid or vector encodes a 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 may be introduced into cells ex vivo and then introduced into the subject, or the nucleic acid or vector may be introduced into the subject's cells in vivo. In some embodiments, the subject has received immune effector cells, such as autologous T cells (CAR-T cells), genetically modified to express a chimeric antigen receptor protein (CAR)-degron tag fusion protein, and is experiencing an adverse immune response (e.g., cytokine release syndrome or neurotoxicity) as a result of the treatment. In other embodiments, the gene therapy includes gene knock-in, administration of a viral vector, or CRISPR (clustered regularly interspaced short palindromic repeats)-mediated knock-in.

[0149]

[0010] A further aspect of the present invention is directed to a method for reducing overexpression of a gene in a subject, comprising: introducing into one or more relevant cells of the subject a nucleic acid sequence encoding a degron tag inserted genomic in-frame with the nucleic acid sequence of an endogenous protein associated with a disease resulting from 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 resulting from a gain-of-function mutation, amplification or increased expression, a monogenic disease, a proteopathy, or a combination thereof.

[0150] A further aspect of the present invention is directed 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 into one or more relevant cells a nucleic acid sequence encoding a degron tag integrated in-frame with the nucleic acid sequence of an endogenous protein suspected to be associated with the disease; and contacting the cells with an effective amount of an IMiD or CELMoD. This method can be performed in vivo (e.g., in an animal model) or in vitro (e.g., in cell culture).

[0151] Any of the methods of the invention may involve contacting a cell with or administering to a subject an IMiD or CELMoD that is thalidomide, pomalidomide, lenalidomide, CC-122, CC-220, or CC-885.

[0152] 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 Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this invention. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, preferred methods and materials are described herein. Accordingly, the terms defined below are more fully described by reference to the specification in its entirety. Also, as used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. It will be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the circumstances employed by those skilled in the art. Aspects of the present invention are illustrated by the following non-limiting examples. [Example]

[0153] Example Thalidomide-like derivatives (immunomodulatory drugs IMiDs, or more recently called cereblon E3 ligase modulators CELMoDs) inhibit the Cullin4-RING E3 ubiquitin ligase CRL4 CRBN CRBN is a substrate receptor for CRL4, and is a degradation inducer that binds to a conserved tryptophan cage on the surface of cereblon (CRBN). CRBNThis induces a change in substrate specificity, leading to the recruitment, ubiquitination, and subsequent proteasomal degradation of neo-substrates such as Ikaros (IKZF1), Aiolos (IKZF3), casein kinase 1α (CK1a or CSNK1A), G1-S phase transition protein 1 (GSPT1), and zinc finger protein 91 (ZFP91) (Table 1). 7,11-16 . [Table 3]

[0154] Neo-substrates interact with the degron-binding surface of CRBN through a recognition motif known as a degron. This motif is characterized by a common β-hairpin loop with a conserved glycine at its apex and is crucial for the interaction of the degron with the degron-binding compound. The amino acid sequence adjacent to the sentinel glycine of the β-hairpin loop is variable among neo-substrates and is important for the specificity of neo-substrate recruitment and degradation by specific CRBN-binding agents. In the case of the neo-substrates Ikaros, Aiolos, and ZFP91, the degron motif is located within the C2H2 zinc finger (ZF) domain, and the first half of the domain has a CxxCG sequence signature, including a conserved glycine immediately following the second cysteine ​​of the ZF. 7,8 .

[0155] Generation of DCD-EGFP fusion construct To address the limitations of currently available protein regulation tools, we developed a novel and robust system for targeted protein degradation that utilizes an IMiD / CeLMOD recognition sequence fused to a protein of interest. By systematically evaluating a matrix of 23 degron-containing domains (DCDs) from various ZF and non-ZF proteins with the IMiD / CeLMOD small molecule panel (see Figure 1), we identified a DCD tag (DCD23) based on a chimeric sequence of Ikaros and ZFP91, which induced acute targeted degradation in vitro and in vivo (Figure 1). This 60-amino acid sequence is referred to herein as iTAG1(DCD23).

[0156] Based on structural analysis of the DCD23 sequence, mutations were introduced to evaluate whether an improved degron sequence could be identified. Many different constructs were tested (e.g., iTAG2v1, iTAG2v2, iTAG2), and the optimal construct was selected and designated DCD23mut or iTAG2 (Figure 2).

[0157] We found that fusion of iTAG2 to an EGFP construct induces degradation similar to that of iTAG1 (DCD23) (Figure 3a) and abolishes nuclear accumulation (Figure 3b). Variations of iTAG2 contemplated herein are also shown in Figure 4.

[0158] Generation of iTAG2-chimeric antigen receptor (CAR) fusion constructs Therefore, we cloned iTAG2 and iTAG1 (DCD23) into the C-terminus of a second-generation anti-B7H3 chimeric antigen receptor (CAR) containing the CD28 and CD3zeta endodomains, respectively. The tagged CARs were co-expressed from a gammaretroviral vector via the 2A sequence with the CD34 marker gene (Figure 5). We then examined whether the two tagged CARs were successfully degraded after exposing cells expressing the tagged CARs to IMiD drugs.

[0159] In the first experiment, we evaluated expression and degradation in human Jurrat T-cell leukemia cells or human 293T cells stably transfected with untagged, iTAG1 (DCD23)-tagged, or iTAG2-tagged anti-B7H3 CAR-T cells. Here, the relative expression of the CAR and CD34 marker genes was determined by direct staining and flow cytometry. In both target cell lines, iTAG2-CAR and untagged CARs expressed similar amounts of CD34 and CAR, whereas iTAG1 (DCD23)-tagged CARs expressed less CD34 than CD34, demonstrating relatively poor cell surface expression (Figure 6). These data indicate that the iTAG2 sequence conferred superior cell surface expression of the chimeric antigen receptor compared to the iTAG1 (DCD23) sequence.

[0160] Next, we evaluated the downregulation of iTAG2-tagged CARs in transfected Jurkat cells after the addition of various concentrations of IMiD drugs (lenalidomide, pomalidomide, and iverdomide). Both iTAG1 (DCD23)-tagged CARs and iTAG2-tagged CARs showed similar downregulation patterns after drug addition, with the maximal decrease in surface expression detectable 4 hours after drug treatment. Iverdomide demonstrated the greatest potency against degradation, with near-maximal downregulation observed at a drug concentration of 0.01 micromolar (Figure 7).

[0161] After demonstrating expression and drug-induced degradation in Jurkat cell lines, we 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 before assessing CAR expression after 24 hours of 10 μM iberdomide addition. Interestingly, in primary T cells after 24 hours, iberdomide treatment almost completely abolished the expression of the brightly expressing CAR population, although dim expression was still observed (Figure 8).

[0162] To assess whether the brighter CAR expression of iTAG2-tagged CARs compared with DCD23-tagged CARs translates to improved effector function, CAR-T populations from two independent donors were cocultured overnight with human SupT1 leukemia target cells isogenic for the target antigen B7H3. B7H3 expression was not detected in wild-type SupT1. After overnight incubation of T cells with the targets, culture supernatants were collected and assayed for the inflammatory cytokines interferon gamma and interleukin-2 by ELISA (Figure 9). The data indicate that the DCD23 fusion protein exhibits approximately five-fold reduced effector function for interferon gamma secretion and approximately 30-fold reduced effector function for IL-2 secretion compared with the iTAG2 fusion protein (Figure 9). The effector function of iTAG2-tagged CARs is not significantly different from that of untagged CARs.

[0163] We compared the surface expression of both the 41BB and CD28 versions of the TE9 CAR, and the surface expression of the iTAG2 fusion protein with both DCD23 and a previously published superdegron (referred to herein as superdegron). In transduced Jurkat cells, surface expression of DCD23- and superdegron-tagged CARs was similarly weak, whereas in this experiment, iTAG2-tagged CARs showed expression equivalent to that of wild-type (untagged) CARs. Contrary to published reports that superdegrons only act on 41BB CARs, we found no evidence of a difference in surface expression between the two costimulatory arrangements (Figure 10). Thus, the data presented here demonstrate that the tested DCD sequences (iTAG2, DCD23, and super-degrader) function with both the 41BB and CD28 versions of second-generation CARs.

[0164] Aiolos peptide-based TR-FRET assay to measure the relative affinities of iTAG1 and iTAG2 for complexes formed by CRBN / DDB1 (DNA damage binding protein 1) and various IMiDs A schematic diagram of the aiolopeptide-based time-resolved Förster resonance energy transfer (TR-FRET) assay is shown in Figure 11. We used this assay to evaluate the ability of iTAG1 and iTAG2 to displace a fluorescently labeled aiolopeptide probe from complexes formed between CRBN, DDB1, and various IMiDs (lenalidomide, pomalidomide, and iverdomide), thereby allowing us to measure the relative affinities of iTAG1 and iTAG2 for these complexes. Results are reported as IC values ​​calculated from the TR-FRET curves. 50 The IC obtained with iTAG2 is shown in Figure 12. 50 is the IC obtained with iTAG1 50 The relative affinities obtained for the three compounds showed the same trend for iTAG1 and iTAG2, with iverdomide showing the strongest binding, followed by pomalidomide and lenalidomide.

[0165] Superdegron-tagged GFP localizes primarily to the nucleus iTAG2-tagged GFP shows cytoplasmic localization, whereas iTAG1 is predominantly nuclear. To determine the localization of a "superdegron" with some homology to iTAG1, we cloned the superdegron into an expression vector, replacing iTAG1 with a GFP-fused degron. After transient transfection into HMECs (human mammary epithelial cells), the superdegron tag was shown to result in strong nuclear localization of GFP, as shown in Figure 13.

[0166] Confirmation of complex formation between CRBN / DDB1 and iTAG2 in the presence of iverdimide by size-exclusion chromatography Biochemical evidence for the IMiD-dependent direct interaction between iTAG2 and the CRBN / DDB1 complex was obtained using size-exclusion chromatography (SEC). Purified CRBN / DDB1 protein complexes and iTAG2 proteins were mixed and subjected to SEC in the presence or absence of iberdomide, as shown in Figure 14. SEC peak 1, corresponding to high-molecular-weight species, shifted to a lower elution time in the presence of iberdomide, along with a slight increase in optical density at 280 nm, consistent with the formation of a larger complex in the presence of iberdomide (Figure 14A). SDS-PAGE analysis of representative fractions from the SEC analysis in the absence of iberdomide confirmed that SEC peak 1 contained only DDB1 and CRBN proteins, while iTAG2 was present exclusively in SEC peak 2 (Figure 14B). In the presence of iberdomide, SEC peak 1 contained all three proteins, confirming that DDB1, CRBN, and iTAG2 formed a complex in the presence of iberdomide (Figure 14C).

[0167] iTAG2-tagged human CAR-T cells rapidly re-emerge after washing the cells and discontinuing the IMiD drug Re-expression of cleaved chimeric antigen receptors after removal of the degradation inducer is important for enabling CAR-T cells to switch between active and quiescent states, thereby enabling further therapeutic manipulation and fine-tuning. To determine reversibility, human PBMC-derived TE9-28Z-iTAG2 CAR-T cells were treated with various concentrations of iberdomide to reduce CAR-T surface expression, then washed twice and cultured in fresh medium lacking iberdomide for 24 hours, demonstrating re-expression of cell surface CAR, as shown in Figure 15A and B.

[0168] iTAG2-tagged anti-B7H3 CAR: Expression and function in lentiviral vectors To evaluate the general functionality of the TE9-28Z-ITAG2 anti-B7H3 CAR-T construct when expressed from a different viral vector driven by a different promoter, the construct was cloned into the third-generation lentivector pCLL2 backbone and viral supernatants were generated by transiently transfecting 293T cells with VSV-G-pseudotyped virus. Two versions of the TE9-28Z-ITAG2 sequence were evaluated in this format, with and without codon optimization and removal of the cloning scar. There is no difference in the amino acid sequence of iTAG2 between the original version (Construct 3) and the codon-optimized version (Construct 4) (Figures 16A and B). To investigate degradation after codon optimization of the CAR and iTAG2 sequences, CAR-T cells generated from three independent donors using pCCL2 lentiviral vector-transduced GMP-like conditions were expanded to 9 days post-stimulation and treated with 100 nM iberdomide overnight before direct staining of both the CAR and co-expressed RQR8 marker genes by flow cytometry. The logarithmic reduction in CAR expression, expressed as median geometric fluorescence intensity (gMFI), was highly consistent. We found that iTAG2 from the codon-optimized lentiviral backbone exhibited similar susceptibility to iberdomide-induced degradation as non-codon-optimized iTAG2 (Figure 16C). TE9-28z-ITAG2-expressing codon-optimized lentiviral vectors exhibited enhanced cytotoxicity against antigen-positive target cells and secretion of inflammatory cytokines (Figure 16D-F). The lack of codon-optimized TE9-ITAG2 cytotoxicity against the B7H3-negative SupT1 target was confirmed using an overnight luminescence-based assay at an E:T ratio of 1:2 (data shown in Figure 16G for three independent CAR-T donors).

[0169] method: iTAG2 Design We generated three mutant forms of iTAG1: - iTAG2V1, with only the central ZFP91 / Ikaros chimeric zinc finger mutated: TIFF2025537708000007.tif19164 - iTAG2V2, with one additional mutation in the linker between ZF1 and ZF2 of Ikaros: TIFF2025537708000008.tif19164 - iTAG2, in which several residues present in the construct are mutated: The TIFF2025537708000009.tif19164 sequence was commercially synthesized (GeneArt, ThermoFischer Scientific) and inserted into the pLVX-TetOne-Puro lentiviral vector.

[0170] Evaluation of EGFP-ITAG(1 / 2) constructs Viral transduction and cell line generation For viral particle production, HEK293T cells were seeded 24 h before transfection and transfected with pLVX-TetOne-Puro lentiviral vector, psPAX2 (packaging plasmid), and pMD.2G (ThermoFisher) using Lipofectamine. (商標) Transfection was performed using a 3000. Virus-containing supernatants were collected 48 hours after transfection, filtered through a 0.45 μm filter (Sartorius Stedim), and stored at −80°C in cryovials (ThermoFischer Scientific).

[0171] For cell transduction, HMEC cells were infected at an MOI of 3 (viral particle titer was determined using the Lenti-X™ GoStix™ Kit (Clontech)) and polybrene (4 μg / ml) was used to enhance transduction efficiency. Cells were cultured for at least two passages before construct expression was induced with 1 μg / ml doxycycline (DOX) for 24 hours. Fluorescence-activated cell sorting (FACS) was used to select the top 30% of the cell population with the highest EGFP expression.

[0172] Flow cytometry degradation test Expression of the constructs was induced by treating cells with 1 μg / ml DOX for 24 hours. Cells were treated with IMiD / CELMoD at the concentrations and time points indicated for each test setup. Cells were collected on ice into sterile 5 ml polystyrene round-bottom tubes. As a marker of cell viability, DAPI (4',6-diamino-2-phenylindole; ThermoFisher Scientific) was added to the cell suspension at a ratio of 1:1000. EGFP signals were measured using a BD LSR II flow cytometer (BD Biosciences). Further processing of the results was performed using FlowJo 10 software (FlowJo).

[0173] Nuclear localization assay by immunofluorescence HMEC cells were seeded at 1,000 cells / well in glass-bottom 96-well plates (Perkin Elmer) and treated with 1 μg / ml DOX for 24 hours to induce the constructs. Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at 37°C and permeabilized with 0.05% Ryton-X-100 detergent for 10 minutes at room temperature. Prior to cell imaging, cells were incubated with 1 μg / ml DAPI (ThermoFisher Scientific) for 1 minute to stain the cell nuclei. Cells were visualized using a Zeiss Axio Vert.A1 FL-LED inverted epifluorescence microscope (Zeiss), and images were captured using ZEN Blue software (Zeiss).

[0174] Evaluation of the CAR-ITAG(1 / 2) construct Construct generation and cloning The degradation tags iTAG1 (DCD23) and iTAG2 were synthesized as gene blocks (IDT) and cloned into the MMLV-derived gammaretroviral vector SFG using standard restriction site ligation. This vector drives transcription of a single transcript containing the RQR8 epitope and the CAR construct (anti-B7H3 scFv (TE9), CD8 transmembrane domain, CD28 costimulatory domain, and CD3 signaling domain) separated by a T2A ribosomal skip site. The degradation tag is inserted immediately after the CD3 signaling domain, without any spacer sequence.

[0175] Virus production Phoenix ampho packaging cells were grown to approximately 80% confluence using standard protocols and transfected with CAR + / - tagged viral constructs using GeneJuice Transfection Reagent. Viral supernatants were harvested 48 and 72 hours post-transfection and pooled before being flash-frozen.

[0176] Transduction of primary cells and cell lines: Jurkat and 293T cell lines were cultured in DMEM supplemented with 10% fetal calf serum and transduced with constructs containing CAR degradation tags. Transduction efficiency was analyzed 72 hours post-transduction by flow cytometry staining for the CD34 epitope encoded by the RQR8 gene.

[0177] PBMC-derived T cells from two healthy donors were cultured after CD56 depletion using Miltenyi CD56 depletion beads (day 0) and magnetic negative selection using LD columns according to the manufacturer's instructions. After depletion, PBMCs were stimulated with CD3+CD28 antibody beads (day 1) and IL-2 activation at 100 iU / ml (day 3). Cells were transfected with CAR+ / - cleavage inducer-tagged virus on day 4. PBMCs were transfected with undiluted viral supernatant, while cell lines were transfected with diluted virus at a multiplicity of infection of 1–5. Transduction efficiency was assessed on day 4 by flow cytometry staining for RQR8 and CAR expression.

[0178] IMiD-induced CAR degradation and CAR functionality testing: Transduced Jurkat cell lines were treated with pomalidomide, iverdomide, or lenalidomide at 10 μM, 1 μM, or 0.01 μM, or with DMSO, for 2, 4, or 24 hours in a split-cell assay. CAR degradation was assessed by flow cytometry. After 24 hours, CAR expression was assessed by flow cytometry in T cells derived from PBMCs lysed with iverdomide at 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, or 0 μM. To assess the functionality of the CAR + / - degradation inducer tag, transduced T cells were cultured 1:1 with SUPT1 + / - B7H3 for 24 hours. Supernatants were collected, and IL-2 and IFNγ levels were measured by ELISA.

[0179] Flow cytometry analysis: CAR surface expression for transfection efficiency and degradation studies in primary cells and cell lines was quantified using a combination of his-tagged B7H3 protein and an anti-his-tagged fluorescent antibody (J095G45, BioLegend) and a directly fluorescently labeled anti-CD34 antibody (QBEnd10, R&D) on an LSR2 cytometer. Analysis was performed using Flo-Jo software.

[0180] Aiolos peptide-based TR-FRET assay: The protein production and purification methods used in the TR-FRET assay were described in our paper on the discovery of iTAG (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 is described in.

[0181] All TR-FRET assays were performed in a black 384-well ProxiPlate Plus (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. A final concentration range of 82 μM to 2.05 nM for iTAG1 and 27 μM to 0.68 nM for iTAG2 was achieved using an Echo E550 (Beckman Coulter, USA) acoustic liquid dispenser. Final concentrations of 5 nM WT full-length CRBN / DDB1 complex, 750 nM Sulfo-Cy5 fluorescent aiolic acid-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 to a final concentration of 10 μM using an echo. The plate was sealed, centrifuged at 200 g for 1 min, and stored at 4 °C overnight. TR-FRET signals were read using a PHERAstar FSX plate reader (BMG Labtech, Germany). The final signal was measured as the ratio: TR-FRET signal = Channel 1 / Channel 2 Channel 1 represents positive FRET at 665 nm, and channel 2 represents terbium emission when no FRET occurs at 620 nm.

[0182] Size Exclusion Chromatography: The protein production and purification methods used to reconstitute the complex formed between CRBN / DDB1, iverdomid, and iTAG2 by size-exclusion chromatography (SEC) were published in our publication on the discovery of iTAG (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 is described in.

[0183] SEC was performed using a Superose 6 5 / 150 GL column (Cytiva Life Sciences) attached to an Agilent 1260 Infinity II LC System HPLC system. Purified CRBN / DDB1 and iTAG2 were mixed at 15 μM and 50 μM, respectively, in a buffer containing 20 mM HEPES pH 7.0, 200 mM NaCl, and 1 mM TCEP to prepare two 60 μL samples. 100 μM iberdomide was added to one sample, and both samples were incubated on ice for 1 h and then centrifuged at 21,000 g for 10 min before injection to remove potential aggregates. The Superose 6 column was pre-equilibrated with 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 before injection of 50 μL of each sample. Fractions of 100 μL were collected and subjected to SDS-PAGE on precast NuPAGE 4-12% Bis-Tris gels (Invitrogen) using standard protocols, with SeeBlue Plus2 stained protein standards (Invitrogen) loaded into the first lane of each gel.

[0184] Attention is directed to all papers and documents related to this application, filed contemporaneously or prior to this application, and in the public domain herewith, and the contents of all such papers and documents are hereby incorporated by reference.

[0185] All features disclosed in this specification (including the accompanying claims, abstract and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0186] Each feature disclosed in this specification (including the accompanying claims, abstract, and drawings), unless expressly stated otherwise, may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

[0187] The invention is not limited to the details of the above embodiments, and extends to any novel or novel combination of features disclosed in this specification (including the accompanying claims, abstract and drawings), or any novel or novel combination of method or process steps so disclosed.

[0188] References 1. Bonger, KM, Chen, LC, Liu, CW & Wandless, TJ Small-molecule displacement of a cryptic degron causes conditional protein degradation. Nat. Chem. Biol. 7, 531-537 (2011). 2. Chung, HK et al. Tunable and reversible drug control of protein production via a self-excising degron. Nat Chem Biol 11, 713-720 (2015). 3. Buckley, DL et al. HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins. ACS Chem Biol 10, 1831-1837 (2015). 4. Nabet, B. et al. Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat. Commun. 11, 1-8 (2020). 5. Nabet, B. et al. The dTAG system for immediate and target-specific protein degradation. Nat. Chem. Biol. 14, 431-441 (2018). 6. Ege, N., Bouguenina, H., Tatari, M. & Chopra, R. Phenotypic screening with target identification and validation in the discovery and development of E3 ligase modulators. Cell Chem. Biol. 28, 283-299 (2021). 7. An, J. et al. PSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4 CRBN ubiquitin ligase. Nat. Commun. 8, 1-11 (2017). 8. Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, (2018). 9. Weber, E. W. et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 372, (2021). 10. Jan, M. et al. Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide. Sci. Transl. Med. 13, (2021). 11. Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345-1350 (2010). 12. Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of ikaros proteins. Science 343, 305-309 (2014). 13. Kroenke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301-305 (2014). 14. Kronke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature 523, 183-188 (2015). 15. Gandhi, A. K. et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4CRBN. Br. J. Haematol. 164, 811-821 (2014). 16. Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252-257 (2016). 17. Kroenke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301-305 (2014). 18. Kroenke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523, 183-188 (2015). 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. LQCEICGFTCX １ QX ２ GNLLX ３ HIX ４ A degron tag comprising the amino acid sequence of LH (SEQ ID NO: 66), wherein X １ , X ２ , X ３ and X ４ is not K, R or H.

2. LQCEICGFTCX １ QX ２ GNLLX ３ HIX ４ The degron tag of claim 1, comprising the amino acid sequence of LH (SEQ ID NO: 2), wherein: X １ is E or a conservative amino acid substitution thereof; X ２ is A or a conservative amino acid substitution thereof; X ３ is N or a conservative amino acid substitution thereof; and X ４ is E or a conservative amino acid substitution thereof.

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

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

5. The degron tag of any one of claims 1 to 4, wherein the degron tag comprises an additional N-terminal zinc finger alpha-helix subdomain and an additional C-terminal zinc finger beta-hairpin subdomain adjacent to the amino acid sequence of any one of SEQ ID NOs: 66, 2, 5, 12 or 13.

6. 6. The degron tag of claim 5, wherein the additional C-terminal zinc finger beta-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. An additional N-terminal zinc finger alpha helix subdomain has the amino acid sequence PNVLMVHX ５ X ６ SH (SEQ ID NO: 71) or FNVLMVHX ５ X ６ 7. The delong tag of claim 5 or 6, comprising: XSH (SEQ ID NO: 72); ５ and X ６ is not R, K or H.

8. An additional N-terminal zinc finger alpha helix subdomain has the amino acid sequence PNVLMVHX ５ X ６ SH (SEQ ID NO: 16) or FNVLMVHX ５ X ６ 8. The degron tag of claim 7, comprising: SH (SEQ ID NO: 17); X ５ is N or a conservative amino acid substitution thereof; and X ６ is E or a conservative amino acid substitution thereof.

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

10. The degron tag of claim 8 or 9, wherein the degron tag comprises the following amino acid sequence: PNVLMVHX ５ X ６ SHTGEX ７ PLQCEICGFTCX １ QX ２ GNLLX ３ HIX ４ LHSGEX ８ PFKCHLCNYACRRRDAL (SEQ ID NO: 73), or FNVLMVHX ５ X ６ SHTGEX ７ PLQCEICGFTCX １ QX ２ GNLLX ３ HIX ４ LHTGEX ８ PFKCHLCNYACQRRDAL (SEQ ID NO: 74); X ７ and X ８ is not R, K or H.

11. The degron tag of claim 10, wherein the degron tag comprises the following amino acid sequence: PNVLMVHX ５ X ６ SHTGEX ７ PLQCEICGFTCX １ QX ２ GNLLX ３ HIX ４ LHSGEX ８ PFKCHLCNYACRRRDAL (SEQ ID NO: 24), or FNVLMVHX ５ X ６ SHTGEX ７ PLQCEICGFTCX １ QX ２ GNLLX ３ HIX ４ LHTGEX ８ PFKCHLCNYACQRRDAL (SEQ ID NO: 45); X ７ is I or a conservative amino acid substitution thereof; and X ８ is I or a conservative amino acid substitution thereof.

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

13. The degron tag of any one of claims 1 to 12, wherein the degron tag has a length of about 23 to about 70 amino acids.

14. The degron tag of claim 10, wherein the degron 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 of claim 15, wherein the degron tag is located at the C-terminus of the protein of interest.

17. 17. The fusion protein of 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 coupler (TAC), a chimeric autoantibody receptor (CAAR), or an antibody-binding T cell receptor (ACTR).

18. The CAR fusion protein is arranged in the order of N-terminus to C-terminus as follows: 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 18. The fusion protein of claim 17, comprising:

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

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

21. A vector comprising the nucleic acid sequence of claim 20.

22. The vector of claim 21, wherein the vector is a viral vector, and optionally the viral vector is selected from the group consisting of a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, a vaccinia viral vector, a picornavirus vector, and an alphavirus vector.

23. 23. A cell expressing the nucleic acid sequence of claim 20 or the vector of claim 21 or 22.

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

25. 25. The cell of 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 lymphocytes, 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. 26. A 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. 27. A pharmaceutical composition comprising a degron tag, fusion protein, nucleotide sequence, vector or cell according to any one of claims 1 to 26, and a pharmaceutically acceptable excipient, carrier, adjuvant and / or diluent.

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

29. 28. The pharmaceutical composition of claim 27 for use in immune cell therapy.

30. 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 any one of claims 15 to 19. A method for degrading a target protein, comprising:

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

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

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