Compositions and methods for selective protein degradation

Abstract

To provide methods to modulate the expression level of therapeutic proteins, e.g., to modulate the levels of therapeutic proteins to increase efficacy and / or decrease side effects.SOLUTION: The invention provides compositions including a fusion polypeptide and methods for making a fusion polypeptide that includes a COF1 / CRBN-binding polypeptide, COF2 / CRBN-binding polypeptide, or COF3 / CRBN-binding polypeptide and a heterologous polypeptide of interest.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 574,188, filed October 18, 2017, the contents of which are incorporated by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on October 17, 2018, is named N2067-7136WO_SL.txt and is 2,052,554 bytes in size. [Background technology]

[0003] Many therapeutic proteins have been developed as important drugs to prevent or treat diseases. Side effects may occur during or after treatment and range from loss of efficacy to severe toxicity. It is desirable to develop strategies to regulate the expression level of therapeutic proteins, e.g., to regulate the level of therapeutic proteins to increase efficacy and / or reduce side effects. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides, at least in part, a fusion polypeptide comprising a compound of formula (I) (COF1) / CRBN binding polypeptide, a compound of formula (II) (COF2) / CRBN binding polypeptide, or a compound of formula (III) (COF3) / CRBN binding polypeptide for targeted protein inactivation. In some embodiments, the fusion polypeptide comprises one or more COF1 / CRBN, COF2 / CRBN, or COF3 / CRBN binding polypeptides and one or more heterologous polypeptides, such as a polypeptide of interest. The COF1 / CRBN, COF2 / CRBN, or COF3 / CRBN binding polypeptides can be operably linked to the heterologous polypeptide, for example, via a linker. In some embodiments, in the presence of COF1 or COF2 (such as thalidomide and its derivatives (e.g., lenalidomide, pomalidomide, and thalidomide)) or in the presence of COF3 (e.g., compounds disclosed in Table 29), the COF1 / CRBN, COF2 / CRBN, or COF3 / CRBN binding polypeptide alters the level and / or activity of the fusion polypeptide; e.g., increases degradation, e.g., proteasomal degradation, of the fusion polypeptide. In some embodiments, degradation of the fusion polypeptide is ubiquitin-dependent.

[0005] Without being bound by theory, in some embodiments, the COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide provides an amino acid sequence and / or structural motif that results in post-translational modification (e.g., ubiquitination) of the fusion polypeptide in the presence of COF1 or COF2 (such as thalidomide and its derivatives (e.g., lenalidomide, pomalidomide and thalidomide)) or in the presence of COF3 (e.g., compounds disclosed in Table 29) to result in a modified, e.g., ubiquitinated, fusion polypeptide. For example, one or more amino acids, e.g., lysine or methionine, in the fusion polypeptide can be ubiquitinated in the presence of COF1, COF2 or COF3. In some embodiments, the ubiquitinated fusion polypeptide is selectively degraded. In some embodiments, the post-translational modification of the fusion polypeptide increases degradation of the fusion polypeptide (e.g., increasing the level and / or rate of degradation). In some embodiments, the level and / or rate of degradation is increased by at least 1.5, 2, 3, 4, 5, 10, 20, 30, 40 or 50 fold relative to the level and / or rate of degradation of a reference polypeptide, e.g., a fusion polypeptide in the absence of COF1, COF2 or COF3, a heterologous polypeptide, or a fusion of a heterologous polypeptide without a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide, or with a moiety other than a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide.

[0006] In one aspect, provided herein is a fusion polypeptide comprising a compound of formula (I) (COF1) / CRBN binding polypeptide and a heterologous polypeptide, wherein the compound of formula (I) is [ka] (In the formula, X is O or S; R 1 is C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2~C 6 Alkynyl, C 1 ~C 6 heteroalkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which may be one or more R 4 independently and optionally replaced by; R 2a and R 2b Each of the groups is independently hydrogen or C 1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 3 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; Each R 4 is independently 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 6 is independently 1 ~C 6 Alkyl, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7 are independently halo, oxo, cyano, -OR B , -N(R C )(R D), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; n is 0, 1, 2, 3 or 4; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, ester, hydrate, solvate or tautomer thereof.

[0007] In some embodiments, the heterologous polypeptide is a heterologous mammalian polypeptide. In some embodiments, the heterologous polypeptide is a heterologous bacterial polypeptide. In some embodiments, the heterologous polypeptide is a heterologous viral polypeptide. In some embodiments, the heterologous polypeptide comprises an amino acid sequence from or derived from a mammalian polypeptide, a bacterial polypeptide, a viral polypeptide, a plant polypeptide, a yeast polypeptide, a fungal polypeptide, an archaeal polypeptide, a fish, e.g., a zebrafish polypeptide. In some embodiments, the heterologous polypeptide comprises a polypeptide in Table 2, e.g., a cytoplasmic and / or nuclear polypeptide or a transmembrane polypeptide as set forth in Table 2.

[0008] In some embodiments, the COF1 / CRBN binding polypeptide is fused to a heterologous polypeptide. In some embodiments, the COF1 / CRBN binding polypeptide and the heterologous polypeptide are linked by a peptide bond. In some embodiments, the COF1 / CRBN binding polypeptide and the heterologous polypeptide are linked by a bond other than a peptide bond. In some embodiments, the heterologous polypeptide is directly linked to the COF1 / CRBN binding polypeptide. In some embodiments, the heterologous polypeptide is indirectly linked to the COF1 / CRBN binding polypeptide. In some embodiments, the COF1 / CRBN binding polypeptide and the heterologous polypeptide are operably linked via a linker, e.g., a glycine-serine linker, e.g., a linker comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:28, 37, 38, 39, and 99.

[0009] In some embodiments, the COF1 / CRBN binding polypeptide is linked to the C-terminus of the heterologous polypeptide. In some embodiments, the COF1 / CRBN binding polypeptide is linked to the N-terminus of the heterologous polypeptide. In some embodiments, the COF1 / CRBN binding polypeptide is in the middle of the heterologous polypeptide.

[0010] In some embodiments, the association of the COF1 / CRBN binding polypeptide with cereblon (CRBN) in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15%, or 20% or less of the association of the COF1 / CRBN binding polypeptide with CRBN in the presence of COF1, for example, an excess amount of COF1, as measured, for example, by an assay described herein, for example, by immunoprecipitation. In some embodiments, the COF1 / CRBN binding polypeptide does not bind to CRBN in the absence of COF1. In some embodiments, the association of the fusion polypeptide with CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15%, or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF1, for example, an excess amount of COF1, as measured, for example, by an assay described herein, for example, by immunoprecipitation. In some embodiments, the fusion polypeptide does not bind to CRBN in the absence of COF1. In some embodiments, the association or binding is as measured in a mammalian cell, such as a human cell.

[0011] In some embodiments, the ubiquitination of the heterologous polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF1, e.g., an excess amount of COF1, as measured, for example, by an assay described herein. In some embodiments, the ubiquitination of the fusion polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF1, e.g., an excess amount of COF1, as measured, for example, by an assay described herein. In some embodiments, the heterologous polypeptide or fusion polypeptide is ubiquitinated at one or more lysine or methionine residues in the presence of COF1. In some embodiments, ubiquitination is as measured in a mammalian cell, such as a human cell.

[0012] In some embodiments, the degradation of the fusion polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less than the degradation of the fusion polypeptide in the presence of COF1, for example, an excess amount of COF1, as measured, for example, by an assay described herein, for example, Western blot analysis or flow cytometry analysis. In some embodiments, the degradation of the fusion polypeptide is mediated by ubiquitination in the presence of COF1. In some embodiments, the degradation of the fusion polypeptide is mediated by lysosomes. In some embodiments, the degradation is as measured in mammalian cells, for example human cells.

[0013] In some embodiments, the fusion polypeptide is a cell surface polypeptide. In some embodiments, the recycling rate of the fusion polypeptide from the cell surface to an intracellular compartment in the absence of COF1 is, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or less than the recycling rate of the fusion polypeptide from the cell surface to an intracellular compartment in the presence of COF1, e.g., an excess of COF1, as measured, e.g., by an assay described herein. In some embodiments, the recycling is as measured in mammalian cells, e.g., human cells.

[0014] In some embodiments, the COF1 / CRBN binding polypeptide is 10 to 95 amino acid residues in length, 15 to 90 amino acid residues in length, 20 to 85 amino acid residues in length, 25 to 80 amino acid residues in length, 30 to 75 amino acid residues in length, 35 to 70 amino acid residues in length, 40 to 65 amino acid residues in length, 45 to 60 amino acid residues in length, 50 to 65 amino acid residues in length, or 55 to 65 amino acid residues in length.

[0015] In some embodiments, the COF1 / CRBN binding polypeptide comprises a β-turn. In some embodiments, the COF1 / CRBN binding polypeptide comprises a β-turn of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF1 / CRBN binding polypeptide comprises a β-hairpin. In some embodiments, the COF1 / CRBN binding polypeptide comprises a β-hairpin of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF1 / CRBN binding polypeptide comprises a β-strand. In some embodiments, the COF1 / CRBN binding polypeptide comprises a β-strand of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF1 / CRBN binding polypeptide comprises an α-helix. In some embodiments, the COF1 / CRBN binding polypeptide comprises an α-helix of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF1 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, and a first α-helix. In some embodiments, the COF1 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, and a first α-helix of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF1 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, a first α-helix, and a second α-helix. In some embodiments, a COF1 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, a first α-helix, and a second α-helix of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the β-hairpin and the second α-helix are separated by no more than 60, 50, 40, or 30 amino acid residues.

[0016] In some embodiments, the COF1 / CRBN binding polypeptide comprises a COF1 / CRBN binding sequence derived from a naturally occurring polypeptide or a COF1 / CRBN binding variant thereof. In some embodiments, the COF1 / CRBN binding polypeptide comprises a COF1 / CRBN binding sequence derived from a naturally occurring IKZF polypeptide or a COF1 / CRBN binding variant thereof. In some embodiments, the COF1 / CRBN binding polypeptide comprises a COF1 / CRBN binding sequence derived from a naturally occurring IKZF1, IKZF2, IKZF3, IKZF4, or IKZF5, or a COF1 / CRBN binding variant thereof. In some embodiments, the COF1 / CRBN binding sequence comprises two or more non-contiguous sequences derived from a naturally occurring polypeptide, such as a naturally occurring IKZF polypeptide, such as a naturally occurring IKZF1, IKZF2, IKZF3, IKZF4, or IKZF5.

[0017] In some embodiments, the COF1 / CRBN binding polypeptide comprises an IKZF polypeptide or a structural motif thereof.

[0018] In some embodiments, the IKZF polypeptide is an IKZF1 polypeptide, an IKZF3 polypeptide, an IKZF2 polypeptide having an H141Q substitution (numbered according to SEQ ID NO:21), or an IKZF4 polypeptide having an H188Q substitution (numbered according to SEQ ID NO:22).

[0019] In some embodiments, the COF1 / CRBN binding polypeptide is i) the association between a COF1 / CRBN binding polypeptide and CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association between a COF1 / CRBN binding polypeptide and CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF1, e.g., an excess amount of COF1, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It contains sufficient amino acid sequence and / or structural motifs derived from an IKZF (eg, IKZF1 or IKZF3).

[0020] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0021] In some embodiments, the COF1 / CRBN binding polypeptide comprises about 10 to about 95 amino acid residues, about 15 to about 90 amino acid residues, about 20 to about 85 amino acid residues, about 25 to about 80 amino acid residues, about 30 to about 75 amino acid residues, about 35 to about 70 amino acid residues, about 40 to about 65 amino acid residues, about 45 to about 65 amino acid residues, about 50 to about 65 amino acid residues, or about 55 to about 65 amino acid residues of IKZF1 or IKZF3.

[0022] In some embodiments, the COF1 / CRBN binding polypeptide is i) the association between a COF1 / CRBN binding polypeptide and CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association between a COF1 / CRBN binding polypeptide and CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF1, e.g., an excess amount of COF1, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 180 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 180 of SEQ ID NO:19).

[0023] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0024] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 136-180 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:5) or a sequence that differs from amino acid residues 136-180 (numbered according to SEQ ID NO:19) of IKZF3 by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid residues (e.g., a sequence that differs from amino acid residues 136-180 of SEQ ID NO:19 by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid residues) (e.g., a sequence that has 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid substitutions from amino acid residues 136-180 of SEQ ID NO:19).

[0025] In some embodiments, the COF1 / CRBN binding polypeptide is i) the association between a COF1 / CRBN binding polypeptide and CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association between a COF1 / CRBN binding polypeptide and CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF1, e.g., an excess amount of COF1, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 170 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 170 of SEQ ID NO:19).

[0026] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0027] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 136-170 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:6) or a sequence that differs from amino acid residues 136-170 (numbered according to SEQ ID NO:19) of IKZF3 by 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 or fewer amino acid residues (e.g., a sequence that differs from amino acid residues 136-170 of SEQ ID NO:19 by 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 or fewer amino acid residues) (e.g., a sequence that has 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 or fewer amino acid substitutions from amino acid residues 136-170 of SEQ ID NO:19).

[0028] In some embodiments, one, two, three or all of the following amino acid residues remain unchanged: glutamine at position 147, cysteine ​​at position 148, glutamine at position 150, glycine at position 152, leucine at position 161 or leucine at position 162, numbered according to SEQ ID NO:19. In some embodiments, the glutamine at position 147, numbered according to SEQ ID NO:19, remains unchanged. In some embodiments, the cysteine ​​at position 148, numbered according to SEQ ID NO:19, remains unchanged. In some embodiments, the glutamine at position 150, numbered according to SEQ ID NO:19, remains unchanged. In some embodiments, the glycine at position 152, numbered according to SEQ ID NO:19, remains unchanged. In some embodiments, the leucine at position 161, numbered according to SEQ ID NO:19, remains unchanged. In some embodiments, the leucine at position 162, numbered according to SEQ ID NO:19, remains unchanged.

[0029] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 136-139 of IKZF3 (numbered according to SEQ ID NO:19), e.g., the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:40.

[0030] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO:19). In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:77. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:5. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:5.

[0031] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 136-170 of IKZF3 (numbered according to SEQ ID NO:19). In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:78. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:6. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:78.

[0032] In some embodiments, the COF1 / CRBN binding polypeptide is i) the association between a COF1 / CRBN binding polypeptide and CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association between a COF1 / CRBN binding polypeptide and CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF1, e.g., an excess amount of COF1, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 236 to 249 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 236 to 249 of SEQ ID NO:19).

[0033] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0034] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 236-249 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:11) or a sequence that differs from amino acid residues 236-249 (numbered according to SEQ ID NO:19) of IKZF3 by no more than 1, 2, 3, 4, 5, 6, or 7 amino acid residues (e.g., a sequence that differs from amino acid residues 236-249 of SEQ ID NO:19 by no more than 1, 2, 3, 4, 5, 6, or 7 amino acid residues) (e.g., a sequence that has no more than 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions from amino acid residues 236-249 of SEQ ID NO:19).

[0035] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 236-249 of IKZF3 (numbered according to SEQ ID NO: 19). In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:11.

[0036] In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:91.

[0037] In some embodiments, the COF1 / CRBN binding polypeptide is i) the association between a COF1 / CRBN binding polypeptide and CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association between a COF1 / CRBN binding polypeptide and CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF1, e.g., an excess amount of COF1, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF1 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF1 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF1, e.g., an excess of COF1, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136-180 and 236-249 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136-180 and 236-249 of SEQ ID NO:19).

[0038] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0039] In some embodiments, the COF1 / CRBN-binding polypeptide comprises a first sequence that includes amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO:19) (e.g., a first sequence that includes the amino acid sequence of SEQ ID NO:5) or a first sequence that differs from amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO:19) by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid residues (e.g., a first sequence that includes the amino acid sequence of SEQ ID NO:5) a first sequence that differs by not more than 30, 35 or 40 amino acid residues; and a second sequence comprising amino acid residues 236 to 249 of IKZF3 (numbered according to SEQ ID NO:19) (e.g., a second sequence comprising the amino acid sequence of SEQ ID NO:11) or a second sequence that differs by not more than 1, 2, 3, 4, 5, 6 or 7 amino acid residues from amino acid residues 236 to 249 of IKZF3 (numbered according to SEQ ID NO:19) (e.g., a second sequence that differs by not more than 1, 2, 3, 4, 5, 6 or 7 amino acid residues from amino acid residues 236 to 249 of SEQ ID NO:19).

[0040] In some embodiments, the COF1 / CRBN binding polypeptide comprises amino acid residues 136-180 and 236-249 of IKZF3 (numbered according to SEQ ID NO:19). In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:1. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:3.

[0041] In some embodiments, the COF1 / CRBN binding polypeptide comprises a first sequence comprising amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO: 19) and a second sequence comprising the amino acid sequence of MALEKMALEKMALE (SEQ ID NO: 91). In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 85.

[0042] In some embodiments, the COF1 / CRBN binding polypeptide comprises a first sequence comprising amino acid residues 136-170 of IKZF3 (numbered according to SEQ ID NO: 19) and a second sequence comprising the amino acid sequence of MALEKMALEKMALE (SEQ ID NO: 91). In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 86.

[0043] In some embodiments, the COF1 / CRBN binding polypeptide comprises at least one less lysine than the corresponding native sequence. In some embodiments, one or more lysine residues in the corresponding native sequence are replaced by a different amino acid, e.g., arginine. In some embodiments, the COF1 / CRBN binding polypeptide comprises less than 1, 2, 3, 4 or 5 lysine residues. In some embodiments, the COF1 / CRBN binding polypeptide does not comprise a lysine residue. In some embodiments, the COF1 / CRBN binding polypeptide is not ubiquitinated, e.g., in the presence of COF1, e.g., as measured by an assay described herein, optionally with ubiquitination as measured in a mammalian cell, e.g., a human cell.

[0044] In some embodiments, the COF1 / CRBN binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 41, 42, and 43. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 43.

[0045] In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the COF1 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the COF1 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 4.

[0046] In some embodiments, COF1 is an immunomodulatory imide drug (IMiD) or a pharma- ceutically acceptable salt thereof.

[0047] In some embodiments, COF1 has formula (Ia): [ka] (In the formula, Ring A is carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which independently and optionally is represented by one or more R 4 Replaced with; M is not present or C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl or C 1 ~C 6 and heteroalkyl, each alkyl, alkenyl, alkynyl, and heteroalkyl independently and optionally includes one or more R 4 Replaced with; R 2a and R 2b Each of the groups is independently hydrogen or C 1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 3a is hydrogen, C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O)x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; R 3 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; Each R 4 is independently 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D), -C(O)N(R C )(R D ), -N(R C )C(O)R A , S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 6 is independently 1 ~C 6 Alkyl, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7 are independently halo, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, cyano, -ORB , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; n is 0, 1, 2 or 3; o is 0, 1, 2, 3, 4 or 5; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, ester, hydrate or tautomer thereof.

[0048] In some embodiments of Formula (Ia), X is O. In some embodiments, M is absent. In some embodiments, Ring A is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as 2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione). In some embodiments, R 4 is oxo or OR B (For example, -OCH 3 or -OCH 2 CH 3 ) and o is 0, 1, or 2. In some embodiments, R 2a and R 2b Each of is independently hydrogen or R 2a and R 2b taken together with the carbon atom to which they are attached form a carbonyl group. In some embodiments, R 3a is heteroalkyl (e.g., -CH 2 NHC(O)CH 2 ), -N(R C )(R D ) (e.g., -NH 2 ) or -N(R C )C(O)R A (For example, -NHC(O)CH 3 In one embodiment, n is 0.

[0049] In some embodiments, COF1 is thalidomide or an analog or pharma- ceutically acceptable salt thereof.

[0050] In some embodiments, COF1 is selected from the group consisting of lenalidomide, pomalidomide, thalidomide, and 2-(4-(tert-butyl)phenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)acetamide or a pharma- ceutically acceptable salt thereof.

[0051] In some embodiments, COF1 is [ka] or a pharma- ceutically acceptable salt thereof.

[0052] In some embodiments, COF1 is lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof. In some embodiments, COF1 is lenalidomide or an analog or a pharma- ceutically acceptable salt thereof. In some embodiments, COF1 is lenalidomide or a pharma- ceutically acceptable salt thereof.

[0053] In some embodiments, the fusion polypeptide further comprises a degradation domain, wherein the degradation domain is separated from the COF1 / CRBN binding polypeptide and the heterologous polypeptide by a heterologous protease cleavage site.

[0054] In some embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus: i) degradation domains, heterologous protease cleavage sites, heterologous polypeptides and COF1 / CRBN binding polypeptides; ii) a degradation domain, a heterologous protease cleavage site, a COF1 / CRBN binding polypeptide and a heterologous polypeptide; iii) COF1 / CRBN binding polypeptides, heterologous polypeptides, heterologous protease cleavage sites and degradation domains; or iv) Heterologous Polypeptides and COF1 / CRBN Binding Polypeptides, Heterologous Protease Cleavage Sites and Degradation Domains Includes.

[0055] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, a degradation domain, a heterologous protease cleavage site, a heterologous polypeptide, and a COF1 / CRBN binding polypeptide.

[0056] In some embodiments, the degradation domain has a first state associated with a first level of expression of the fusion polypeptide and a second state associated with a second level of expression of the fusion polypeptide, the second level being increased, e.g., at least 2, 3, 4, 5, 10, 20, or 30-fold above the first level in the presence of a stabilizing compound.

[0057] In some embodiments, in the absence of a stabilizing compound, the fusion polypeptide is degraded by cellular degradative pathways, e.g., at least 50%, 60%, 70%, 80%, 90% or more of the fusion polypeptide is degraded, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis.

[0058] In some embodiments, the level of expression and / or degradation is as measured in a mammalian cell, such as a human cell.

[0059] In some embodiments, in the presence of a stabilizing compound, i) the degradation domain adopts a conformation that is more resistant to cellular degradation relative to the conformation in the absence of the stabilizing compound; or ii) the conformation of the fusion polypeptide is more tolerant to cleavage at the heterologous protease cleavage site relative to the conformation in the absence of the stabilizing compound.

[0060] In some embodiments, the degradation domain is selected from an estrogen receptor (ER) domain, an FKB protein (FKBP) domain, or a dihydrofolate reductase (DHFR) domain.

[0061] In some embodiments, the degradation domain is an estrogen receptor (ER) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 46 or 48. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 48.

[0062] In some embodiments, the stabilizing compound is bazedoxifene or 4-hydroxytamoxifen (4-OHT) or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain is an estrogen receptor (ER) domain and the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 46 and the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof.

[0063] In some embodiments, the degradation domain is an FKB protein (FKBP) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 50. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the stabilizing compound is Shield-1 or a pharma- ceutically acceptable salt thereof.

[0064] In some embodiments, the degradation domain is a dihydrofolate reductase (DHFR) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 51. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the stabilizing compound is trimethoprim or a pharma- ceutically acceptable salt thereof.

[0065] In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian intracellular protease. In some embodiments, the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, granzyme B, factor XA, enterokinase, genenase, sortase, precision protease, thrombin, TEV protease, and elastase 1 ... selected from the group consisting of a RX(K / R)R consensus motif (wherein X can be any amino acid; SEQ ID NO: 52), a RXXX[KR]R consensus motif (wherein X can be any amino acid; SEQ ID NO: 53), a RRX consensus motif (SEQ ID NO: 54), an IEPDX consensus motif (SEQ ID NO: 55), an Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 56), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 57), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 58), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 59), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 60), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 61), an Asp-Asp- In some embodiments, the heterologous protease cleavage site is cleaved by furin. In some embodiments, the heterologous protease cleavage site comprises a furin cleavage site selected from the group consisting of RTKR (SEQ ID NO: 123); GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125); GTGAEDPRPSRKRR (SEQ ID NO: 127); LQWLEQQVAKRRTKR (SEQ ID NO: 129); GTGAEDPRPSRKRRSLGG (SEQ ID NO: 131); GTGAEDPRPSRKRRSLG (SEQ ID NO: 133); SLNLTESHNSRKKR (SEQ ID NO: 135); CKINGYPKRGRKRR (SEQ ID NO: 137); and SARNRQKR (SEQ ID NO: 34).In some embodiments, the heterologous protease cleavage site does not comprise the amino acid sequence of SARNRQKR (SEQ ID NO: 34). In some embodiments, the heterologous protease cleavage site comprises a furin cleavage site of GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125). In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian extracellular protease. In some embodiments, the mammalian extracellular protease is selected from the group consisting of factor XA, enterokinase, genenase, sortase, precision protease, thrombin, TEV protease, and elastase 1. In some embodiments, the heterologous protease cleavage site comprises an amino acid sequence selected from the group consisting of Ile-Glu / Asp-Gly-Arg (SEQ ID NO:56), Asp-Asp-Asp-Asp-Lys (SEQ ID NO:57), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO:58), the LPXTG / A consensus motif (SEQ ID NO:59), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO:60), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:61), ENLYFQG (SEQ ID NO:62), and [AGSV]-X (where X can be any amino acid; SEQ ID NO:63).

[0066] In one aspect, provided herein is a fusion polypeptide comprising a first domain and a second domain separated by a heterologous protease cleavage site, the first domain comprising a degradation domain and the second domain comprising a compound of formula (II) (COF2) / CRBN binding polypeptide and a heterologous polypeptide, such as a heterologous mammalian, bacterial or viral polypeptide, the compound of formula (II) being [ka] (In the formula, X is O or S; R 1 is C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C6 Alkynyl, C 1 ~C 6 heteroalkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which may be one or more R 4 independently and optionally replaced by; R 2a and R 2b Each of the groups is independently hydrogen or C 1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 10 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E or L-tag; each alkyl, alkenyl, alkynyl and heteroalkyl is independently and optionally selected from one or more R 11 Replaced with; Each R 4 is independently 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, C(O)R A , -C(O)OR B , OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 11 is independently 1 ~C 6 Alkyl, halo, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7 are independently halo, oxo, cyano, -OR B , -N(R C )(RD ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, halo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each L is independently 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, -C(O)R A1 , -C(O)OR B1 , -OR B1 , -N(R C1 )(R D1 ), -C(O)N(R C1 )(R D1 ), -N(R C1 )C(O)R A1 , -S(O) x R E1 , -S(O) x N(R C1 )(R D1 ) or -N(R C1 )S(O) x R E1 wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 12 Replaced with; Each tag is a targeting moiety capable of binding to a target protein; R A1 , R B1 , R C1 , R D1 and R E1 each independently represents hydrogen, C 1 ~C6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 heteroalkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 12 Replaced with; Each R 12 is independently 1 ~C 6 alkyl, halo, cyano, carbocyclyl or heterocyclyl; n is 0, 1, 2, 3 or 4; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, ester, hydrate, tautomer or prodrug thereof.

[0067] In some embodiments, the degradation domain has a first state associated with a first level of expression of the fusion polypeptide and a second state associated with a second level of expression of the fusion polypeptide, the second level being increased, e.g., at least 2, 3, 4, 5, 10, 20, or 30-fold above the first level in the presence of a stabilizing compound.

[0068] In some embodiments, in the absence of a stabilizing compound, the fusion polypeptide is degraded by cellular degradative pathways, e.g., at least 50%, 60%, 70%, 80%, 90% or more of the fusion polypeptide is degraded, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis.

[0069] In some embodiments, the level of expression and / or degradation is as measured in a mammalian cell, such as a human cell.

[0070] In some embodiments, in the presence of a stabilizing compound, i) the degradation domain adopts a conformation that is more resistant to cellular degradation relative to the conformation in the absence of the stabilizing compound; or ii) the conformation of the fusion polypeptide is more tolerant to cleavage at the heterologous protease cleavage site relative to the conformation in the absence of the stabilizing compound.

[0071] In some embodiments, the degradation domain is selected from an estrogen receptor (ER) domain, an FKB protein (FKBP) domain, or a dihydrofolate reductase (DHFR) domain.

[0072] In some embodiments, the degradation domain is an estrogen receptor (ER) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 46 or 48. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 48.

[0073] In some embodiments, the stabilizing compound is bazedoxifene or 4-hydroxytamoxifen (4-OHT) or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain is an estrogen receptor (ER) domain and the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 46 and the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof.

[0074] In some embodiments, the degradation domain is an FKB protein (FKBP) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 50. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the stabilizing compound is Shield-1 or a pharma- ceutically acceptable salt thereof.

[0075] In some embodiments, the degradation domain is a dihydrofolate reductase (DHFR) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 51. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the stabilizing compound is trimethoprim or a pharma- ceutically acceptable salt thereof.

[0076] In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian intracellular protease. In some embodiments, the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, granzyme B, factor XA, enterokinase, genenase, sortase, precision protease, thrombin, TEV protease, and elastase 1 ... selected from the group consisting of a RX(K / R)R consensus motif (wherein X can be any amino acid; SEQ ID NO: 52), a RXXX[KR]R consensus motif (wherein X can be any amino acid; SEQ ID NO: 53), a RRX consensus motif (SEQ ID NO: 54), an IEPDX consensus motif (SEQ ID NO: 55), an Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 56), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 57), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 58), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 59), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 60), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 61), an Asp-Asp- In some embodiments, the heterologous protease cleavage site is cleaved by furin. In some embodiments, the heterologous protease cleavage site comprises a furin cleavage site selected from the group consisting of RTKR (SEQ ID NO: 123); GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125); GTGAEDPRPSRKRR (SEQ ID NO: 127); LQWLEQQVAKRRTKR (SEQ ID NO: 129); GTGAEDPRPSRKRRSLGG (SEQ ID NO: 131); GTGAEDPRPSRKRRSLG (SEQ ID NO: 133); SLNLTESHNSRKKR (SEQ ID NO: 135); CKINGYPKRGRKRR (SEQ ID NO: 137); and SARNRQKR (SEQ ID NO: 34).In some embodiments, the heterologous protease cleavage site does not comprise the amino acid sequence of SARNRQKR (SEQ ID NO: 34). In some embodiments, the heterologous protease cleavage site comprises a furin cleavage site of GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125). In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian extracellular protease. In some embodiments, the mammalian extracellular protease is selected from the group consisting of factor XA, enterokinase, genenase, sortase, precision protease, thrombin, TEV protease, and elastase 1. In some embodiments, the heterologous protease cleavage site comprises an amino acid sequence selected from the group consisting of Ile-Glu / Asp-Gly-Arg (SEQ ID NO:56), Asp-Asp-Asp-Asp-Lys (SEQ ID NO:57), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO:58), the LPXTG / A consensus motif (SEQ ID NO:59), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO:60), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:61), ENLYFQG (SEQ ID NO:62), and [AGSV]-X (where X can be any amino acid; SEQ ID NO:63).

[0077] In some embodiments, the degradation domain is fused to a heterologous protease cleavage site, which is further fused to a second domain.

[0078] In some embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus: i) degradation domains, heterologous protease cleavage sites, heterologous polypeptides and COF2 / CRBN binding polypeptides; ii) a degradation domain, a heterologous protease cleavage site, a COF2 / CRBN binding polypeptide and a heterologous polypeptide; iii) COF2 / CRBN binding polypeptides, heterologous polypeptides, heterologous protease cleavage sites and degradation domains; or iv) Heterologous Polypeptides and COF2 / CRBN Binding Polypeptides, Heterologous Protease Cleavage Sites and Degradation Domains Includes.

[0079] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, a degradation domain, a heterologous protease cleavage site, a heterologous polypeptide, and a COF2 / CRBN binding polypeptide. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, a degradation domain, a heterologous protease cleavage site, a COF2 / CRBN binding polypeptide, and a heterologous polypeptide. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, a COF2 / CRBN binding polypeptide, a heterologous polypeptide, a heterologous protease cleavage site, and a degradation domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, a heterologous polypeptide and a COF2 / CRBN binding polypeptide, a heterologous protease cleavage site, and a degradation domain.

[0080] In some embodiments, the association of the COF2 / CRBN binding polypeptide with cereblon (CRBN) in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the COF2 / CRBN binding polypeptide with CRBN in the presence of COF2, for example, an excess of COF2, as measured, for example, by an assay described herein, for example, by immunoprecipitation. In some embodiments, the COF2 / CRBN binding polypeptide does not bind to CRBN in the absence of COF2. In some embodiments, the association of the fusion polypeptide with CRBN in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF2, for example, an excess of COF2, as measured, for example, by an assay described herein, for example, by immunoprecipitation. In some embodiments, the fusion polypeptide does not bind to CRBN in the absence of COF2. In some embodiments, the association and / or binding is as measured in a mammalian cell, e.g., a human cell.

[0081] In some embodiments, the ubiquitination of the heterologous polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the ubiquitination of the heterologous polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein. In some embodiments, the ubiquitination of the fusion polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the ubiquitination of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein. In some embodiments, the heterologous polypeptide or fusion polypeptide is ubiquitinated at one or more lysine or methionine residues in the presence of COF2. In some embodiments, ubiquitination is as measured in a mammalian cell, such as a human cell.

[0082] In some embodiments, the degradation of the fusion polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. In some embodiments, the degradation of the fusion polypeptide is mediated by ubiquitination in the presence of COF2. In some embodiments, the degradation is as measured in mammalian cells, e.g., human cells.

[0083] In some embodiments, the COF2 / CRBN binding polypeptide is 10 to 95 amino acid residues in length, 15 to 90 amino acid residues in length, 20 to 85 amino acid residues in length, 25 to 80 amino acid residues in length, 30 to 75 amino acid residues in length, 35 to 70 amino acid residues in length, 40 to 65 amino acid residues in length, 45 to 60 amino acid residues in length, 50 to 65 amino acid residues in length, or 55 to 65 amino acid residues in length.

[0084] In some embodiments, the COF2 / CRBN binding polypeptide comprises a β-turn. In some embodiments, the COF2 / CRBN binding polypeptide comprises a β-turn of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF2 / CRBN binding polypeptide comprises a β-hairpin. In some embodiments, the COF2 / CRBN binding polypeptide comprises a β-hairpin of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF2 / CRBN binding polypeptide comprises a β-strand. In some embodiments, the COF2 / CRBN binding polypeptide comprises a β-strand of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF2 / CRBN binding polypeptide comprises an α-helix. In some embodiments, the COF2 / CRBN binding polypeptide comprises an α-helix of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF2 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, and a first α-helix. In some embodiments, the COF2 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, and a first α-helix of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the COF2 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, a first α-helix, and a second α-helix. In some embodiments, a COF2 / CRBN binding polypeptide comprises, from N-terminus to C-terminus, a first β-strand, a β-hairpin, a second β-strand, a first α-helix, and a second α-helix of IKZF1 or IKZF3 (e.g., human IKZF1 or IKZF3). In some embodiments, the β-hairpin and the second α-helix are separated by no more than 60, 50, 40, or 30 amino acid residues.

[0085] In some embodiments, the COF2 / CRBN binding polypeptide comprises a COF2 / CRBN binding sequence derived from a naturally occurring polypeptide or a COF2 / CRBN binding variant thereof. In some embodiments, the COF2 / CRBN binding polypeptide comprises a COF2 / CRBN binding sequence derived from a naturally occurring IKZF polypeptide or a COF2 / CRBN binding variant thereof. In some embodiments, the COF2 / CRBN binding polypeptide comprises a COF2 / CRBN binding sequence derived from a naturally occurring IKZF1, IKZF2, IKZF3, IKZF4, or IKZF5, or a COF2 / CRBN binding variant thereof. In some embodiments, the COF2 / CRBN binding sequence comprises two or more non-contiguous sequences derived from a naturally occurring polypeptide, such as a naturally occurring IKZF polypeptide, such as a naturally occurring IKZF1, IKZF2, IKZF3, IKZF4, or IKZF5.

[0086] In some embodiments, the COF2 / CRBN binding polypeptide comprises an IKZF polypeptide or a structural motif thereof.

[0087] In some embodiments, the IKZF polypeptide is an IKZF1 polypeptide, an IKZF3 polypeptide, an IKZF2 polypeptide having an H141Q substitution (numbered according to SEQ ID NO:21), or an IKZF4 polypeptide having an H188Q substitution (numbered according to SEQ ID NO:22).

[0088] In some embodiments, the COF2 / CRBN binding polypeptide is i) the association of a COF2 / CRBN binding polypeptide with CRBN in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of a COF2 / CRBN binding polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It contains sufficient amino acid sequence and / or structural motifs derived from an IKZF (eg, IKZF1 or IKZF3).

[0089] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0090] In some embodiments, the COF2 / CRBN binding polypeptide comprises about 10 to about 95 amino acid residues, about 15 to about 90 amino acid residues, about 20 to about 85 amino acid residues, about 25 to about 80 amino acid residues, about 30 to about 75 amino acid residues, about 35 to about 70 amino acid residues, about 40 to about 65 amino acid residues, about 45 to about 65 amino acid residues, about 50 to about 65 amino acid residues, or about 55 to about 65 amino acid residues of IKZF1 or IKZF3.

[0091] In some embodiments, the COF2 / CRBN binding polypeptide is i) the association of a COF2 / CRBN binding polypeptide with CRBN in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of a COF2 / CRBN binding polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 180 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 180 of SEQ ID NO:19).

[0092] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0093] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 136-180 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:5) or a sequence that differs from amino acid residues 136-180 (numbered according to SEQ ID NO:19) of IKZF3 by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid residues (e.g., a sequence that differs from amino acid residues 136-180 of SEQ ID NO:19 by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid residues) (e.g., a sequence that has 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid substitutions from amino acid residues 136-180 of SEQ ID NO:19).

[0094] In some embodiments, the COF2 / CRBN binding polypeptide is i) the association of a COF2 / CRBN binding polypeptide with CRBN in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of a COF2 / CRBN binding polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 170 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136 to 170 of SEQ ID NO:19).

[0095] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0096] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 136-170 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:6) or a sequence that differs from amino acid residues 136-170 (numbered according to SEQ ID NO:19) of IKZF3 by 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 or fewer amino acid residues (e.g., a sequence that differs from amino acid residues 136-170 of SEQ ID NO:19 by 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 or fewer amino acid residues) (e.g., a sequence that has 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 or fewer amino acid substitutions from amino acid residues 136-170 of SEQ ID NO:19).

[0097] In some embodiments, one, two, three or all of the following amino acid residues remain unchanged: glutamine at position 147, cysteine ​​at position 148, glutamine at position 150, glycine at position 152, leucine at position 161 or leucine at position 162, numbered according to SEQ ID NO: 19. In some embodiments, the glutamine at position 147, numbered according to SEQ ID NO: 19, remains unchanged. In some embodiments, the cysteine ​​at position 148, numbered according to SEQ ID NO: 19, remains unchanged. In some embodiments, the glutamine at position 150, numbered according to SEQ ID NO: 19, remains unchanged. In some embodiments, the glycine at position 152, numbered according to SEQ ID NO: 19, remains unchanged. In some embodiments, the leucine at position 161, numbered according to SEQ ID NO: 19, remains unchanged. In some embodiments, the leucine at position 162, numbered according to SEQ ID NO: 19, remains unchanged.

[0098] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 136-139 of IKZF3 (numbered according to SEQ ID NO:19), e.g., the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:40.

[0099] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO:19). In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:77. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:5. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:5.

[0100] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 136-170 of IKZF3 (numbered according to SEQ ID NO:19). In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:78. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:6. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:78.

[0101] In some embodiments, the COF2 / CRBN binding polypeptide is i) the association of a COF2 / CRBN binding polypeptide with CRBN in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of a COF2 / CRBN binding polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 236 to 249 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 236 to 249 of SEQ ID NO:19).

[0102] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0103] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 236-249 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:11) or a sequence that differs from amino acid residues 236-249 (numbered according to SEQ ID NO:19) of IKZF3 by no more than 1, 2, 3, 4, 5, 6, or 7 amino acid residues (e.g., a sequence that differs from amino acid residues 236-249 of SEQ ID NO:19 by no more than 1, 2, 3, 4, 5, 6, or 7 amino acid residues) (e.g., a sequence that has no more than 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions from amino acid residues 236-249 of SEQ ID NO:19).

[0104] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 236-249 of IKZF3 (numbered according to SEQ ID NO: 19). In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:11.

[0105] In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:91.

[0106] In some embodiments, the COF2 / CRBN binding polypeptide is i) the association of a COF2 / CRBN binding polypeptide with CRBN in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of a COF2 / CRBN binding polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the heterologous polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; iv) ubiquitination of the fusion polypeptide in the absence of COF2 is, e.g., 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, e.g., by an assay described herein; or v) degradation of the fusion polypeptide in the absence of COF2 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or less than 70% of the degradation of the fusion polypeptide in the presence of COF2, e.g., an excess of COF2, as measured, for example, by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; It includes a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136-180 and 236-249 (numbered according to SEQ ID NO:19) of IKZF3 (e.g., a sufficient amino acid sequence and / or structural motif derived from amino acid residues 136-180 and 236-249 of SEQ ID NO:19).

[0107] In some embodiments, the association, ubiquitination and / or degradation is as measured in a mammalian cell, such as a human cell.

[0108] In some embodiments, the COF2 / CRBN-binding polypeptide comprises a first sequence that includes amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO:19) (e.g., a first sequence that includes the amino acid sequence of SEQ ID NO:5) or a first sequence that differs from amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO:19) by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid residues (e.g., a first sequence that differs from amino acid residues 136-180 of SEQ ID NO:19 by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 or fewer amino acid residues). a first sequence that differs by not more than 30, 35 or 40 amino acid residues; and a second sequence comprising amino acid residues 236 to 249 of IKZF3 (numbered according to SEQ ID NO:19) (e.g., a second sequence comprising the amino acid sequence of SEQ ID NO:11) or a second sequence that differs by not more than 1, 2, 3, 4, 5, 6 or 7 amino acid residues from amino acid residues 236 to 249 of IKZF3 (numbered according to SEQ ID NO:19) (e.g., a second sequence that differs by not more than 1, 2, 3, 4, 5, 6 or 7 amino acid residues from amino acid residues 236 to 249 of SEQ ID NO:19).

[0109] In some embodiments, the COF2 / CRBN binding polypeptide comprises amino acid residues 136-180 and 236-249 of IKZF3 (numbered according to SEQ ID NO:19). In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:1. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO:3.

[0110] In some embodiments, the COF2 / CRBN binding polypeptide comprises a first sequence comprising amino acid residues 136-180 of IKZF3 (numbered according to SEQ ID NO: 19) and a second sequence comprising the amino acid sequence of MALEKMALEKMALE (SEQ ID NO: 91). In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 85.

[0111] In some embodiments, the COF2 / CRBN binding polypeptide comprises a first sequence comprising amino acid residues 136-170 of IKZF3 (numbered according to SEQ ID NO: 19) and a second sequence comprising the amino acid sequence of MALEKMALEKMALE (SEQ ID NO: 91). In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 86.

[0112] In some embodiments, the COF2 / CRBN binding polypeptide comprises at least one less lysine than the corresponding native sequence. In some embodiments, one or more lysine residues in the corresponding native sequence are replaced by a different amino acid, e.g., arginine. In some embodiments, the COF2 / CRBN binding polypeptide comprises less than 1, 2, 3, 4 or 5 lysine residues. In some embodiments, the COF2 / CRBN binding polypeptide does not comprise a lysine residue. In some embodiments, the COF2 / CRBN binding polypeptide is not ubiquitinated, e.g., in the presence of COF2, e.g., as measured by an assay described herein, optionally with ubiquitination as measured in a mammalian cell, e.g., a human cell.

[0113] In some embodiments, the COF2 / CRBN binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 41, 42, and 43. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 43.

[0114] In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the COF2 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the COF2 / CRBN binding polypeptide consists of the amino acid sequence of SEQ ID NO: 4.

[0115] In some embodiments, COF2 is an immunomodulatory imide drug (IMiD) or a pharma- ceutically acceptable salt thereof.

[0116] In some embodiments, the COF2 has formula (I): [ka] (In the formula, X is O or S; R 1 is C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 heteroalkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which may be one or more R 4 independently and optionally replaced by; R 2a and R 2b Each of the groups is independently hydrogen or C 1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 3 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(RC )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; Each R 4 is independently 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 6 is independently 1 ~C 6 Alkyl, oxo, cyano, -OR B , -N(R C )(R D), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7 are independently halo, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; n is 0, 1, 2, 3 or 4; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, ester, hydrate, solvate or tautomer thereof.

[0117] In some embodiments, the COF2 has the formula (Ia): [ka] (In the formula, Ring A is carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which independently and optionally is represented by one or more R 4 Replaced with; M is not present or C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6Alkynyl or C 1 ~C 6 and heteroalkyl, each alkyl, alkenyl, alkynyl, and heteroalkyl independently and optionally includes one or more R 4 Replaced with; R 2a and R 2b Each of the groups is independently hydrogen or C 1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 3a is hydrogen, C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; R 3 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; Each R 4 is independently 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 6 is independently 1 ~C 6 Alkyl, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7 are independently halo, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; n is 0, 1, 2 or 3; o is 0, 1, 2, 3, 4 or 5; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, ester, hydrate or tautomer thereof.

[0118] In some embodiments, COF2 is thalidomide or an analog or pharma- ceutically acceptable salt thereof. In some embodiments, COF2 is selected from the group consisting of lenalidomide, pomalidomide, thalidomide, and 2-(4-(tert-butyl)phenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)acetamide or a pharma- ceutically acceptable salt thereof.

[0119] In some embodiments, the COF2 is [ka] or a pharma- ceutically acceptable salt thereof.

[0120] In some embodiments, COF2 is lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof. In some embodiments, COF2 is lenalidomide or an analog or pharma- ceutically acceptable salt thereof. In some embodiments, COF2 is lenalidomide or a pharma- ceutically acceptable salt thereof.

[0121] In some embodiments, the COF2 has formula (I): [ka] (In the formula, X is O or S; R 1 is C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 heteroalkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which may be one or more R 4 independently and optionally replaced by; R 2a and R 2b Each of the groups is independently hydrogen or C1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 3 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; Each R 4 is independently 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(RC )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 6 is independently 1 ~C 6 Alkyl, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7 are independently halo, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, cyano, -OR B , -N(R C )(RD ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; n is 0, 1, 2, 3 or 4; and x is 0, 1 or 2. or the structure of a pharma- ceutically acceptable salt, ester, hydrate, solvate or tautomer thereof.

[0122] In some embodiments, the COF2 has the formula (Ia): [ka] (In the formula, Ring A is carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which independently and optionally is represented by one or more R 4 Replaced with; M is not present or C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl or C 1 ~C 6 and heteroalkyl, each alkyl, alkenyl, alkynyl, and heteroalkyl independently and optionally includes one or more R 4 Replaced with; R 2a and R 2b Each of the groups is independently hydrogen or C 1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 3a is hydrogen, C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; R 3 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; Each R 4 is independently1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 6 is independently 1 ~C 6 Alkyl, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7 are independently halo, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; n is 0, 1, 2 or 3; o is 0, 1, 2, 3, 4 or 5; and x is 0, 1 or 2. or the structure of a pharma- ceutically acceptable salt, ester, hydrate or tautomer thereof.

[0123] In some embodiments, COF2 comprises an immunomodulatory imide drug (IMiD) or a pharma- ceutically acceptable salt thereof. In some embodiments, COF2 comprises thalidomide or an analog or pharma- ceutically acceptable salt thereof. In some embodiments, COF2 comprises lenalidomide, pomalidomide, thalidomide, and 2-(4-(tert-butyl)phenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)acetamide or a pharma- ceutically acceptable salt thereof.

[0124] In some embodiments, the COF2 is [ka] or a pharma- ceutically acceptable salt thereof.

[0125] In some embodiments, COF2 comprises lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof. In some embodiments, COF2 comprises lenalidomide or an analog or a pharma- ceutically acceptable salt thereof. In some embodiments, COF2 comprises lenalidomide or a pharma- ceutically acceptable salt thereof.

[0126] In some embodiments, the COF2 is a ligand (e.g., R 10 In some embodiments, R of formula (II) further comprises an L-tag. 10 is an L-tag, L is a linker selected from the linkers disclosed in WO 2017 / 024318 (e.g., Figures 28-31), and the tag is selected from the dTAG targeting ligands disclosed in WO 2017 / 024318 (e.g., Table T, pages 119-129). In some embodiments, COF2 comprises an IMiD (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) and a ligand, and the IMiD (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) is linked to the ligand, e.g., via a linker. In some embodiments, the COF2 / CRBN binding polypeptide binds to a ligand, and the binding between the COF2 / CRBN binding polypeptide and an IMiD (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) in the absence of the ligand is 0.0001, 0.001, 0.01, 0.1, 1 or 10% or less of the binding between the COF2 / CRBN binding polypeptide and the ligand, e.g., the COF2 / CRBN binding polypeptide does not bind to an IMiD (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof), and optionally the COF2 / CRBN binding polypeptide is selected from the dTAGs disclosed in WO2017 / 024318 (e.g., pages 36-65).

[0127] In one aspect, provided herein is a fusion polypeptide comprising a compound of formula (III) (COF3) / CRBN binding polypeptide and a heterologous polypeptide, the compound of formula (III) being [ka] (In the formula, X 1 CR 3 and; [ka] X 1 CR 3 and R 3 is optionally a double bond when not present; Each R 1 is independently 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 1 ~C 6 hydroxyalkyl or halo, or The Two R's 1 together with the carbon atom to which they are attached form a 5- or 6-membered heterocyclyl ring, or The Two R's 1 If they are on adjacent atoms, they are combined with the atom to which they are bonded. 6 ~C 10 forming an aryl or a 5- or 6-membered heteroaryl ring containing 1 to 3 heteroatoms selected from O, N and S; R 2 is hydrogen, C 1 ~C 6 Alkyl, -C(O)C 1 ~C 6 Alkyl, -C(O)(CH 2 ) 0~3 -C 6 ~C 10 Aryl, -C(O)O(CH 2 ) 0~3 -C 6 ~C 10 Aryl, C 6 ~C 10aryl or 5- or 6-membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S; C 3 ~C 8 carbocyclyl or 5-7-heterocyclyl containing 1-3 heteroatoms selected from O, N and S, and alkyl optionally contains one or more R 4 and aryl, heteroaryl, carbocyclyl and heterocyclyl are optionally substituted with one or more R 5 or R 1 and R 2 when on adjacent atoms, form a 5- or 6-membered heterocyclyl ring together with the atom to which they are attached; R 3 is hydrogen, or [ka] When is a double bond, R 3 does not exist; Each R 4 is -C(O)OR 6 , -C(O)NR 6 R 6’ , -NR 6 C(O)R 6’ , halo, -OH, -NH 2 , Cyano, C. 6 ~C 10 aryl, 5- or 6-membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S; C 3 ~C 8 and aryl, heteroaryl, carbocyclyl, and heterocyclyl are independently selected from 5-7 membered heterocyclyl rings containing 1-3 heteroatoms selected from O, N, and S, and the aryl, heteroaryl, carbocyclyl, and heterocyclyl are optionally selected from one or more R 7 Replaced with; Each R 5 is C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6Alkoxy, C 1 ~C 6 Haloalkyl, C 1 ~C 6 Haloalkoxy, C 1 ~C 6 Hydroxyalkyl, halo, -OH, -NH 2 , Cyano, C. 3 ~C 7 carbocyclyl, 5- to 7-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, C 6 ~C 10 aryl and 5- or 6-membered heteroaryl containing 1-3 heteroatoms selected from O, N and S; or The Two R's 5 If they are on adjacent atoms, they are combined with the atom to which they are bonded. 6 ~C 10 aryl or a 5- or 6-membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S, optionally with one or more R 10 or The Two R's 5 If they are on adjacent atoms, they are combined with the atom to which they are bonded. 5 ~C 7 carbocyclyl or a 5- to 7-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, and optionally one or more R 10 Replaced with; R 6 and R 6’ are each independently hydrogen, C 1 ~C 6 Alkyl or C 6 ~C 10 is aryl; Each R 7 is C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Haloalkyl, C1 ~C 6 Haloalkoxy, -C(O)R 8 , -(CH 2 ) 0~3 C(O)OR 8 , -C(O)NR 8 R 9 , -NR 8 C(O)R 9 , -NR 8 C(O)OR 9 , -S(O) p NR 8 R 9 , -S(O) p R 12 , (C 1 ~C 6 ) hydroxyalkyl, halo, -OH, -O(CH 2 ) 1~3 CN, -NH 2 , cyano, -O(CH 2 ) 0~3 -C 6 ~C 10 -O(CH) containing 1 to 3 heteroatoms selected from aryl, adamantyl, O, N, and S 2 ) 0~3 - 5- or 6-membered heteroaryl, C 6 ~C 10 aryl, monocyclic or bicyclic 5-10 membered heteroaryl containing 1-3 heteroatoms selected from O, N and S; C 3 ~C 7 carbocyclyl and 5-7 membered heterocyclyl containing 1-3 heteroatoms selected from O, N and S, and alkyl is optionally selected from one or more R 11 and aryl, heteroaryl and heterocyclyl are optionally substituted with halogen, C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl and C 1 ~C 6 substituted with one or more substituents each independently selected from alkoxy; The Two R's 7 form a=(O) together with the carbon atom to which they are attached, or The Two R's 7 If they are on adjacent atoms, they are combined with the atom to which they are bonded. 6 ~C 10 aryl or a 5- or 6-membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S, optionally with one or more R 10 or The Two R's 7 C along with the atoms to which they are attached 5 ~C 7 carbocyclyl or a 5- to 7-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, and optionally one or more R 10 Replaced with; R 8 and R 9 are each independently hydrogen or C 1 ~C 6 is alkyl; Each R 10 is C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Haloalkyl, C 1 ~C 6 Haloalkoxy, C 1 ~C 6 Hydroxyalkyl, halo, -OH, -NH 2 and cyano; or The Two R's 10 form a=(O) together with the carbon atom to which they are attached; Each R 11 Cyano, C 1 ~C 6 Alkoxy, C 6 ~C 10 aryl and 5-7 membered heterocyclyl containing 1-3 heteroatoms selected from O, N and S, each aryl and heterocyclyl optionally being selected from C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C6 Haloalkyl, C 1 ~C 6 Haloalkoxy, C 1 ~C 6 Hydroxyalkyl, halo, -OH, -NH 2 and cyano; R 12 is C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 6 ~C 10 aryl or a 5-7 membered heterocyclyl containing 1-3 heteroatoms selected from O, N and S; R x is hydrogen or deuterium; p is 0, 1 or 2; n is 0, 1 or 2; y is 1 or 2, where n+y≦3; and q is 0, 1, 2, 3 or 4. or a pharma- ceutically acceptable salt, ester, hydrate, solvate or tautomer thereof.

[0128] In one embodiment, the compound of formula (III) has the formula (III-b): [ka] (In the formula, X 1 , R 1 , R 2 , n, q and subvariables thereof are defined as described above for formula (III). or a pharma- ceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0129] In one embodiment, the compound of formula (III) has the formula (III-d): [ka] (In the formula, R 1 , R 2, q and its subvariables are defined as described above for formula (III). or a pharma- ceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0130] In one embodiment, the COF3 / CRBN binding polypeptide is fused to a heterologous polypeptide. In one embodiment, the COF3 / CRBN binding polypeptide and the heterologous polypeptide are linked by a peptide bond. In one embodiment, the COF3 / CRBN binding polypeptide and the heterologous polypeptide are linked by a bond other than a peptide bond. In one embodiment, the heterologous polypeptide is directly linked to the COF3 / CRBN binding polypeptide. In one embodiment, the heterologous polypeptide is indirectly linked to the COF3 / CRBN binding polypeptide. In one embodiment, the COF3 / CRBN binding polypeptide and the heterologous polypeptide are operably linked via a linker, e.g., a glycine-serine linker, e.g., a linker comprising the amino acid sequence of SEQ ID NO: 28. In one embodiment, the COF3 / CRBN binding polypeptide is linked to the C-terminus of the heterologous polypeptide. In one embodiment, the COF3 / CRBN binding polypeptide is linked to the N-terminus of the heterologous polypeptide.

[0131] In one embodiment, the association of the fusion polypeptide with CRBN in the absence of COF3 is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15% or 20% or less of the association of the fusion polypeptide with CRBN in the presence of COF3, for example, an excess of COF3, as measured, for example, by an assay described herein, for example, by immunoprecipitation. In one embodiment, the fusion polypeptide does not bind to CRBN in the absence of COF3. In one embodiment, the ubiquitination of the fusion polypeptide in the absence of COF3 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less of the ubiquitination of the fusion polypeptide in the presence of COF3, for example, an excess of COF3, as measured, for example, by an assay described herein. In one embodiment, the fusion polypeptide is ubiquitinated at one or more lysine or methionine residues in the presence of COF3. In one embodiment, the degradation of the fusion polypeptide in the absence of COF3 is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less than the degradation of the fusion polypeptide in the presence of COF3, for example, an excess of COF3, as measured, for example, by an assay described herein, for example, Western blot analysis or flow cytometry analysis. In one embodiment, the degradation of the fusion polypeptide is mediated by ubiquitination in the presence of COF3. In one embodiment, the association, ubiquitination and / or degradation is as measured in mammalian cells, for example human cells.

[0132] In one embodiment, the COF3 / CRBN binding polypeptide is 10 to 95 amino acid residues long, 15 to 90 amino acid residues long, 20 to 85 amino acid residues long, 25 to 80 amino acid residues long, 30 to 75 amino acid residues long, 35 to 70 amino acid residues long, 40 to 65 amino acid residues long, 45 to 60 amino acid residues long, 50 to 65 amino acid residues long, or 55 to 65 amino acid residues long. In one embodiment, the COF3 / CRBN binding polypeptide is 59 amino acid residues long.

[0133] In one embodiment, the COF3 / CRBN binding polypeptide comprises a β-turn. In one embodiment, the COF3 / CRBN binding polypeptide comprises a β-hairpin. In one embodiment, the COF3 / CRBN binding polypeptide comprises a β-strand. In one embodiment, the COF3 / CRBN binding polypeptide comprises an α-helix. In one embodiment, the COF3 / CRBN binding polypeptide comprises, from the N-terminus to the C-terminus, a first β-strand, a β-hairpin, a second β-strand, and a first α-helix. In one embodiment, the COF3 / CRBN binding polypeptide comprises, from the N-terminus to the C-terminus, a first β-strand, a β-hairpin, a second β-strand, a first α-helix, and a second α-helix. In one embodiment, the β-hairpin and the second α-helix are separated by no more than 60, 50, 40, or 30 amino acid residues.

[0134] In one embodiment, the COF3 / CRBN binding polypeptide comprises a COF3 / CRBN binding sequence derived from a naturally occurring polypeptide or a COF3 / CRBN binding variant thereof. In one embodiment, the COF3 / CRBN binding polypeptide comprises a COF3 / CRBN binding sequence derived from a naturally occurring IKZF polypeptide or a COF3 / CRBN binding variant thereof. In one embodiment, the COF3 / CRBN binding polypeptide comprises a COF3 / CRBN binding sequence derived from a naturally occurring IKZF2 or a COF3 / CRBN binding variant thereof. In one embodiment, the COF3 / CRBN binding sequence comprises two or more non-contiguous sequences derived from a naturally occurring IKZF polypeptide, such as a naturally occurring IKZF2.

[0135] In one embodiment, the COF3 / CRBN binding polypeptide comprises amino acid residues 130-174 of IKZF2 (numbered according to SEQ ID NO:21). In one embodiment, the COF3 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:113. In one embodiment, the COF3 / CRBN binding polypeptide comprises a sequence that differs from amino acid residues 130-174 of IKZF2 (numbered according to SEQ ID NO:21) by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 amino acid residues. In one embodiment, the COF3 / CRBN binding polypeptide comprises a sequence that differs from amino acid residues 130-174 of SEQ ID NO:21 by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 amino acid residues. In one embodiment, the COF3 / CRBN binding polypeptide comprises a sequence from amino acid residues 130-174 of SEQ ID NO:21 with up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 amino acid substitutions.

[0136] In one embodiment, the COF3 / CRBN binding polypeptide comprises amino acid residues 230-243 of IKZF2 (numbered according to SEQ ID NO:21). In one embodiment, the COF3 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:114. In one embodiment, the COF3 / CRBN binding polypeptide comprises a sequence that differs from amino acid residues 230-243 of IKZF2 (numbered according to SEQ ID NO:21) by 1, 2, 3, 4, or no more than 10 amino acid residues. In one embodiment, the COF3 / CRBN binding polypeptide comprises a sequence that differs from amino acid residues 230-243 of SEQ ID NO:21 by 1, 2, 3, 4, or no more than 10 amino acid residues. In one embodiment, the COF3 / CRBN binding polypeptide comprises a sequence having 1, 2, 3, 4, or no more than 10 amino acid substitutions from amino acid residues 230-243 of SEQ ID NO:21.

[0137] In one embodiment, the histidine at position 141, numbered according to SEQ ID NO:21, remains unchanged.

[0138] In one embodiment, the COF3 / CRBN binding polypeptide comprises amino acid residues 130-174 of IKZF2 (numbered according to SEQ ID NO:21). In one embodiment, the COF3 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:113. In one embodiment, the COF3 / CRBN binding polypeptide comprises amino acid residues 230-243 of IKZF2 (numbered according to SEQ ID NO:21). In one embodiment, the COF3 / CRBN binding polypeptide comprises the amino acid sequence of SEQ ID NO:114. In one embodiment, the COF3 / CRBN binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:109.

[0139] In some embodiments, the fusion polypeptide further comprises a degradation domain, wherein the degradation domain is separated from the COF3 / CRBN binding polypeptide and the heterologous polypeptide by a heterologous protease cleavage site.

[0140] In some embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus: i) degradation domains, heterologous protease cleavage sites, heterologous polypeptides and COF3 / CRBN binding polypeptides; ii) a degradation domain, a heterologous protease cleavage site, a COF3 / CRBN binding polypeptide and a heterologous polypeptide; iii) COF3 / CRBN binding polypeptides, heterologous polypeptides, heterologous protease cleavage sites and degradation domains; or iv) Heterologous Polypeptides and COF3 / CRBN Binding Polypeptides, Heterologous Protease Cleavage Sites and Degradation Domains Includes.

[0141] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, a degradation domain, a heterologous protease cleavage site, a heterologous polypeptide, and a COF3 / CRBN binding polypeptide.

[0142] In some embodiments, the degradation domain has a first state associated with a first level of expression of the fusion polypeptide and a second state associated with a second level of expression of the fusion polypeptide, the second level being increased, e.g., at least 2, 3, 4, 5, 10, 20, or 30-fold above the first level in the presence of a stabilizing compound.

[0143] In some embodiments, in the absence of a stabilizing compound, the fusion polypeptide is degraded by cellular degradative pathways, e.g., at least 50%, 60%, 70%, 80%, 90% or more of the fusion polypeptide is degraded, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis.

[0144] In some embodiments, the level of expression and / or degradation is as measured in a mammalian cell, such as a human cell.

[0145] In some embodiments, in the presence of a stabilizing compound, i) the degradation domain adopts a conformation that is more resistant to cellular degradation relative to the conformation in the absence of the stabilizing compound; or ii) the conformation of the fusion polypeptide is more tolerant to cleavage at the heterologous protease cleavage site relative to the conformation in the absence of the stabilizing compound.

[0146] In some embodiments, the degradation domain is selected from an estrogen receptor (ER) domain, an FKB protein (FKBP) domain, or a dihydrofolate reductase (DHFR) domain.

[0147] In some embodiments, the degradation domain is an estrogen receptor (ER) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 46 or 48. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 48.

[0148] In some embodiments, the stabilizing compound is bazedoxifene or 4-hydroxytamoxifen (4-OHT) or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain is an estrogen receptor (ER) domain and the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 46 and the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof.

[0149] In some embodiments, the degradation domain is an FKB protein (FKBP) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 50. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the stabilizing compound is Shield-1 or a pharma- ceutically acceptable salt thereof.

[0150] In some embodiments, the degradation domain is a dihydrofolate reductase (DHFR) domain. In some embodiments, the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 51. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the stabilizing compound is trimethoprim or a pharma- ceutically acceptable salt thereof.

[0151] In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian intracellular protease. In some embodiments, the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, granzyme B, factor XA, enterokinase, genenase, sortase, precision protease, thrombin, TEV protease, and elastase 1 ... selected from the group consisting of a RX(K / R)R consensus motif (wherein X can be any amino acid; SEQ ID NO: 52), a RXXX[KR]R consensus motif (wherein X can be any amino acid; SEQ ID NO: 53), a RRX consensus motif (SEQ ID NO: 54), an IEPDX consensus motif (SEQ ID NO: 55), an Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 56), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 57), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 58), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 59), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 60), an Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 61), an Asp-Asp- In some embodiments, the heterologous protease cleavage site is cleaved by furin. In some embodiments, the heterologous protease cleavage site comprises a furin cleavage site selected from the group consisting of RTKR (SEQ ID NO: 123); GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125); GTGAEDPRPSRKRR (SEQ ID NO: 127); LQWLEQQVAKRRTKR (SEQ ID NO: 129); GTGAEDPRPSRKRRSLGG (SEQ ID NO: 131); GTGAEDPRPSRKRRSLG (SEQ ID NO: 133); SLNLTESHNSRKKR (SEQ ID NO: 135); CKINGYPKRGRKRR (SEQ ID NO: 137); and SARNRQKR (SEQ ID NO: 34).In some embodiments, the heterologous protease cleavage site does not comprise the amino acid sequence of SARNRQKR (SEQ ID NO: 34). In some embodiments, the heterologous protease cleavage site comprises a furin cleavage site of GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125). In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian extracellular protease. In some embodiments, the mammalian extracellular protease is selected from the group consisting of factor XA, enterokinase, genenase, sortase, precision protease, thrombin, TEV protease, and elastase 1. In some embodiments, the heterologous protease cleavage site comprises an amino acid sequence selected from the group consisting of Ile-Glu / Asp-Gly-Arg (SEQ ID NO:56), Asp-Asp-Asp-Asp-Lys (SEQ ID NO:57), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO:58), the LPXTG / A consensus motif (SEQ ID NO:59), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO:60), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:61), ENLYFQG (SEQ ID NO:62), and [AGSV]-X (where X can be any amino acid; SEQ ID NO:63).

[0152] In certain embodiments of the foregoing aspects, the heterologous polypeptide is selected from a cytoplasmic and / or nuclear polypeptide or a transmembrane polypeptide, e.g., a heterologous polypeptide in Table 2. In some embodiments, the cytoplasmic and / or nuclear polypeptide is selected from the group consisting of a component of the apoptotic pathway (e.g., caspase 9), a component of the CRISPR / Cas system (e.g., Cas9), a transcription factor (e.g., MITF, c-Myc, STAT3, STAT5, NF-kappaB, beta-catenin, Notch, GLI, or c-JUN), Tet methylcytosine dioxygenase 2 (TET2), FKBP, and tau. In some embodiments, the transmembrane polypeptide is selected from the group consisting of CD62L, CCR1, CCR2, CCR5, CCR7, CCR10, CXCR2, CXCR3, CXCR4, CXCR6, CTLA4, PD1, BTLA, VISTA, CD137L, CD80, CD86, TIGIT, CD3, CD8, CD19, CD22, CD20, BCMA, and chimeric antigen receptor (CAR). In some embodiments, the heterologous polypeptide is selected from the group consisting of a chimeric antigen receptor (CAR), a component of a CRISPR / Cas system (e.g., Cas9), CD8, CD19, and CD22.

[0153] In some embodiments, the heterologous polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises, from N-terminal to C-terminal, an antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain comprises one or more primary signaling domains. In some embodiments, the intracellular signaling domain comprises one or more costimulatory signaling domains. In some embodiments, one of the one or more primary signaling domains comprises a CD3-zeta stimulatory domain. In some embodiments, one or more of the costimulatory signaling domains are intracellular domains derived from a costimulatory protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds to CD83. In some embodiments, one or more of the costimulatory signaling domains comprises a 4-1BB costimulatory domain. In some embodiments, one or more of the costimulatory signaling domains comprises a CD28 costimulatory domain. In some embodiments, the antigen binding domain is an scFv.

[0154] In some embodiments, the antigen binding domain is selected from the group consisting of CD19; CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule-1, CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family members; B-cell maturation antigen; Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2; mesothelin; interleukin interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific fetal antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor mutated 2 (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta, 9 (LMP2); glycoprotein 100 (gp100); breakpoint cluster region (BCR) and ephB2 (EBV) oncogene polypeptide consisting of Luson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial cell marker 1 (TEM1 / CD248);tumor endothelial cell marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary glandular differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB 3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survival; telomerase; prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells; rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma metastasis breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1);CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukemia immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the antigen is selected from CD19, CD22, BCMA, CD20, CD123, EGFRvIII, or mesothelin. In some embodiments, the antigen is CD19. In some embodiments, the antigen is CD22. In some embodiments, the antigen is BCMA. In some embodiments, the antigen is CD20. In some embodiments, the antigen is CD123. In some embodiments, the antigen is EGFRvIII.

[0155] In one aspect, provided herein is a nucleic acid molecule encoding the fusion polypeptide disclosed herein.In another aspect, provided herein is a vector comprising the nucleic acid molecule.In some embodiments, the vector is a viral vector.In some embodiments, the vector is a lentiviral vector.In another aspect, provided herein is a viral particle comprising the vector.

[0156] In another aspect, provided herein is a cell, e.g., a host cell, comprising a fusion polypeptide disclosed herein, a nucleic acid molecule disclosed herein, or a vector disclosed herein. In some embodiments, the cell, e.g., the host cell, is a mammalian cell, e.g., a human cell, e.g., a human effector cell, e.g., a human T cell or a human NK cell.

[0157] In some embodiments, the cell, e.g., the host cell, is a CAR-expressing cell, e.g., a CAR-T cell. In some embodiments, the cell, e.g., the host cell, comprises components of a CRISPR / Cas system. In some embodiments, the cell, e.g., the host cell, is a human cancer cell, e.g., a human tumor cell.

[0158] In some embodiments, the cell, e.g., a host cell, comprises a ubiquitin ligase complex, e.g., an E3 ubiquitin ligase complex, and the ubiquitin ligase complex comprises CRBN.

[0159] In some embodiments, the cells comprise a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN binding polypeptide and a heterologous polypeptide), and when the cells are contacted with COF1, e.g., an excess of COF1, i) the association of the COF1 / CRBN binding polypeptide with CRBN is increased by at least, e.g., 10, 50, 100, 1000, or 10000 fold compared to the association of the COF1 / CRBN binding polypeptide with CRBN when the cell is not contacted with COF1, as measured, e.g., by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN is increased by at least, e.g., 10, 50, 100, 1000, or 10,000 fold compared to the association of the fusion polypeptide with CRBN when the cell is not contacted with COF1, e.g., as measured by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to ubiquitination of the heterologous polypeptide when the cell is not contacted with COF1, e.g., as measured by an assay described herein; iv) ubiquitination of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to ubiquitination of the fusion polypeptide when the cell is not contacted with COF1, e.g., as measured by an assay described herein; v) degradation of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to degradation of the fusion polypeptide when the cell is not contacted with COF1, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; or vi) The expression level of the fusion polypeptide is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less compared to the expression level of the fusion polypeptide when the cell is not contacted with COF1, as measured, for example, by an assay described herein, such as Western blot analysis or flow cytometry analysis.

[0160] In some embodiments, the cells further comprise a COF1, such as lenalidomide or pomalidomide, or a pharma- ceutically acceptable salt thereof.

[0161] In some embodiments, the cells comprise a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF3 / CRBN binding polypeptide and a heterologous polypeptide), and when the cells are contacted with COF3, e.g., an excess of COF3, i) the association of the COF3 / CRBN binding polypeptide with CRBN is increased by at least, e.g., 10, 50, 100, 1000 or 10000 fold compared to the association of the COF3 / CRBN binding polypeptide with CRBN when the cell is not contacted with COF3, as measured, e.g., by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN is increased by at least, e.g., 10, 50, 100, 1000, or 10000 fold compared to the association of the fusion polypeptide with CRBN when the cell is not contacted with COF3, e.g., as measured by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40 or 50 fold compared to ubiquitination of the heterologous polypeptide when the cell is not contacted with COF3, e.g., as measured by an assay described herein; iv) ubiquitination of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to ubiquitination of the fusion polypeptide when the cell is not contacted with COF3, e.g., as measured by an assay described herein; v) degradation of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to degradation of the fusion polypeptide when the cell is not contacted with COF3, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; or vi) The expression level of the fusion polypeptide is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less compared to the expression level of the fusion polypeptide when the cell is not contacted with COF3, as measured, for example, by an assay described herein, such as Western blot analysis or flow cytometry analysis.

[0162] In some embodiments, the cells comprise a COF3, such as a compound disclosed in Table 29, or a pharma- ceutically acceptable salt thereof.

[0163] In some embodiments, the cells comprise a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide, a heterologous polypeptide and a degradation domain), and in the absence of a stabilizing compound, the fusion polypeptide is degraded by a cellular degradative pathway, e.g., at least 50%, 60%, 70%, 80%, 90% or more of the fusion polypeptide is degraded, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis.

[0164] In some embodiments, the fusion polypeptide further comprises a heterologous protease cleavage site. In some embodiments, the cell further comprises a protease capable of cleaving the heterologous protease cleavage site.

[0165] In some embodiments, a cell comprises a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide, a heterologous polypeptide, and a degradation domain), and when the cell is contacted with a stabilizing compound, e.g., an excess of the stabilizing compound, i) does the degradation domain adopt a conformation that is more resistant to cellular degradation relative to the conformation in the absence of the stabilizing compound; ii) the conformation of the fusion polypeptide is more tolerant to cleavage at the heterologous protease cleavage site relative to the conformation in the absence of the stabilizing compound; or iii) the expression level of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40 or 50 fold compared to the expression level of the fusion polypeptide when the cell is not contacted with the stabilizing compound, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis.

[0166] In some embodiments, the cell further comprises a stabilizing compound. In some embodiments, the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO:46.

[0167] In some embodiments, a cell comprises a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide, a heterologous polypeptide and a degradation domain), and when the cell is contacted with a stabilizing compound, e.g., an excess of both the stabilizing compound and COF1, COF2 or COF3, e.g., an excess of COF1, COF2 or COF3, i) the association of a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide with CRBN is increased by at least, e.g., 10, 50, 100, 1000 or 10000 fold compared to the association of a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide with CRBN when the cells are contacted with a stabilizing compound alone and not with COF1, COF2 or COF3, e.g., as measured by an assay described herein, e.g., immunoprecipitation; ii) the association of the fusion polypeptide with CRBN is increased by at least, e.g., 10, 50, 100, 1000, or 10000 fold compared to the association of the fusion polypeptide with CRBN when the cell is contacted with the stabilizing compound alone and not with COF1, COF2 or COF3, e.g., as measured by an assay described herein, e.g., immunoprecipitation; iii) ubiquitination of the heterologous polypeptide is increased, e.g., by at least 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to ubiquitination of the heterologous polypeptide when the cell is contacted with the stabilizing compound alone and not with COF1, COF2 or COF3, e.g., as measured by an assay described herein; iv) ubiquitination of the fusion polypeptide is increased, e.g., by at least 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to ubiquitination of the fusion polypeptide when the cell is contacted with the stabilizing compound alone and not with COF1, COF2 or COF3, e.g., as measured by an assay described herein; v) degradation of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to degradation of the fusion polypeptide when the cells are contacted with the stabilizing compound alone and not with COF1, COF2, or COF3, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis; or vi) the expression level of the fusion polypeptide is, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or less compared to the expression level of the fusion polypeptide when the cells are contacted with only the stabilizing compound and not with COF1, COF2 or COF3, as measured, for example, by an assay described herein, for example, Western blot analysis or flow cytometry analysis.

[0168] In some embodiments, the cells further comprise COF1 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof), COF2 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof), or COF3 (e.g., a compound disclosed in Table 29 or a pharma- ceutically acceptable salt thereof).

[0169] In some embodiments, the heterologous polypeptide is a chimeric antigen receptor (CAR), optionally comprising, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains.

[0170] In one aspect, disclosed herein is a pharmaceutical composition comprising a fusion polypeptide disclosed herein or a cell disclosed herein and a pharma- ceutically acceptable carrier, excipient, or stabilizer.

[0171] In one aspect, disclosed herein is a method of making the cells disclosed herein.

[0172] In one aspect, disclosed herein is a method of degrading a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide)), comprising contacting the fusion polypeptide or a cell comprising said fusion polypeptide with COF1 (e.g., lenalidomide or pomalidomide or a pharmaceutically acceptable salt thereof). In some embodiments, in the presence of COF1 (e.g., lenalidomide or pomalidomide or a pharmaceutically acceptable salt thereof), the expression level of said fusion polypeptide is substantially reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of said fusion polypeptide in the absence of COF1 (e.g., lenalidomide or pomalidomide or a pharmaceutically acceptable salt thereof), e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. In some embodiments, the fusion polypeptide or cell is contacted with COF1 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) ex vivo. In some embodiments, the fusion polypeptide or cell is contacted with COF1 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) in vivo.

[0173] In one aspect, provided herein is a method of controlling expression of a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN or COF2 / CRBN binding polypeptide, a heterologous polypeptide (e.g., a CAR polypeptide), and a degradation domain), comprising: i) contacting the fusion polypeptide or a cell comprising the fusion polypeptide with a stabilizing compound, optionally in the presence of the stabilizing compound, a) does the degradation domain adopt a conformation that is more resistant to cellular degradation relative to the conformation in the absence of the stabilizing compound; b) the conformation of the fusion polypeptide is more tolerant to cleavage at the heterologous protease cleavage site relative to the conformation in the absence of the stabilizing compound; or c) the expression level of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to the expression level of the fusion polypeptide in the absence of a stabilizing compound, as measured, e.g., by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. A method is disclosed that includes:

[0174] In some embodiments, the method further comprises, after step i), ii) contacting the fusion polypeptide or a cell comprising the fusion polypeptide with COF1 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) or COF2 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof), optionally wherein in the presence of COF1 or COF2, the expression level of the fusion polypeptide is substantially reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide after step i) and before step ii), e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. Further includes:

[0175] In some embodiments, the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof.In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO:46.

[0176] In some embodiments, the fusion polypeptide or cell is contacted with COF1 or COF2 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) and / or a stabilizing compound ex vivo. In some embodiments, the fusion polypeptide or cell is contacted with COF1 or COF2 (e.g., lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof) and / or a stabilizing compound in vivo.

[0177] In some embodiments, the heterologous polypeptide is a chimeric antigen receptor (CAR). In some embodiments, a CAR comprises, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains.

[0178] In one aspect, provided herein is a method of producing a cell, comprising: i) providing a cell comprising a nucleic acid molecule encoding a fusion polypeptide comprising a compound of formula 1 (COF1) / CRBN-binding polypeptide and a chimeric antigen receptor (CAR), optionally the CAR comprising, from the N-terminus to the C-terminus, an antigen-binding domain, a transmembrane domain and one or more intracellular signaling domains; and ii) contacting the cells ex vivo with COF1, optionally wherein in the presence of COF1, the expression level of the fusion polypeptide is substantially reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide in the absence of COF1, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. and the compound of formula (I) comprises [ka] (In the formula, X is O or S; R 1 is C 1 ~C 6Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 heteroalkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which may be one or more R 4 independently and optionally replaced by; R 2a and R 2b Each of the groups is independently hydrogen or C 1 ~C 6 alkyl; or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or thiocarbonyl group; R 3 Each of the above may be independently selected from the group consisting of C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ) or -N(R C )S(O) x R E wherein each alkyl, alkenyl, alkynyl, and heteroalkyl is independently and optionally selected from one or more R 6 Replaced with; Each R 4 is independently 1 ~C 6 Alkyl, C2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 1 ~C 6 Heteroalkyl, halo, cyano, oxo, -C(O)R A , -C(O)OR B , -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , -S(O) x R E , -S(O) x N(R C )(R D ), -N(R C )S(O) x R E , carbocyclyl, heterocyclyl, aryl, or heteroaryl, each of which is independently and optionally selected from one or more R 7 Replaced with; R A , R B , R C , R D and R E Each of the groups is independently hydrogen or C 1 ~C 6 is alkyl; Each R 6 is independently 1 ~C 6 Alkyl, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ), -N(R C )C(O)R A , aryl, or heteroaryl, each aryl and heteroaryl being independently and optionally selected from one or more R 8 Replaced with; Each R 7are independently halo, oxo, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; Each R 8 is independently 1 ~C 6 Alkyl, cyano, -OR B , -N(R C )(R D ), -C(O)N(R C )(R D ) or -N(R C )C(O)R A and; n is 0, 1, 2, 3 or 4; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof.

[0179] In some embodiments, after contacting the cells with COF1 ex vivo, proliferation of the cells is increased by at least, e.g., 1.2, 1.5, 2, 5, or 10 fold relative to proliferation of the cells prior to contacting with COF1. In some embodiments, COF1 is lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof. In some embodiments, the COF1 / CRBN binding polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100 (e.g., the COF1 / CRBN binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3).

[0180] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) contacting a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF1 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide)) with COF1 ex vivo, optionally wherein in the presence of COF1, the expression level of the fusion polypeptide is reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide before the cell was contacted with COF1 ex vivo; and ii) administering an effective amount of the cells to a subject. and optionally after step i) and before step ii), Reducing the amount of COF1 in contact with the cell, e.g., within and / or around the cell, thereby treating the disease. A method is provided further comprising:

[0181] In some embodiments, the method further comprises, after step ii), iii) administering to the subject an effective amount of COF1, optionally wherein the administration of COF1 reduces the expression level of the fusion polypeptide, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide after step ii) and before step iii), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) administration of COF1 is in response to the occurrence of an adverse reaction in a subject or in response to a prediction of the occurrence of an adverse reaction in a subject; and / or c) administration of COF1 reduces or prevents adverse effects; Further includes:

[0182] In some embodiments, the method further comprises, after step iii), iv) discontinuing administration of COF1, optionally wherein discontinuing administration of COF1 increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold relative to the expression level of the fusion polypeptide after step iii) and before step iv) (e.g., discontinuing administration of COF1 restores the expression level of the fusion polypeptide to the expression level after step ii) and before step iii)), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) discontinuing administration of COF1 is in response to tumor recurrence in the subject or in response to a prediction of recurrence in the subject; and / or c) discontinuing administration of COF1 treats or prevents tumor recurrence. Further includes:

[0183] In some embodiments, the method further comprises, after step iv), v) repeating steps iii) and / or iv), thereby treating the disease. Further includes:

[0184] In some embodiments, COF1 is lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, and optionally, the COF1 / CRBN binding polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100 (e.g., the COF1 / CRBN binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3). In some embodiments, COF1 is lenalidomide or a pharma- ceutically acceptable salt thereof, and optionally, the lenalidomide or a pharma- ceutically acceptable salt thereof is administered, for example, at 2.5 mg, 5 mg, 10 mg, 15 mg, or 25 mg per day.

[0185] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) administering to a subject an effective amount of a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF1 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide)), optionally contacting the cells with COF1 ex vivo prior to administration, and optionally in the presence of COF1, the expression level of the fusion polypeptide is reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide before the cells are contacted with COF1 ex vivo, and optionally the amount of COF1 contacting the cells, e.g., within and / or around the cells, is reduced after the cells are contacted with COF1 ex vivo and before the cells are administered to the subject, thereby treating the disease. A method is provided that includes:

[0186] In some embodiments, the cells are not contacted with COF1 ex vivo prior to administration.

[0187] In some embodiments, the method further comprises, after step i), ii) administering to the subject an effective amount of COF1, optionally wherein the administration of COF1 reduces the expression level of the fusion polypeptide by, e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide after step i) and before step ii), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) administration of COF1 is in response to the occurrence of an adverse reaction in a subject or in response to a prediction of the occurrence of an adverse reaction in a subject; and / or c) administration of COF1 reduces or prevents adverse effects; Further includes:

[0188] In some embodiments, the method further comprises, after step ii), iii) discontinuing administration of COF1, optionally wherein discontinuing administration of COF1 increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold relative to the expression level of the fusion polypeptide after step ii) and before step iii) (e.g., discontinuing administration of COF1 restores the expression level of the fusion polypeptide to the expression level after step i) and before step ii)), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) discontinuing administration of COF1 is in response to tumor recurrence in the subject or in response to prediction of recurrence in the subject; and / or c) discontinuing administration of COF1 treats or prevents tumor recurrence. Further includes:

[0189] In some embodiments, the method further comprises, after step iii), iv) repeating steps ii) and / or iii), thereby treating the disease. Further includes:

[0190] In some embodiments, COF1 is lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, and optionally, the COF1 / CRBN binding polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100 (e.g., the COF1 / CRBN binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3). In some embodiments, COF1 is lenalidomide or a pharma- ceutically acceptable salt thereof, and optionally, the lenalidomide or a pharma- ceutically acceptable salt thereof is administered, for example, at 2.5 mg, 5 mg, 10 mg, 15 mg, or 25 mg per day.

[0191] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) administering an effective amount of COF1 to a subject, the subject comprising a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF1 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide), and optionally, administration of COF1 reduces the expression level of the fusion polypeptide, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide prior to administration of COF1, and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) administration of COF1 is in response to the occurrence of an adverse reaction in a subject or in response to a prediction of the occurrence of an adverse reaction in a subject; and / or c) administration of COF1 reduces or prevents adverse effects; A method is provided that includes:

[0192] In some embodiments, the method further comprises, after step i), ii) discontinuing administration of COF1, optionally wherein the discontinuing administration of COF1 increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold relative to the expression level of the fusion polypeptide after step i) and before step ii) (e.g., the discontinuing administration of COF1 restores the expression level of the fusion polypeptide to the expression level before administration of COF1), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) discontinuing administration of COF1 is in response to tumor recurrence in the subject or in response to a prediction of recurrence in the subject; and / or c) discontinuing administration of COF1 treats or prevents tumor recurrence. Further includes:

[0193] In some embodiments, the method further comprises, after step ii), iii) repeating steps i) and / or ii), thereby treating the disease. Further includes:

[0194] In some embodiments, COF1 is lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, and optionally, the COF1 / CRBN binding polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100 (e.g., the COF1 / CRBN binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3). In some embodiments, COF1 is lenalidomide or a pharma- ceutically acceptable salt thereof, and optionally, the lenalidomide or a pharma- ceutically acceptable salt thereof is administered, for example, at 2.5 mg, 5 mg, 10 mg, 15 mg, or 25 mg per day.

[0195] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) To the subject, (1) a stabilizing compound, and (2) an effective amount of a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF1 / CRBN or COF2 / CRBN binding polypeptide, a heterologous polypeptide (e.g., a CAR polypeptide), and a degradation domain). wherein, optionally, the expression level of the fusion polypeptide in the presence of the stabilizing compound is, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of the fusion polypeptide in the absence of the stabilizing compound, thereby treating the disease. A method is provided that includes:

[0196] In some embodiments, the method further comprises, after step i), ii) discontinuing administration of the stabilizing compound, optionally wherein discontinuing administration of the stabilizing compound reduces the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold, relative to the expression of the fusion polypeptide after step i) and before step ii), and optionally a) the subject responds to the treatment of step i) (e.g., the subject has a complete response to the treatment of step i), the subject shows a reduction in tumor mass, the subject shows a reduction in tumor cells, or the treatment of step i) is effective in the subject); and / or b) discontinuing administration of the stabilizing compound is responsive to the subject's response to the treatment of step i) (e.g., the subject has a complete response to the treatment of step i), the subject shows a reduction in tumor mass, the subject shows a reduction in tumor cells, or the treatment of step i) is effective in the subject); Further includes:

[0197] In some embodiments, the method further comprises, after step i), iii) discontinuing administration of the stabilizing compound, optionally wherein discontinuing administration of the stabilizing compound reduces the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold, relative to the expression of the fusion polypeptide after step i) and before step ii), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) discontinuing administration of the stabilized compound is in response to the occurrence of an adverse reaction in the subject or in response to a prediction of the occurrence of an adverse reaction in the subject; and / or c) discontinuing administration of the stabilized compound reduces or prevents adverse effects. Further includes:

[0198] In some embodiments, the method further comprises, after step i), iv) discontinuing administration of the stabilizing compound and administering an effective amount of COF1 or COF2 to the subject, optionally wherein step iv) reduces the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50-fold relative to the expression of the fusion polypeptide after step i) and before step iv), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) step iv) is responsive to the occurrence of an adverse reaction in a subject or responsive to a prediction of the occurrence of an adverse reaction in a subject; and / or c) step iv) reduces or prevents adverse effects; In some embodiments, the adverse effect is acute toxicity.

[0199] In some embodiments, the method further comprises, after step iv), v) discontinuing administration of COF1 or COF2, e.g., for 1 day, 5 days, 10 days, or 15 days, after the amount of cells expressing the fusion polypeptide on their surface falls below a predetermined value. Further includes:

[0200] In some embodiments, the method further comprises, after step ii), iii), iv), or v), vi) administering an effective amount of a stabilizing compound, optionally wherein the administration of the stabilizing compound increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold, relative to the expression level of the fusion polypeptide after step ii), iii), iv), or v) and before step vi), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) administration of the stabilized compound is in response to tumor recurrence in the subject or in response to a prediction of recurrence in the subject; and / or c) administering the stabilized compound treats or prevents tumor recurrence. Further includes:

[0201] In some embodiments, the method further comprises, after step vi), vii) repeating steps iii), iv), v) or vi), thereby treating the disease. Further includes:

[0202] In some embodiments, the method further comprises, prior to step i), viii) contacting the cells ex vivo with a stabilizing compound, optionally wherein the expression level of the fusion polypeptide in the presence of the stabilizing compound is, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of the fusion polypeptide in the absence of the stabilizing compound. Further includes:

[0203] In some embodiments, the cells are not contacted with a stabilizing compound ex vivo prior to administration. In some embodiments, the cells are not contacted with any of the stabilizing compounds, COF1, or COF2 ex vivo prior to administration.

[0204] In some embodiments, the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof, and optionally the degradation domain comprises the amino acid sequence of SEQ ID NO:46.

[0205] In some embodiments, COF1 is lenalidomide or pomalidomide, or a pharma- ceutically acceptable salt thereof, and optionally, the COF1 / CRBN binding polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100 (e.g., the COF1 / CRBN binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3).

[0206] In some embodiments, COF2 is lenalidomide or pomalidomide, or a pharma- ceutically acceptable salt thereof, and optionally, the COF2 / CRBN binding polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100 (e.g., the COF1 / CRBN binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3).

[0207] In some embodiments, COF1 or COF2 is lenalidomide or a pharma- ceutically acceptable salt thereof, optionally wherein lenalidomide or a pharma- ceutically acceptable salt thereof is administered, for example, at 2.5 mg, 5 mg, 10 mg, 15 mg or 25 mg per day.

[0208] In one aspect, disclosed herein is a method of degrading a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF3 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide)), comprising contacting the fusion polypeptide or a cell comprising said fusion polypeptide with COF3 (e.g., a compound disclosed in Table 29 or a pharma- ceutically acceptable salt thereof). In one embodiment, in the presence of COF3, the expression level of said fusion polypeptide is substantially reduced, for example, by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent, relative to the expression level of said fusion polypeptide in the absence of COF3, as measured, for example, by an assay described herein, for example, Western blot analysis or flow cytometry analysis. In one embodiment, the fusion polypeptide or cell is contacted with COF3 ex vivo. In one embodiment, the fusion polypeptide or cell is contacted with COF3 in vivo.

[0209] In one aspect, provided herein is a method of controlling expression of a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF3 / CRBN binding polypeptide, a heterologous polypeptide (e.g., a CAR polypeptide), and a degradation domain), comprising: i) contacting the fusion polypeptide or a cell comprising the fusion polypeptide with a stabilizing compound, optionally in the presence of the stabilizing compound, a) does the degradation domain adopt a conformation that is more resistant to cellular degradation relative to the conformation in the absence of the stabilizing compound; b) the conformation of the fusion polypeptide is more tolerant to cleavage at the heterologous protease cleavage site relative to the conformation in the absence of the stabilizing compound; or c) the expression level of the fusion polypeptide is increased by at least, e.g., 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold compared to the expression level of the fusion polypeptide in the absence of a stabilizing compound, as measured, e.g., by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. A method is disclosed that includes:

[0210] In some embodiments, the method further comprises, after step i), ii) contacting the fusion polypeptide or a cell comprising the fusion polypeptide with a COF3 (e.g., a compound disclosed in Table 29 or a pharma- ceutically acceptable salt thereof), optionally wherein in the presence of COF3, the expression level of the fusion polypeptide is substantially reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide after step i) and before step ii), e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. Further includes:

[0211] In some embodiments, the fusion polypeptide or cell is contacted with COF3 ex vivo. In some embodiments, the fusion polypeptide or cell is contacted with COF3 in vivo. In some embodiments, the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof. In some embodiments, the degradation domain comprises the amino acid sequence of SEQ ID NO:46.

[0212] In one aspect, provided herein is a method of producing a cell, comprising: i) providing a cell comprising a nucleic acid molecule encoding a fusion polypeptide comprising a COF3 / CRBN binding polypeptide and a chimeric antigen receptor (CAR), optionally the CAR comprising, from N-terminal to C-terminal direction, an antigen binding domain, a transmembrane domain and one or more intracellular signaling domains; and ii) contacting the cells ex vivo with COF3, optionally wherein in the presence of COF3, the expression level of the fusion polypeptide is substantially reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide in the absence of COF3, e.g., as measured by an assay described herein, e.g., Western blot analysis or flow cytometry analysis. A method is disclosed that includes:

[0213] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) contacting a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF3 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide)) with COF3 ex vivo, optionally wherein in the presence of COF3, the expression level of the fusion polypeptide is reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide before the cell was contacted with COF3 ex vivo; and ii) administering an effective amount of the cells to a subject. and optionally after step i) and before step ii), Reducing the amount of COF3 in contact with the cell, e.g., within and / or around the cell, thereby treating the disease. A method is provided further comprising:

[0214] In some embodiments, the method further comprises, after step ii), iii) administering to the subject an effective amount of COF3, optionally wherein administration of COF3 reduces the expression level of the fusion polypeptide by, e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide after step ii) and before step iii), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) administration of COF3 is in response to the occurrence of an adverse reaction in a subject or in response to a prediction of the occurrence of an adverse reaction in a subject; and / or c) administration of COF3 reduces or prevents adverse effects; Further includes:

[0215] In some embodiments, the method further comprises, after step iii), iv) discontinuing administration of COF3, optionally wherein discontinuing administration of COF3 increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold relative to the expression level of the fusion polypeptide after step iii) and before step iv) (e.g., discontinuing administration of COF3 restores the expression level of the fusion polypeptide to the expression level after step ii) and before step iii)), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) discontinuing administration of COF3 is in response to tumor recurrence in the subject or in response to a prediction of recurrence in the subject; and / or c) discontinuing administration of COF3 treats or prevents tumor recurrence. Further includes:

[0216] In some embodiments, the method further comprises, after step iv), v) repeating steps iii) and / or iv), thereby treating the disease. Further includes:

[0217] In some embodiments, the COF3 is a compound disclosed in Table 29 or a pharma- ceutically acceptable salt thereof. In some embodiments, the COF3 / CRBN binding polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 109, or an amino acid sequence having at least 80, 85, 90, or 95% identity thereto.

[0218] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) administering to a subject an effective amount of a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF3 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide)), optionally contacting the cells with COF3 ex vivo prior to administration, and optionally in the presence of COF3, the expression level of the fusion polypeptide is reduced, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide before the cells are contacted with COF3 ex vivo, and optionally the amount of COF3 contacting the cells, e.g., within and / or around the cells, is reduced after the cells are contacted with COF3 ex vivo and before the cells are administered to the subject, thereby treating the disease. A method is provided that includes:

[0219] In some embodiments, the cells are not contacted with COF3 ex vivo prior to administration.

[0220] In some embodiments, the method further comprises, after step i), ii) administering to the subject an effective amount of COF3, optionally wherein administration of COF3 reduces the expression level of the fusion polypeptide by, e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide after step i) and before step ii), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) administration of COF3 is in response to the occurrence of an adverse reaction in a subject or in response to a prediction of the occurrence of an adverse reaction in a subject; and / or c) administration of COF3 reduces or prevents adverse effects; Further includes:

[0221] In some embodiments, the method further comprises, after step ii), iii) discontinuing administration of COF3, optionally wherein discontinuing administration of COF3 increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold, relative to the expression level of the fusion polypeptide after step ii) and before step iii) (e.g., discontinuing administration of COF3 restores the expression level of the fusion polypeptide to the expression level after step i) and before step ii)), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) discontinuing administration of COF3 is in response to tumor recurrence in the subject or in response to a prediction of recurrence in the subject; and / or c) discontinuing administration of COF3 treats or prevents tumor recurrence. Further includes:

[0222] In some embodiments, the method further comprises, after step iii), iv) repeating steps ii) and / or iii), thereby treating the disease. Further includes:

[0223] In some embodiments, the COF3 is a compound disclosed in Table 29 or a pharma- ceutically acceptable salt thereof. In some embodiments, the COF3 / CRBN binding polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 109, or an amino acid sequence having at least 80, 85, 90, or 95% identity thereto.

[0224] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) administering an effective amount of COF3 to a subject, the subject comprising a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF3 / CRBN binding polypeptide and a heterologous polypeptide (e.g., a CAR polypeptide), and optionally, administration of COF3 reduces the expression level of the fusion polypeptide, e.g., by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent relative to the expression level of the fusion polypeptide prior to administration of COF3, and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) administration of COF3 is in response to the occurrence of an adverse reaction in a subject or in response to a prediction of the occurrence of an adverse reaction in a subject; and / or c) administration of COF3 reduces or prevents adverse effects; A method is provided that includes:

[0225] In some embodiments, the method further comprises, after step i), ii) discontinuing administration of COF3, optionally wherein discontinuing administration of COF3 increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold relative to the expression level of the fusion polypeptide after step i) and before step ii) (e.g., discontinuing administration of COF3 restores the expression level of the fusion polypeptide to the expression level before administration of COF3), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) discontinuing administration of COF3 is in response to tumor recurrence in the subject or in response to a prediction of recurrence in the subject; and / or c) discontinuing administration of COF3 treats or prevents tumor recurrence. Further includes:

[0226] In some embodiments, the method further comprises, after step ii), iii) repeating steps i) and / or ii), thereby treating the disease. Further includes:

[0227] In some embodiments, the COF3 is a compound disclosed in Table 29 or a pharma- ceutically acceptable salt thereof. In some embodiments, the COF3 / CRBN binding polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 109, or an amino acid sequence having at least 80, 85, 90, or 95% identity thereto.

[0228] In one aspect, provided herein is a method of treating a subject having a disease associated with expression of a tumor antigen, comprising: i) To the subject, (1) a stabilizing compound, and (2) an effective amount of a cell disclosed herein (e.g., a cell comprising a fusion polypeptide comprising a COF3 / CRBN binding polypeptide, a heterologous polypeptide (e.g., a CAR polypeptide), and a degradation domain); wherein, optionally, the expression level of the fusion polypeptide in the presence of the stabilizing compound is, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of the fusion polypeptide in the absence of the stabilizing compound, thereby treating the disease. A method is provided that includes:

[0229] In some embodiments, the method further comprises, after step i), ii) discontinuing administration of the stabilizing compound, optionally wherein discontinuing administration of the stabilizing compound reduces the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold, relative to the expression of the fusion polypeptide after step i) and before step ii), and optionally a) the subject responds to the treatment of step i) (e.g., the subject has a complete response to the treatment of step i), the subject shows a reduction in tumor mass, the subject shows a reduction in tumor cells, or the treatment of step i) is effective in the subject); and / or b) discontinuing administration of the stabilizing compound is responsive to the subject's response to the treatment of step i) (e.g., the subject has a complete response to the treatment of step i), the subject shows a reduction in tumor mass, the subject shows a reduction in tumor cells, or the treatment of step i) is effective in the subject); Further includes:

[0230] In some embodiments, the method further comprises, after step i), iii) discontinuing administration of the stabilizing compound, optionally wherein discontinuing administration of the stabilizing compound reduces the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold, relative to the expression of the fusion polypeptide after step i) and before step ii), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) discontinuing administration of the stabilized compound is in response to the occurrence of an adverse reaction in the subject or in response to a prediction of the occurrence of an adverse reaction in the subject; and / or c) discontinuing administration of the stabilized compound reduces or prevents adverse effects. Further includes:

[0231] In some embodiments, the method further comprises, after step i), iv) discontinuing administration of the stabilizing compound and administering an effective amount of COF3 to the subject, optionally wherein step iv) reduces the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50-fold relative to the expression of the fusion polypeptide after step i) and before step iv), and optionally a) the subject has experienced, is experiencing, or is predicted to experience an adverse reaction; b) step iv) is responsive to the occurrence of an adverse reaction in a subject or responsive to a prediction of the occurrence of an adverse reaction in a subject; and / or c) step iv) reduces or prevents adverse effects; In some embodiments, the adverse effect is acute toxicity.

[0232] In some embodiments, the method further comprises, after step iv), v) discontinuing administration of COF3, for example for 1 day, 5 days, 10 days, or 15 days, after the amount of cells expressing the fusion polypeptide on their surface falls below a predetermined value. Further includes:

[0233] In some embodiments, the method further comprises, after step ii), iii), iv), or v), vi) administering an effective amount of a stabilizing compound, optionally wherein the administration of the stabilizing compound increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold, relative to the expression level of the fusion polypeptide after step ii), iii), iv), or v) and before step vi), and optionally a) the subject has relapsed, is relapsed, or is predicted to relapse; b) administration of the stabilized compound is in response to tumor recurrence in the subject or in response to a prediction of recurrence in the subject; and / or c) administering the stabilized compound treats or prevents tumor recurrence. Further includes:

[0234] In some embodiments, the method further comprises, after step vi), vii) repeating steps iii), iv), v) or vi), thereby treating the disease. Further includes:

[0235] In some embodiments, the method further comprises, prior to step i), viii) contacting the cells ex vivo with a stabilizing compound, optionally wherein the expression level of the fusion polypeptide in the presence of the stabilizing compound is, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of the fusion polypeptide in the absence of the stabilizing compound. Further includes:

[0236] In some embodiments, the cells are not contacted with the stabilizing compound ex vivo prior to administration.

[0237] In some embodiments, the stabilizing compound is bazedoxifene or a pharma- ceutically acceptable salt thereof, and optionally the degradation domain comprises the amino acid sequence of SEQ ID NO:46.

[0238] In some embodiments, the COF3 is a compound disclosed in Table 29 or a pharma- ceutically acceptable salt thereof. In some embodiments, the COF3 / CRBN binding polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 109, or an amino acid sequence having at least 80, 85, 90, or 95% identity thereto.

[0239] In some embodiments, the heterologous polypeptide of the fusion polypeptide is a chimeric antigen receptor (CAR), optionally comprising, from N-terminal to C-terminal, an antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains.

[0240] In one aspect, the present invention also provides a fusion polypeptide, a nucleic acid molecule, a vector, a viral particle, a cell or a pharmaceutical composition as disclosed herein for use as a medicament. In one aspect, the present invention also provides a fusion polypeptide, a nucleic acid molecule, a vector, a viral particle, a cell or a pharmaceutical composition as disclosed herein for use in treating a subject having a disease associated with expression of a tumor antigen.

[0241] In certain embodiments of the foregoing methods, the disease associated with expression of a tumor antigen is cancer.

[0242] In some embodiments, the cancer is mesothelioma (e.g., malignant pleural mesothelioma), for example in a subject who has progressed against at least one prior standard of care; lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer); pancreatic cancer (e.g., pancreatic ductal adenocarcinoma or metastatic pancreatic ductal adenocarcinoma (PDA) in a subject who has progressed against at least one prior standard of care); esophageal adenocarcinoma, ovarian cancer (e.g., serous ovarian epithelial carcinoma in a subject who has progressed after at least one prior standard of care regimen), breast cancer, colorectal cancer, bladder cancer, or any combination thereof.

[0243] In some embodiments, the disease associated with expression of a tumor antigen is a hematological cancer, such as a hematological cancer selected from leukemia or lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndromes. , non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / splenic leukemia, diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, nodal marginal zone lymphoma, pediatric nodal Selected from marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma.

[0244] In some embodiments, the cancer is selected from MCL, CLL, ALL, Hodgkin's lymphoma, AML, or multiple myeloma.

[0245] In certain embodiments of the above method, the cell is autologous to the subject. In certain embodiments of the above method, the cell is allogeneic to the subject. In some embodiments, the cell is a CAR-expressing cell, e.g., a CART cell.

[0246] In some embodiments, a subject is administered cells expressing at least one fusion polypeptide disclosed herein prior to administration of COF1, COF2, or COF3.

[0247] In one aspect, provided herein is a method for identifying genetic factors associated with a particular biological phenotype, such as genetic factors associated with cancer onset and / or progression, comprising: i) modulating expression of a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN or COF3 / CRBN binding polypeptide and a heterologous polypeptide) in a cell by exposing the cell to COF1 or COF3, e.g., lenalidomide or a pharma- ceutically acceptable salt thereof; (ii) selecting cells having a phenotype of interest, e.g., a phenotype associated with the development and / or progression of cancer; and (iii) identifying said fusion polypeptides that induce said phenotype of interest. Including, Methods are disclosed wherein exposing the cell to COF1 or COF3, e.g., lenalidomide or a pharma- ceutically acceptable salt thereof, reduces the expression level of the fusion polypeptide, e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 percent, relative to the expression level of the fusion polypeptide before exposure to COF1 or COF3, e.g., lenalidomide or a pharma- ceutically acceptable salt thereof.

[0248] In one aspect, provided herein is a method for identifying genetic factors associated with a particular biological phenotype, such as genetic factors associated with cancer onset and / or progression, comprising: i) modulating expression of a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide, a heterologous polypeptide and a degradation domain) in a cell by exposing the cell to a stabilizing compound, such as bazedoxifene or a pharma- ceutically acceptable salt thereof, followed by COF1, COF2 or COF3, such as lenalidomide or a pharma- ceutically acceptable salt thereof; (ii) selecting cells having a phenotype of interest, e.g., a phenotype associated with the development and / or progression of cancer; and (iii) identifying said fusion polypeptides that induce said phenotype of interest. Including, Exposure of the cells to a stabilizing compound, e.g., bazedoxifene or a pharma- ceutically acceptable salt thereof, increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold relative to the expression level of the fusion polypeptide prior to exposure to the stabilizing compound, e.g., bazedoxifene or a pharma- ceutically acceptable salt thereof, and exposure of the cells to a COF1, COF2, or COF3, e.g., lenalidomide or a pharma- ceutically acceptable salt thereof, increases the expression level of the fusion polypeptide, e.g., at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 fold relative to the expression level of the fusion polypeptide prior to exposure to the stabilizing compound, e.g., bazedoxifene or a pharma- ceutically acceptable salt thereof, Methods are disclosed that reduce the expression level of the fusion polypeptide by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 percent relative to the expression level of the fusion polypeptide after exposure to a compound and before exposure to COF1 or COF2 (such as thalidomide and its derivatives (e.g., lenalidomide, pomalidomide and thalidomide)) or COF3 (e.g., a compound disclosed in Table 29).

[0249] In certain embodiments of the foregoing aspects, the heterologous polypeptide is a chimeric antigen receptor (CAR) polypeptide. In some embodiments, the CAR polypeptide comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in Table 3. In some embodiments, the CAR polypeptide is an anti-CD19 CAR polypeptide and comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in any of Tables 5, 6, 7, and 30. In some embodiments, the CAR polypeptide is an anti-CD123 CAR polypeptide and comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in any of Tables 8, 9, 10, 11, 12, 13, and 14. In some embodiments, the CAR polypeptide is an anti-BCMA CAR polypeptide and comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in any of Tables 15, 16, 17, 18, and 31. In some embodiments, the CAR polypeptide is an anti-CD22 CAR polypeptide and comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in any of Tables 19 and 20. In some embodiments, the CAR polypeptide is an anti-CD20 CAR polypeptide and comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in Table 32. In some embodiments, the CAR polypeptide is an anti-EGFRvIII CAR polypeptide and comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in Table 33. In some embodiments, the CAR polypeptide is an anti-mesothelin CAR polypeptide and comprises an amino acid sequence disclosed herein, such as an amino acid sequence disclosed in Table 34.

[0250] In certain embodiments of the foregoing aspects, the fusion polypeptide comprises an amino acid sequence disclosed herein, for example, an amino acid sequence disclosed in Table 4 or Table 28.

[0251] In certain embodiments of the foregoing aspects, the COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide comprises an amino acid sequence disclosed herein, for example, an amino acid sequence disclosed in Table 1.

[0252] In certain embodiments of the foregoing aspects, the degradation domain comprises an amino acid sequence disclosed herein, e.g., an amino acid sequence disclosed in Table 22.

[0253] In certain embodiments of the foregoing aspects, the heterologous protease cleavage site comprises an amino acid sequence disclosed herein, e.g., an amino acid sequence disclosed in Table 23. [Brief description of the drawings]

[0254] [Figure 1A] FIG. 1 is a schematic diagram of HilD-tag 136-180 and 236-249 degrons fused to nanoluciferase via a 16 glycine-serine linker. [Figure 1B] FIG. 13 is a graph showing the level of luminescence measured from HEK293T cells reverse transfected with 50 ng of pNL1.1CMV construct encoding nanoluciferase linked to IKZF3 136-180 and 236-249. IKZF3 136-180 and 236-249 promoted a reduction in luminescence in cells treated with 1 μM, 10 μM or 100 μM lenalidomide for 6 hours compared to cells treated with DMSO only. MG132 treatment blocked lenalidomide-dependent degradation of nanoluciferase.

[0255] [Diagram 2]Western blot showing that IKZF3 136-180 and 236-249 promoted lenalidomide-dependent degradation of nanoluciferase in HEK293GT cells transfected with pNL1.1CMV constructs encoding IKZF3 136-180 and 236-249 tagged nanoluciferase (IC50=10 nM). Lenalidomide-dependent degradation was not observed in similarly transfected HEK293GT cereblon (CRBN) KO cells. Treatment with the proteasome inhibitor MG132 blocked the ability of IKZF3 136-180 and 236-249 to promote lenalidomide-dependent degradation.

[0256] [Figure 3A] A schematic showing 136-180 of IKZF3, which contains 236-249 of IKZF3 predicted as a hairpin and two β-sheets adjacent to the α-helix, as well as an additional α-helix. The bottom schematic is a shortened version of the 136-180 degron of IKZF3 removing amino acids at the N- and C-terminus (SEQ ID NOS: 3, 5, and 7-10, respectively, in order of appearance). [Figure 3B]1 is a Western blot showing results from a study testing lenalidomide-dependent degradation of nanoluciferase fused to various IKZF3-based degradation tags. IKZF3-based degradation tags were fused to the N-terminus of nanoluciferase, cloned into pNL1.1CMV vector, and transfected into HEK293T cells. Transfected cells were treated with either DMSO or 10 μM lenalidomide for 4 hours and then analyzed by Western blot. Two types of exposure (long and short exposure) are shown for nanoluciferase ("Nanoluc"). IKZF3 136-180 and 236-249, IKZF3 136-180 and 236-249 K245R, IKZF3 136-180 and 236-249 K245S, IKZF3 136-180 MALEK, and IKZF3 136-170 MALEK all promoted lenalidomide-dependent degradation, whereas IKZF3 140-170 MALEK, IKZF3 141-163 MALEK, and IKZF3 145-155 MALEK did not mediate lenalidomide-induced degradation.

[0257] [Figure 4A] Graphs of Western blots showing lenalidomide-dependent degradation of IKZF3136-180 tagged nanoluciferase (FIG. 4A) or IKZF3136-170MALEK tagged nanoluciferase (FIG. 4B) in HEK293T cells with an IC50 of approximately 100 nM in both cases upon 2 hours of lenalidomide treatment. Tagged nanoluciferase fusions were expressed using the pNL1.1CMV construct. [Figure 4B] Graphs of Western blots showing lenalidomide-dependent degradation of IKZF3136-180 tagged nanoluciferase (FIG. 4A) or IKZF3136-170MALEK tagged nanoluciferase (FIG. 4B) in HEK293T cells with an IC50 of approximately 100 nM in both cases upon 2 hours of lenalidomide treatment. Tagged nanoluciferase fusions were expressed using the pNL1.1CMV construct. [Figure 4C]Western blot showing the time course of lenalidomide-dependent degradation of 136-180 tagged nanoluciferase of IKZF3 in HEK293T cells, showing immediate degradation at 1 h and nearly complete degradation at 4 h. Tagged nanoluciferase fusions were expressed using the pNL1.1CMV construct.

[0258] [Figure 5A] Western blots showing lenalidomide-dependent degradation of IKZF3 136-180 and 236-249 tagged melanogenesis-related transcription factor (MITF) (left panel) and IKZF3 136-180 tagged MITF (right panel). Tagged MITF fusions were transfected into HEK293T using the pNL1.1CMV construct. Degradation of IKZF3 136-180 and 236-249 tagged MITF shows an IC50 of approximately 100 nM. This degradation was dependent on proteasome activity, as degradation was blocked by MG132 treatment. IKZF3 136-180 also mediated lenalidomide-dependent degradation, albeit to a lesser extent than IKZF3 136-180 and 236-249. [Figure 5B] Western blots showing lenalidomide-dependent degradation of IKZF3 136-180 and 236-249 tagged MITF (left panel) and IKZF3 136-180 tagged MITF (right panel) after cells expressing these fusion proteins were treated with 10 μM lenalidomide for various times, with 4 hours of treatment showing the greatest amount of degradation among the time points tested.

[0259] [Figure 6A]Figure 6A is a graph of a Western blot showing lenalidomide-dependent degradation of MITF tagged with IKZF3 136-180 and 236-249 (Figure 6A) or IKZF3 136-180 and 236-249 in which all lysine residues in the tag have been mutated to arginine ("lysine-free IKZF3 136-180 and 236-249") (Figure 6B). HEK293T cells expressing tagged MITF fusions using the pNL1.1CMV construct were treated with various concentrations of lenalidomide for 24 hours. The IC50 is approximately 10 nM for IKZF3 136-180 and 236-249 tagged MITF (Figure 6A) and less than 100 nM for lysine-free IKZF3 136-180 and 236-249 tagged MITF (Figure 6B). In both cases, lenalidomide-dependent degradation was dependent on the proteasome, as degradation could be blocked by the proteasome inhibitor MG132, suggesting that MITF, but not the IKZF3 degron tag, was ubiquitinated. [Figure 6B] Figure 6A is a graph of a Western blot showing lenalidomide-dependent degradation of MITF tagged with IKZF3 136-180 and 236-249 (Figure 6A) or IKZF3 136-180 and 236-249 in which all lysine residues in the tag have been mutated to arginine ("lysine-free IKZF3 136-180 and 236-249") (Figure 6B). HEK293T cells expressing tagged MITF fusions using the pNL1.1CMV construct were treated with various concentrations of lenalidomide for 24 hours. The IC50 is approximately 10 nM for IKZF3 136-180 and 236-249 tagged MITF (Figure 6A) and less than 100 nM for lysine-free IKZF3 136-180 and 236-249 tagged MITF (Figure 6B). In both cases, lenalidomide-dependent degradation was dependent on the proteasome, as degradation could be blocked by the proteasome inhibitor MG132, suggesting that MITF, but not the IKZF3 degron tag, was ubiquitinated. [Figure 6C]Western blot showing lenalidomide-dependent degradation of 136-180 and 236-249 tagged MITF on lysine-free IKZF3. HEK293T cells expressing tagged MITF fusions using the pNL1.1CMV construct were treated with 10 μM lenalidomide for 2, 4, 8 or 24 hours. [Figure 6D] Western blots of IKZF3 136-180 and 236-249 tagged MITF (left panel) and lysine-free IKZF3 136-180 and 236-249 tagged MITF (right panel). HEK293T cells expressing tagged MITF fusions using the pNL1.1CMV construct were treated with 10 μM lenalidomide, pomalidomide, thalidomide, a negative control IMiD that can bind CRBN but not IKZF1 or IKZF3, or DMSO for 24 hours before cells were subjected to Western blot analysis. Pomalidomide mediated degradation of tagged MITF to a slightly greater extent than lenalidomide, whereas thalidomide was only very less effective in mediating such degradation.

[0260] [Figure 7] Western blot showing lenalidomide-dependent degradation of 136-180 Q147H-tagged MITF in IKZF3. HEK293T cells transfected with pNL1.1CMV encoding the tagged MITF fusion were treated with various lenalidomide doses for 24 hours. 136-180 Q147H-tagged MITF in IKZF3 showed no degradation in the presence of lenalidomide.

[0261] [Figure 8] Western blot showing lenalidomide-dependent degradation of 136-180 and 236-249 tagged avian myelocytomatosis viral oncogene (MYC) homologues of IKZF3 with an IC50 of approximately 10 nM. HEK293T cells expressing tagged MITF fusions using the pNL1.1CMV construct were treated with various concentrations of lenalidomide for 4 hours.

[0262] [Figure 9A] Western blot showing lenalidomide-dependent degradation of C-terminal degron-tagged single-pass transmembrane proteins, CD3 zeta, CD8 / CD3 zeta chimera, CD8, CD19, and CD22 at 4 h. Jurkat cells were infected with pNGX_LV_V002-CDx-IKZF3 136-180 and 236-249 construct viruses, selected with G418, and treated with 10 μM lenalidomide or DMSO. In FIG. 9A, staining using anti-V5 antibody (both long 1 min exposure and short 1 s exposure are shown) and anti-beta-actin antibody are shown. All constructs were expressed and degraded with 10 μM lenalidomide treatment. The table in FIG. 9A shows the protein molecular weight (MW), number of cytosolic amino acid residues ("cytosolic AA"), and number of cytosolic lysines for each protein. Interestingly, degradation correlates better with the total number of amino acids ("AA") in the cytoplasm than with the number of cytosolic lysine residues. [Figure 9B] 9B is a graph of a Western blot showing lenalidomide-dependent degradation of C-terminally tagged CD19 (FIG. 9B), C-terminally tagged CD3 zeta (FIG. 9C), and C-terminally tagged CD8 / CD3 zeta (FIG. 9D). Cells expressing IKZF3 136-180 and 236-249 tagged CD19, CD3 zeta, or CD8 / CD3 zeta were treated with 10 μM lenalidomide for 6 hours or with various lenalidomide doses for 24 hours. In FIG. 9B, degradation of IKZF3 136-180 and 236-249 tagged CD19 showed an IC50 of approximately 100 nM, with potent degradation detected at 6 hours. The degradation of IKZF3 136-180 and 236-249 tagged CD3 zeta shown in Figure 9C is weaker than that of IKZF3 136-180 and 236-249 tagged CD19. The degradation of tagged CD3 zeta was evident after cells were treated with 10 μM lenalidomide for 24 hours. The degradation of IKZF3 136-180 and 236-249 tagged CD8 / CD3 zeta shown in Figure 9D is stronger than that of IKZF3 136-180 and 236-249 tagged CD3 zeta. [Figure 9C]9B is a graph of a Western blot showing lenalidomide-dependent degradation of C-terminally tagged CD19 (FIG. 9B), C-terminally tagged CD3 zeta (FIG. 9C), and C-terminally tagged CD8 / CD3 zeta (FIG. 9D). Cells expressing IKZF3 136-180 and 236-249 tagged CD19, CD3 zeta, or CD8 / CD3 zeta were treated with 10 μM lenalidomide for 6 hours or with various lenalidomide doses for 24 hours. In FIG. 9B, degradation of IKZF3 136-180 and 236-249 tagged CD19 showed an IC50 of approximately 100 nM, with potent degradation detected at 6 hours. The degradation of IKZF3 136-180 and 236-249 tagged CD3 zeta shown in Figure 9C is weaker than that of IKZF3 136-180 and 236-249 tagged CD19. The degradation of tagged CD3 zeta was evident after cells were treated with 10 μM lenalidomide for 24 hours. The degradation of IKZF3 136-180 and 236-249 tagged CD8 / CD3 zeta shown in Figure 9D is stronger than that of IKZF3 136-180 and 236-249 tagged CD3 zeta. [Figure 9D] 9B is a graph of a Western blot showing lenalidomide-dependent degradation of C-terminally tagged CD19 (FIG. 9B), C-terminally tagged CD3 zeta (FIG. 9C), and C-terminally tagged CD8 / CD3 zeta (FIG. 9D). Cells expressing IKZF3 136-180 and 236-249 tagged CD19, CD3 zeta, or CD8 / CD3 zeta were treated with 10 μM lenalidomide for 6 hours or with various lenalidomide doses for 24 hours. In FIG. 9B, degradation of IKZF3 136-180 and 236-249 tagged CD19 showed an IC50 of approximately 100 nM, with potent degradation detected at 6 hours. The degradation of IKZF3 136-180 and 236-249 tagged CD3 zeta shown in Figure 9C is weaker than that of IKZF3 136-180 and 236-249 tagged CD19. The degradation of tagged CD3 zeta was evident after cells were treated with 10 μM lenalidomide for 24 hours. The degradation of IKZF3 136-180 and 236-249 tagged CD8 / CD3 zeta shown in Figure 9D is stronger than that of IKZF3 136-180 and 236-249 tagged CD3 zeta.

[0263] [Figure 10A] A series of flow cytometry histograms comparing cell surface expression of IKZF3 136-180 and 236-249 tagged CD19 on Jurkat cells treated with 1 μM or 10 μM lenalidomide for 1 hour (FIG. 10A), 6 hours (FIG. 10B), 16 hours (FIG. 10C) or 24 hours (FIG. 10D). Some cells were pretreated with 10 μM MG132 prior to treatment with 10 μM lenalidomide. DMSO served as a solvent control. IKZF3 136-180 and 236-249 were fused to the C-terminus of CD19. [Figure 10B] A series of flow cytometry histograms comparing cell surface expression of IKZF3 136-180 and 236-249 tagged CD19 on Jurkat cells treated with 1 μM or 10 μM lenalidomide for 1 hour (FIG. 10A), 6 hours (FIG. 10B), 16 hours (FIG. 10C) or 24 hours (FIG. 10D). Some cells were pretreated with 10 μM MG132 prior to treatment with 10 μM lenalidomide. DMSO served as a solvent control. IKZF3 136-180 and 236-249 were fused to the C-terminus of CD19. [Figure 10C] A series of flow cytometry histograms comparing cell surface expression of IKZF3 136-180 and 236-249 tagged CD19 on Jurkat cells treated with 1 μM or 10 μM lenalidomide for 1 hour (FIG. 10A), 6 hours (FIG. 10B), 16 hours (FIG. 10C) or 24 hours (FIG. 10D). Some cells were pretreated with 10 μM MG132 prior to treatment with 10 μM lenalidomide. DMSO served as a solvent control. IKZF3 136-180 and 236-249 were fused to the C-terminus of CD19. [Figure 10D]A series of flow cytometry histograms comparing cell surface expression of IKZF3 136-180 and 236-249 tagged CD19 on Jurkat cells treated with 1 μM or 10 μM lenalidomide for 1 hour (FIG. 10A), 6 hours (FIG. 10B), 16 hours (FIG. 10C) or 24 hours (FIG. 10D). Some cells were pretreated with 10 μM MG132 prior to treatment with 10 μM lenalidomide. DMSO served as a solvent control. IKZF3 136-180 and 236-249 were fused to the C-terminus of CD19. [Figure 10E] 10E) or mean fluorescence intensity (MFI) (FIG. 10F) of CD19 positive cells across all lenalidomide doses and time points tested. There was minimal degradation at 1 hour and trace degradation at 6 hours. Degradation was more evident at 16 and 24 hours in both the 1 μM and 10 μM treatment groups, and this degradation could be partially blocked by the proteasome inhibitor MG132. There was an approximately 50% reduction in CD19 positive cells at 16 hours in both the 1 μM and 10 μM treatment groups (FIG. 10E), which was consistent with the reduction in MFI (FIG. 10F). [Figure 10F] 10E) or mean fluorescence intensity (MFI) (FIG. 10F) of CD19 positive cells across all lenalidomide doses and time points tested. There was minimal degradation at 1 hour and trace degradation at 6 hours. Degradation was more evident at 16 and 24 hours in both the 1 μM and 10 μM treatment groups, and this degradation could be partially blocked by the proteasome inhibitor MG132. There was an approximately 50% reduction in CD19 positive cells at 16 hours in both the 1 μM and 10 μM treatment groups (FIG. 10E), which was consistent with the reduction in MFI (FIG. 10F).

[0264] [Figure 11] FIG. 1 is a schematic diagram showing an exemplary fusion protein comprising a degradation domain (degron), a protease cleavage site and a second protein domain (CAR) and the alteration of the degradation of the fusion protein in the presence of a drug, e.g., a stabilizing compound.

[0265] [Figure 12] 12A, 12B, and 12C are schematic diagrams showing the regulation of CAR molecules fused to FurON (FIG. 12A), HilD (FIG. 12B), or both FurON and HilD (FIG. 12C). As shown in FIG. 12A, a CAR fused to FurON can be activated by administering a stabilizing compound (e.g., a small molecule ligand that binds to and stabilizes the degradation domain, e.g., bazedoxifene (BZA)) or inactivated by withdrawing the stabilizing compound. As shown in FIG. 12B, a CAR fused to a HilD tag can be inactivated by administering an IMiD compound (e.g., lenalidomide or pomalidomide) and reactivated by ceasing administration of the IMiD compound. As shown in FIG. 12C, a CAR fused to both FurON and HilD tags can be activated by administering a stabilizing compound, inactivated by withdrawing the stabilizing compound and administering an IMiD compound, and reactivated by withdrawing the IMiD compound and administering a stabilizing compound. Combining the FurON and HilD switches adds an additional layer of control over the expression and activity of the CAR molecule.

[0266] [Figure 13] Western blot graphs showing lenalidomide-dependent degradation of CAR molecules. JNL cells expressing construct 765 (FurON_CAR19) (Figure 13A), construct 766 (FurON_CAR19_16GS_HilD tag_V5) (Figure 13B) or construct 767 (FurON_CAR19_16GS_HilD tag) (Figure 13C) were incubated in the presence of 10 μM lenalidomide ("+") or DMSO ("-") for 24 hours prior to Western blot analysis. All samples received 1 μM bazedoxifene. "A" represents cells transduced with 275 μL of viral supernatant. "B" represents cells transduced with 700 μL of viral supernatant.

[0267] [Figure 14]Figure 14A is a graph of a Western blot showing lenalidomide-dependent degradation of the CAR molecule. JNL cells expressing construct 771 (CAR19_HilD tag_V5) (Figure 14A), construct 769 (CAR19_16GS_HilD tag) (Figure 14B), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 14C) or construct 770 (CAR19_16GS_HilD tag_NoK) were incubated in the presence of 10 μM lenalidomide ("+") or DMSO ("-") for 24 hours prior to Western blot analysis. "A" represents cells transduced with 275 μL of viral supernatant. "B" represents cells transduced with 700 μL of viral supernatant.

[0268] [Figure 15] 15A-B are graphs of Western blots showing lenalidomide-dependent degradation of CAR molecules. JNL cells expressing construct 769 (CAR19_16GS_HilD tag) were incubated with 10 μM lenalidomide or DMSO for 2, 4, 8, 16, or 24 hours (FIG. 15A) or various doses of lenalidomide or DMSO for 24 hours (FIG. 15B) prior to Western blot analysis. FIG. 15A shows the time course of 10 μM lenalidomide treatment. FIG. 15B shows the dose response of lenalidomide at 24 hours.

[0269] [Figure 16A]16A-C are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figure 16A), construct 771 (CAR19_HilD tag_V5) (Figure 16B), construct 6761 (CAR19_16KGS_HilD tag_V5) (Figure 16C), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 16D), construct 770 (CAR19_16GS_HilD tag_NoK) (Figure 16E), construct 773 (HilD tag_CAR19_modSigPep) (Figure 16F), and construct 774 (HilD tag_CAR19) (Figure 16G). JNL cells expressing the indicated constructs were incubated with or without 10 μM lenalidomide for 24 h and then subjected to flow cytometry analysis. [Figure 16B] 16A-C are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figure 16A), construct 771 (CAR19_HilD tag_V5) (Figure 16B), construct 6761 (CAR19_16KGS_HilD tag_V5) (Figure 16C), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 16D), construct 770 (CAR19_16GS_HilD tag_NoK) (Figure 16E), construct 773 (HilD tag_CAR19_modSigPep) (Figure 16F), and construct 774 (HilD tag_CAR19) (Figure 16G). JNL cells expressing the indicated constructs were incubated with or without 10 μM lenalidomide for 24 h and then subjected to flow cytometry analysis. [Figure 16C]16A-C are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figure 16A), construct 771 (CAR19_HilD tag_V5) (Figure 16B), construct 6761 (CAR19_16KGS_HilD tag_V5) (Figure 16C), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 16D), construct 770 (CAR19_16GS_HilD tag_NoK) (Figure 16E), construct 773 (HilD tag_CAR19_modSigPep) (Figure 16F), and construct 774 (HilD tag_CAR19) (Figure 16G). JNL cells expressing the indicated constructs were incubated with or without 10 μM lenalidomide for 24 h and then subjected to flow cytometry analysis. [Figure 16D] 16A-C are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figure 16A), construct 771 (CAR19_HilD tag_V5) (Figure 16B), construct 6761 (CAR19_16KGS_HilD tag_V5) (Figure 16C), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 16D), construct 770 (CAR19_16GS_HilD tag_NoK) (Figure 16E), construct 773 (HilD tag_CAR19_modSigPep) (Figure 16F), and construct 774 (HilD tag_CAR19) (Figure 16G). JNL cells expressing the indicated constructs were incubated with or without 10 μM lenalidomide for 24 h and then subjected to flow cytometry analysis. [Figure 16E]16A-C are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figure 16A), construct 771 (CAR19_HilD tag_V5) (Figure 16B), construct 6761 (CAR19_16KGS_HilD tag_V5) (Figure 16C), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 16D), construct 770 (CAR19_16GS_HilD tag_NoK) (Figure 16E), construct 773 (HilD tag_CAR19_modSigPep) (Figure 16F), and construct 774 (HilD tag_CAR19) (Figure 16G). JNL cells expressing the indicated constructs were incubated with or without 10 μM lenalidomide for 24 h and then subjected to flow cytometry analysis. [Figure 16F] 16A-C are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figure 16A), construct 771 (CAR19_HilD tag_V5) (Figure 16B), construct 6761 (CAR19_16KGS_HilD tag_V5) (Figure 16C), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 16D), construct 770 (CAR19_16GS_HilD tag_NoK) (Figure 16E), construct 773 (HilD tag_CAR19_modSigPep) (Figure 16F), and construct 774 (HilD tag_CAR19) (Figure 16G). JNL cells expressing the indicated constructs were incubated with or without 10 μM lenalidomide for 24 h and then subjected to flow cytometry analysis. [Figure 16G]16A-C are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figure 16A), construct 771 (CAR19_HilD tag_V5) (Figure 16B), construct 6761 (CAR19_16KGS_HilD tag_V5) (Figure 16C), construct 768 (CAR19_16GS_HilD tag_V5) (Figure 16D), construct 770 (CAR19_16GS_HilD tag_NoK) (Figure 16E), construct 773 (HilD tag_CAR19_modSigPep) (Figure 16F), and construct 774 (HilD tag_CAR19) (Figure 16G). JNL cells expressing the indicated constructs were incubated with or without 10 μM lenalidomide for 24 h and then subjected to flow cytometry analysis.

[0270] [Figure 17A] 17A-C are a set of flow cytometry histograms showing surface CAR expression regulated by lenalidomide and / or bazedoxifene (BZA). Constructs tested include construct 765 (FurON_CAR19) (FIG. 17A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 17B), and construct 766 (FurON_CAR19_16GS_HilD tag_V5) (FIG. 17C). JNL cells expressing the indicated constructs were incubated with or without lenalidomide and / or bazedoxifene (BZA) for 24 hours prior to flow cytometry analysis. [Figure 17B]17A-C are a set of flow cytometry histograms showing surface CAR expression regulated by lenalidomide and / or bazedoxifene (BZA). Constructs tested include construct 765 (FurON_CAR19) (FIG. 17A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 17B), and construct 766 (FurON_CAR19_16GS_HilD tag_V5) (FIG. 17C). JNL cells expressing the indicated constructs were incubated with or without lenalidomide and / or bazedoxifene (BZA) for 24 hours prior to flow cytometry analysis. [Figure 17C] 17A-C are a set of flow cytometry histograms showing surface CAR expression regulated by lenalidomide and / or bazedoxifene (BZA). Constructs tested include construct 765 (FurON_CAR19) (FIG. 17A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 17B), and construct 766 (FurON_CAR19_16GS_HilD tag_V5) (FIG. 17C). JNL cells expressing the indicated constructs were incubated with or without lenalidomide and / or bazedoxifene (BZA) for 24 hours prior to flow cytometry analysis.

[0271] [Figure 18A] 18A and 18B are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of various concentrations of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figures 18A and 18C) and construct 770 (CAR19_16GS_HilD tag_NoK) (Figures 18B and 18D). JNL cells expressing the indicated constructs were incubated in the presence or absence of lenalidomide for 4 hours (Figures 18A and 18B) or 20 hours (Figures 18C and 18D) prior to flow cytometry analysis. [Figure 18B]18A and 18B are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of various concentrations of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figures 18A and 18C) and construct 770 (CAR19_16GS_HilD tag_NoK) (Figures 18B and 18D). JNL cells expressing the indicated constructs were incubated in the presence or absence of lenalidomide for 4 hours (Figures 18A and 18B) or 20 hours (Figures 18C and 18D) prior to flow cytometry analysis. [Figure 18C] 18A and 18B are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of various concentrations of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figures 18A and 18C) and construct 770 (CAR19_16GS_HilD tag_NoK) (Figures 18B and 18D). JNL cells expressing the indicated constructs were incubated in the presence or absence of lenalidomide for 4 hours (Figures 18A and 18B) or 20 hours (Figures 18C and 18D) prior to flow cytometry analysis. [Figure 18D] 18A and 18B are a set of flow cytometry histograms showing surface CAR expression in the presence or absence of various concentrations of lenalidomide. Constructs tested include construct 769 (CAR19_16GS_HilD tag) (Figures 18A and 18C) and construct 770 (CAR19_16GS_HilD tag_NoK) (Figures 18B and 18D). JNL cells expressing the indicated constructs were incubated in the presence or absence of lenalidomide for 4 hours (Figures 18A and 18B) or 20 hours (Figures 18C and 18D) prior to flow cytometry analysis. [Figure 18E] 18E) or mean fluorescence intensity (FIG. 18F) for each cell line and each lenalidomide concentration tested. [Figure 18F]18E) or mean fluorescence intensity (FIG. 18F) for each cell line and each lenalidomide concentration tested.

[0272] [Figure 19A] Figure 19A is a set of bar graphs showing a comparison of lenalidomide response between JNL target cell line treatment conditions, length of time of target cell line treatment, time of lenalidomide treatment and number of cells. Figure 19A is a set of graphs showing luminescence signal from a study in which JNL cells (9000 or 12000 cells / well) expressing construct 769 (CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours or 24 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or media (no cells) for 4 hours, 8 hours or 20 hours. FIG. 19B is a set of graphs showing a subset of data from the study described in FIG. 19A: JNL cells (9000 cells / well) expressing construct 769 (CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or medium (no cells) for 20 hours. The y-axis in FIG. 19B shows the luminescence signal after subtraction of the background signal (signal from medium samples). In both FIG. 19A and 19B, the two bars in each graph represent the DMSO-treated sample ("DMSO") and the lenalidomide-treated sample ("Lenalidomide (10 μM)"), respectively. [Figure 19B]Figure 19A is a set of bar graphs showing a comparison of lenalidomide response between JNL target cell line treatment conditions, length of time of target cell line treatment, time of lenalidomide treatment and number of cells. Figure 19A is a set of graphs showing luminescence signal from a study in which JNL cells (9000 or 12000 cells / well) expressing construct 769 (CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours or 24 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or media (no cells) for 4 hours, 8 hours or 20 hours. FIG. 19B is a set of graphs showing a subset of data from the study described in FIG. 19A: JNL cells (9000 cells / well) expressing construct 769 (CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or medium (no cells) for 20 hours. The y-axis in FIG. 19B shows the luminescence signal after subtraction of the background signal (signal from medium samples). In both FIG. 19A and 19B, the two bars in each graph represent the DMSO-treated sample ("DMSO") and the lenalidomide-treated sample ("Lenalidomide (10 μM)"), respectively.

[0273] [Figure 20A]Figure 20A is a set of bar graphs showing a comparison of lenalidomide response between JNL target cell treatment conditions, length of time of target cell treatment, time of lenalidomide treatment and number of cells. Figure 20A is a set of graphs showing luminescence signal from a study in which JNL cells (9000 or 12000 cells / well) expressing construct 767 (FurON_CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours or 24 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or media (no cells) for 4 hours, 8 hours or 20 hours. Figure 20B is a set of graphs showing a subset of data from the study described in Figure 20A: JNL cells (9000 cells / well) expressing construct 767 (FurON_CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or media (no cells) for 20 hours. The y-axis in Figure 20B shows the luminescence signal after subtraction of the background signal (signal from media samples). In both Figures 20A and 20B, the four bars in each graph represent samples treated with neither lenalidomide nor bazedoxifene ("DMSO>>DMSO"), samples treated with bazedoxifene but not lenalidomide ("DMSO>>BZA(1 μM)"), samples treated with lenalidomide but not bazedoxifene ("Lenalidomide(10 μM)>>DMSO"), and samples treated with both lenalidomide and bazedoxifene ("Lenalidomide(10 μM)>>BZA(1 μM)"). [Figure 20B]Figure 20A is a set of bar graphs showing a comparison of lenalidomide response between JNL target cell treatment conditions, length of time of target cell treatment, time of lenalidomide treatment and number of cells. Figure 20A is a set of graphs showing luminescence signal from a study in which JNL cells (9000 or 12000 cells / well) expressing construct 767 (FurON_CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours or 24 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or media (no cells) for 4 hours, 8 hours or 20 hours. Figure 20B is a set of graphs showing a subset of data from the study described in Figure 20A: JNL cells (9000 cells / well) expressing construct 767 (FurON_CAR19_16GS_HilD tag) were treated with 10 μM lenalidomide for 4 hours, followed by incubation with Nalm6 cells, CD19-expressing K562 cells ("K562+CD19"), K562 cells or media (no cells) for 20 hours. The y-axis in Figure 20B shows the luminescence signal after subtraction of the background signal (signal from media samples). In both Figures 20A and 20B, the four bars in each graph represent samples treated with neither lenalidomide nor bazedoxifene ("DMSO>>DMSO"), samples treated with bazedoxifene but not lenalidomide ("DMSO>>BZA(1 μM)"), samples treated with lenalidomide but not bazedoxifene ("Lenalidomide(10 μM)>>DMSO"), and samples treated with both lenalidomide and bazedoxifene ("Lenalidomide(10 μM)>>BZA(1 μM)").

[0274] [Figure 21A]21A-D are graphs showing the dose-response effect of lenalidomide on the NFAT luciferase reporter over three treatment time points. JNL cells expressing construct 765 (FurON_CAR19) (FIG. 21A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 21B), construct 769 (CAR19_16GS_HilD tag) (FIG. 21C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 21D) were incubated with K562 target cells ("K562") or K562 target cells expressing CD19 ("K562+CD19"). Lenalidomide was added 20 hours before target cells were added (44 hours lenalidomide treatment, "20 hours pre-target cells"), 4 hours before target cells were added (28 hours lenalidomide treatment, "4 hours pre-target cells"), or 16 hours after target cells were added (8 hours lenalidomide treatment, "16 hours post-target cells"). JNL cells expressing construct 765 (FurON_CAR19) (Figure 21A) or construct 767 (FurON_CAR19_16GS_HilD tag) (Figure 21B) were also treated with bazedoxifene. In each graph, raw luminescence was plotted against the indicated lenalidomide concentration. [Figure 21B]21A-D are graphs showing the dose-response effect of lenalidomide on the NFAT luciferase reporter over three treatment time points. JNL cells expressing construct 765 (FurON_CAR19) (FIG. 21A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 21B), construct 769 (CAR19_16GS_HilD tag) (FIG. 21C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 21D) were incubated with K562 target cells ("K562") or K562 target cells expressing CD19 ("K562+CD19"). Lenalidomide was added 20 hours before target cells were added (44 hours lenalidomide treatment, "20 hours pre-target cells"), 4 hours before target cells were added (28 hours lenalidomide treatment, "4 hours pre-target cells"), or 16 hours after target cells were added (8 hours lenalidomide treatment, "16 hours post-target cells"). JNL cells expressing construct 765 (FurON_CAR19) (Figure 21A) or construct 767 (FurON_CAR19_16GS_HilD tag) (Figure 21B) were also treated with bazedoxifene. In each graph, raw luminescence was plotted against the indicated lenalidomide concentration. [Figure 21C]21A-D are graphs showing the dose-response effect of lenalidomide on the NFAT luciferase reporter over three treatment time points. JNL cells expressing construct 765 (FurON_CAR19) (FIG. 21A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 21B), construct 769 (CAR19_16GS_HilD tag) (FIG. 21C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 21D) were incubated with K562 target cells ("K562") or K562 target cells expressing CD19 ("K562+CD19"). Lenalidomide was added 20 hours before target cells were added (44 hours lenalidomide treatment, "20 hours pre-target cells"), 4 hours before target cells were added (28 hours lenalidomide treatment, "4 hours pre-target cells"), or 16 hours after target cells were added (8 hours lenalidomide treatment, "16 hours post-target cells"). JNL cells expressing construct 765 (FurON_CAR19) (Figure 21A) or construct 767 (FurON_CAR19_16GS_HilD tag) (Figure 21B) were also treated with bazedoxifene. In each graph, raw luminescence was plotted against the indicated lenalidomide concentration. [Figure 21D]21A-D are graphs showing the dose-response effect of lenalidomide on the NFAT luciferase reporter over three treatment time points. JNL cells expressing construct 765 (FurON_CAR19) (FIG. 21A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 21B), construct 769 (CAR19_16GS_HilD tag) (FIG. 21C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 21D) were incubated with K562 target cells ("K562") or K562 target cells expressing CD19 ("K562+CD19"). Lenalidomide was added 20 hours before target cells were added (44 hours lenalidomide treatment, "20 hours pre-target cells"), 4 hours before target cells were added (28 hours lenalidomide treatment, "4 hours pre-target cells"), or 16 hours after target cells were added (8 hours lenalidomide treatment, "16 hours post-target cells"). JNL cells expressing construct 765 (FurON_CAR19) (Figure 21A) or construct 767 (FurON_CAR19_16GS_HilD tag) (Figure 21B) were also treated with bazedoxifene. In each graph, raw luminescence was plotted against the indicated lenalidomide concentration.

[0275] [Figure 22A]22A, 21B, 21C, and 21D, in which JNL cells were treated with MG132 5 hours prior to treatment with K562+CD19 target cells and with lenalidomide 4 hours prior to treatment with K562+CD19 target cells. Cells tested included JNL cells expressing construct 765 (FurON_CAR19) (FIG. 22A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 22B), construct 769 (CAR19_16GS_HilD tag) (FIG. 22C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 22D). The four bars in each graph represent a sample treated with bazedoxifene (BZA), MG132 and lenalidomide ("BZA, MG132, lenalidomide"), a sample treated with bazedoxifene (BZA) and lenalidomide ("BZA, lenalidomide"), a sample treated with bazedoxifene (BZA) ("BZA") and a sample treated with DMSO only ("DMSO"). The y-axis in each graph represents raw luminescence. [Figure 22B]22A, 21B, 21C, and 21D, in which JNL cells were treated with MG132 5 hours prior to treatment with K562+CD19 target cells and with lenalidomide 4 hours prior to treatment with K562+CD19 target cells. Cells tested included JNL cells expressing construct 765 (FurON_CAR19) (FIG. 22A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 22B), construct 769 (CAR19_16GS_HilD tag) (FIG. 22C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 22D). The four bars in each graph represent a sample treated with bazedoxifene (BZA), MG132 and lenalidomide ("BZA, MG132, lenalidomide"), a sample treated with bazedoxifene (BZA) and lenalidomide ("BZA, lenalidomide"), a sample treated with bazedoxifene (BZA) ("BZA") and a sample treated with DMSO only ("DMSO"). The y-axis in each graph represents raw luminescence. [Figure 22C]22A, 21B, 21C, and 21D, in which JNL cells were treated with MG132 5 hours prior to treatment with K562+CD19 target cells and with lenalidomide 4 hours prior to treatment with K562+CD19 target cells. Cells tested included JNL cells expressing construct 765 (FurON_CAR19) (FIG. 22A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 22B), construct 769 (CAR19_16GS_HilD tag) (FIG. 22C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 22D). The four bars in each graph represent a sample treated with bazedoxifene (BZA), MG132 and lenalidomide ("BZA, MG132, lenalidomide"), a sample treated with bazedoxifene (BZA) and lenalidomide ("BZA, lenalidomide"), a sample treated with bazedoxifene (BZA) ("BZA") and a sample treated with DMSO only ("DMSO"). The y-axis in each graph represents raw luminescence. [Figure 22D]22A, 21B, 21C, and 21D, in which JNL cells were treated with MG132 5 hours prior to treatment with K562+CD19 target cells and with lenalidomide 4 hours prior to treatment with K562+CD19 target cells. Cells tested included JNL cells expressing construct 765 (FurON_CAR19) (FIG. 22A), construct 767 (FurON_CAR19_16GS_HilD tag) (FIG. 22B), construct 769 (CAR19_16GS_HilD tag) (FIG. 22C), or construct 770 (CAR19_16GS_HilD tag_NoK) (FIG. 22D). The four bars in each graph represent a sample treated with bazedoxifene (BZA), MG132 and lenalidomide ("BZA, MG132, lenalidomide"), a sample treated with bazedoxifene (BZA) and lenalidomide ("BZA, lenalidomide"), a sample treated with bazedoxifene (BZA) ("BZA") and a sample treated with DMSO only ("DMSO"). The y-axis in each graph represents raw luminescence.

[0276] [Diagram 23] 1 is a set of schematic diagrams showing HilD-tau fusion constructs. The 0N4R tau isoform was used, which contains the C-terminal repeat domain exon but not the N-terminal exon. Lentiviral constructs were used, but all constructs were introduced by lipofectamine transfection or nucleofection. Fusion products were expressed downstream of the CAG or CMV promoter.

[0277] [Figure 24A]24A-24D are graphs showing the design and results of a study testing the recruitment of the E3 ligase CRBN to HilD-tau fusion proteins. FIG. 24A: Diagram of the experiment. Lenalidomide recruits the E3 ligase cereblon (CRBN) to the IKZF3 β-hairpin, leading to ubiquitination and degradation of associated proteins. To test that this recruitment occurred in HilD-tau fusions, HilD-tau-biotin ligase fusions were made. In the presence of biotin, biotin ligase generates a reactive biotin species that covalently binds to nearby proteins. When lenalidomide is added, CRBN should be recruited to the HilD-tau fusion and should be within range of biotin ligase-mediated biotinylation. FIG. 24B: HEK293T cells were transfected with FLAG-tagged CRBN and HilD-tau-biotin ligase or tau-biotin ligase fusions. 48 hours after transfection, cells were treated with either 50 μM biotin and DMSO or 1 μM lenalidomide for 21 hours. Cells were then washed in PBS and subsequently lysed in ice-cold M-PER buffer and protease inhibitors. Approximately 1 million cells were estimated to be lysed in a volume of 300 μL. Western analysis of cell lysates is shown in the lower blots probed with anti-tau (HT7) or anti-GAPDH antibodies. Biotinylated proteins were immunoprecipitated by incubating 20% ​​cell lysate with 50 μL of streptavidin magnetic beads (Dynabeads M-280) for 30 minutes at room temperature. Biotinylated proteins were eluted from the beads by boiling and subsequently analyzed by Western. When probing for the FLAG signal on FLAG-CRBN, a strong band was observed only in immunoprecipitated material from lenalidomide-treated HEK293T cells containing the HilD tag, but not in DMSO-treated cells or cells treated with lenalidomide but transfected with a tau construct not containing the HilD tag. [Figure 24B]24A-24D are graphs showing the design and results of a study testing the recruitment of the E3 ligase CRBN to HilD-tau fusion proteins. FIG. 24A: Diagram of the experiment. Lenalidomide recruits the E3 ligase cereblon (CRBN) to the IKZF3 β-hairpin, leading to ubiquitination and degradation of associated proteins. To test that this recruitment occurred in HilD-tau fusions, HilD-tau-biotin ligase fusions were made. In the presence of biotin, biotin ligase generates a reactive biotin species that covalently binds to nearby proteins. When lenalidomide is added, CRBN should be recruited to the HilD-tau fusion and should be within range of biotin ligase-mediated biotinylation. FIG. 24B: HEK293T cells were transfected with FLAG-tagged CRBN and HilD-tau-biotin ligase or tau-biotin ligase fusions. 48 hours after transfection, cells were treated with either 50 μM biotin and DMSO or 1 μM lenalidomide for 21 hours. Cells were then washed in PBS and subsequently lysed in ice-cold M-PER buffer and protease inhibitors. Approximately 1 million cells were estimated to be lysed in a volume of 300 μL. Western analysis of cell lysates is shown in the lower blots probed with anti-tau (HT7) or anti-GAPDH antibodies. Biotinylated proteins were immunoprecipitated by incubating 20% ​​cell lysate with 50 μL of streptavidin magnetic beads (Dynabeads M-280) for 30 minutes at room temperature. Biotinylated proteins were eluted from the beads by boiling and subsequently analyzed by Western. When probing for the FLAG signal on FLAG-CRBN, a strong band was observed only in immunoprecipitated material from lenalidomide-treated HEK293T cells containing the HilD tag, but not in DMSO-treated cells or cells treated with lenalidomide but transfected with a tau construct not containing the HilD tag.

[0278] [Diagram 25]25A-C are graphs showing the reduction of toxic tau protein by inducible recruitment of the E3 ligase CRBN. HEK293T cells were transfected with a HilD-tau(P301S)-YFP fusion construct. Tau(P301S) is an aggregation-prone form of tau identified in patients with familial neurodegenerative disease. Upon overnight treatment with lenalidomide, YFP fluorescence was reduced by lenalidomide in a dose-dependent manner as seen in imaging of YFP fluorescence (FIG. 25A). Nine fields per condition are shown. FIG. 25B: YFP fluorescence intensity was quantified after lenalidomide treatment at various doses. FIG. 25C: Toxicity due to overexpression of aggregation-prone tau was observed and quantified by the number of cells identified by division of Hoecht dye fluorescence. Cell death was prevented by lenalidomide treatment and reduced tau levels, indicating that lenalidomide-induced degradation may reveal the cytoprotective effects of targeted proteolysis of toxic proteins.

[0279] [Figure 26A]26A-C are graphs showing quantification of the reduction of tau protein and specific forms of tau in HEK293T cells by inducible recruitment of CRBN. FIG. 26A: HEK293T cells were transfected with a HilD-tau (wild type) fusion construct and treated with various doses of either lenalidomide or DMSO. The top and bottom western blots are representative of experiments repeated in triplicate. The intensity of the tau bands derived from either a polyclonal anti-tau antibody (Dako) or an antibody against the phosphorylated form of tau (AT8) was quantified by normalization to the anti-actin band intensity. Transfection of reduced amounts of DNA in this experiment resulted in a greater reduction in phosphorylated forms of tau (1X DNA: 0.625 micrograms of DNA transfected in 50 μL of Optimem medium with 1.5 μL of Lipofectamine 2000; 0.1X DNA=0.0625 micrograms; for HEK cells in 24-well plates). This suggests that this system can measure the ability of E3 ligase-mediated degradation of tau. In the experiments shown, lenalidomide was administered 4 hours after transfection (for transfections with higher DNA concentrations) or 24 hours after transfection (for transfections with lower DNA concentrations). Figure 26B: Left panel, tau without the HilD tag was not reduced by lenalidomide treatment. Right panel, there was no reduction in tau levels by lenalidomide treatment in HEK293T cells knocked out for cereblon (CRBN). Figure 26C: Quantification of dose response of lenalidomide treatment on YFP intensity in cereblon (CRBN) knockout (KO) cells versus wild type (WT) cells (same data as shown in Figure 26B for wild type cells). Figure 26D: Co-treatment with the NEDD addition inhibitor MLN4924 (1 μM), including 1 hour pretreatment with MLN4924, also prevented tau degradation. Collectively, this data indicates that the E3 ligase function of CRBN is required for lenalidomide-induced HilD-tau fusion degradation. [Figure 26B]26A-C are graphs showing quantification of the reduction of tau protein and specific forms of tau in HEK293T cells by inducible recruitment of CRBN. FIG. 26A: HEK293T cells were transfected with a HilD-tau (wild type) fusion construct and treated with various doses of either lenalidomide or DMSO. The top and bottom western blots are representative of experiments repeated in triplicate. The intensity of the tau bands derived from either a polyclonal anti-tau antibody (Dako) or an antibody against the phosphorylated form of tau (AT8) was quantified by normalization to the anti-actin band intensity. Transfection of reduced amounts of DNA in this experiment resulted in a greater reduction in phosphorylated forms of tau (1X DNA: 0.625 micrograms of DNA transfected in 50 μL of Optimem medium with 1.5 μL of Lipofectamine 2000; 0.1X DNA=0.0625 micrograms; for HEK cells in 24-well plates). This suggests that this system can measure the ability of E3 ligase-mediated degradation of tau. In the experiments shown, lenalidomide was administered 4 hours after transfection (for transfections with higher DNA concentrations) or 24 hours after transfection (for transfections with lower DNA concentrations). Figure 26B: Left panel, tau without the HilD tag was not reduced by lenalidomide treatment. Right panel, there was no reduction in tau levels by lenalidomide treatment in HEK293T cells knocked out for cereblon (CRBN). Figure 26C: Quantification of dose response of lenalidomide treatment on YFP intensity in cereblon (CRBN) knockout (KO) cells versus wild type (WT) cells (same data as shown in Figure 26B for wild type cells). Figure 26D: Co-treatment with the NEDD addition inhibitor MLN4924 (1 μM), including 1 hour pretreatment with MLN4924, also prevented tau degradation. Collectively, this data indicates that the E3 ligase function of CRBN is required for lenalidomide-induced HilD-tau fusion degradation. [Figure 26C]26A-C are graphs showing quantification of the reduction of tau protein and specific forms of tau in HEK293T cells by inducible recruitment of CRBN. FIG. 26A: HEK293T cells were transfected with a HilD-tau (wild type) fusion construct and treated with various doses of either lenalidomide or DMSO. The top and bottom western blots are representative of experiments repeated in triplicate. The intensity of the tau bands derived from either a polyclonal anti-tau antibody (Dako) or an antibody against the phosphorylated form of tau (AT8) was quantified by normalization to the anti-actin band intensity. Transfection of reduced amounts of DNA in this experiment resulted in a greater reduction in phosphorylated forms of tau (1X DNA: 0.625 micrograms of DNA transfected in 50 μL of Optimem medium with 1.5 μL of Lipofectamine 2000; 0.1X DNA=0.0625 micrograms; for HEK cells in 24-well plates). This suggests that this system can measure the ability of E3 ligase-mediated degradation of tau. In the experiments shown, lenalidomide was administered 4 hours after transfection (for transfections with higher DNA concentrations) or 24 hours after transfection (for transfections with lower DNA concentrations). Figure 26B: Left panel, tau without the HilD tag was not reduced by lenalidomide treatment. Right panel, there was no reduction in tau levels by lenalidomide treatment in HEK293T cells knocked out for cereblon (CRBN). Figure 26C: Quantification of dose response of lenalidomide treatment on YFP intensity in cereblon (CRBN) knockout (KO) cells versus wild type (WT) cells (same data as shown in Figure 26B for wild type cells). Figure 26D: Co-treatment with the NEDD addition inhibitor MLN4924 (1 μM), including 1 hour pretreatment with MLN4924, also prevented tau degradation. Collectively, this data indicates that the E3 ligase function of CRBN is required for lenalidomide-induced HilD-tau fusion degradation. [Figure 26D]26A-C are graphs showing quantification of the reduction of tau protein and specific forms of tau in HEK293T cells by inducible recruitment of CRBN. FIG. 26A: HEK293T cells were transfected with a HilD-tau (wild type) fusion construct and treated with various doses of either lenalidomide or DMSO. The top and bottom western blots are representative of experiments repeated in triplicate. The intensity of the tau bands derived from either a polyclonal anti-tau antibody (Dako) or an antibody against the phosphorylated form of tau (AT8) was quantified by normalization to the anti-actin band intensity. Transfection of reduced amounts of DNA in this experiment resulted in a greater reduction in phosphorylated forms of tau (1X DNA: 0.625 micrograms of DNA transfected in 50 μL of Optimem medium with 1.5 μL of Lipofectamine 2000; 0.1X DNA=0.0625 micrograms; for HEK cells in 24-well plates). This suggests that this system can measure the ability of E3 ligase-mediated degradation of tau. In the experiments shown, lenalidomide was administered 4 hours after transfection (for transfections with higher DNA concentrations) or 24 hours after transfection (for transfections with lower DNA concentrations). Figure 26B: Left panel, tau without the HilD tag was not reduced by lenalidomide treatment. Right panel, there was no reduction in tau levels by lenalidomide treatment in HEK293T cells knocked out for cereblon (CRBN). Figure 26C: Quantification of dose response of lenalidomide treatment on YFP intensity in cereblon (CRBN) knockout (KO) cells versus wild type (WT) cells (same data as shown in Figure 26B for wild type cells). Figure 26D: Co-treatment with the NEDD addition inhibitor MLN4924 (1 μM), including 1 hour pretreatment with MLN4924, also prevented tau degradation. Collectively, this data indicates that the E3 ligase function of CRBN is required for lenalidomide-induced HilD-tau fusion degradation.

[0280] [Figure 27]1 is a set of graphs showing an assessment of the aggregation tendency of HilD-Tau(P301S)-YFP fusions expressed in rodent cortical neurons. Rodent cortical neurons were nucleofected with HilD-Tau(P301S)-YFP fusions and subsequently incubated with insoluble tau fractions isolated from in-house produced tau transgenic mice. Actual YFP fluorescence was imaged using an InCell 6000 analyzer. The middle and bottom panels show a magnification of the neurons identified in the top panel. Tau aggregates are clearly visible, as indicated by the intense punctate YFP fluorescence.

[0281] [Figure 28] This is a set of graphs showing lenalidomide-mediated degradation of HilD-tau(P301S)-YFP expressed in rat neurons. Rodent cortical neurons were nucleofected with HilD-tau(P301S)-YFP fusions or tau(P301S)-YFP fusions. Co-transfection with FLAG-tagged CRBN was also tested (top row). Starting on day 9 in vitro, neurons were treated with the indicated doses of lenalidomide. Neurons were live imaged for YFP fluorescence at the indicated intervals. Lenalidomide treatment reduced YFP fluorescence over time for HilD-tau(P301S)-YFP-expressing neurons treated with DMSO or tau(P301S)-YFP-expressing neurons treated with lenalidomide. Degradation occurred with or without co-transfection of human CRBN, indicating that HilD-tau fusions can be degraded by lenalidomide by either rodent or human CRBN.

[0282] [Figure 29]A set of graphs showing lenalidomide-mediated degradation of HilD-Tau(P301S)-YFP expressed in rat neuronal cells. Single cell suspensions of dissociated old human neurospheres, 63 days in vitro derived from embryonic stem cells, were nucleofected with HilD-Tau(P301S)-YFP. Neurospheres contain both neurons and neural progenitor cells. After 10 days of culture, neurons were treated with lenalidomide (total age 73 days in vitro). Images show YFP fluorescence after 20 hours of lenalidomide treatment at the indicated doses. Lenalidomide significantly reduced YFP fluorescence intensity in a dose-dependent manner.

[0283] [Diagram 30] Figure 30A is a set of graphs showing lenalidomide-mediated degradation of CAR19-16GS-HilD tag. Figure 30A is a set of graphs of Western blots of CAR19-HilD tag transduced Jurkat cells treated with a single dose of lenalidomide over time. Samples were tested after compound treatment or after a washout period. Figure 30B is a set of flow cytometry histograms analyzing the same samples used in the Western blot analysis. Anti-CD3 zeta antibody was used in the Western blot analysis, and CD19-PE conjugate was used in the flow cytometry analysis.

[0284] [Figure 31A] A set of flow cytometry histograms analyzing CAR expression under different conditions. Figure 31A is a set of flow cytometry histograms showing CAR expression in primary T cells. The effect of lenalidomide on CAR19 expression at 24 hours is shown in Figure 31B. The effect of lenalidomide on CAR19-HilD expression at 24 or 48 hours is shown in Figure 31C. [Figure 31B]A set of flow cytometry histograms analyzing CAR expression under different conditions. Figure 31A is a set of flow cytometry histograms showing CAR expression in primary T cells. The effect of lenalidomide on CAR19 expression at 24 hours is shown in Figure 31B. The effect of lenalidomide on CAR19-HilD expression at 24 or 48 hours is shown in Figure 31C. [Figure 31C] A set of flow cytometry histograms analyzing CAR expression under different conditions. Figure 31A is a set of flow cytometry histograms showing CAR expression in primary T cells. The effect of lenalidomide on CAR19 expression at 24 hours is shown in Figure 31B. The effect of lenalidomide on CAR19-HilD expression at 24 or 48 hours is shown in Figure 31C.

[0285] [Figure 32A]

[0036] Figure 32 is a set of graphs showing the percent killing mediated by CART cells. Figure 32A is a graph showing the percent killing against CD19 negative cells. Figures 32B and 32C are graphs showing the percent killing of CAR19 T cells (Figure 32B) or CAR19-HilD T cells (Figure 32C) against CD19 positive cells in the presence or absence of 1 μM lenalidomide. [Figure 32B]

[0036] Figure 32 is a set of graphs showing the percent killing mediated by CART cells. Figure 32A is a graph showing the percent killing against CD19 negative cells. Figures 32B and 32C are graphs showing the percent killing of CAR19 T cells (Figure 32B) or CAR19-HilD T cells (Figure 32C) against CD19 positive cells in the presence or absence of 1 μM lenalidomide. [Figure 32C]

[0036] Figure 32 is a set of graphs showing the percent killing mediated by CART cells. Figure 32A is a graph showing the percent killing against CD19 negative cells. Figures 32B and 32C are graphs showing the percent killing of CAR19 T cells (Figure 32B) or CAR19-HilD T cells (Figure 32C) against CD19 positive cells in the presence or absence of 1 μM lenalidomide.

[0286] [Diagram 33] Graph showing levels of IFN-gamma and IL2 secreted from T cells expressing CAR19 or CAR19-HilD in the presence or absence of 1 μM lenalidomide, respectively, with lenalidomide concentration in μM shown on the x-axis.

[0287] [Diagram 34] 1 is a graph showing that lenalidomide abolishes the ability of CART19.HilD to control tumor growth in vivo. Total flux of the ROI is plotted against days after Nalm6 implantation.

[0288] [Diagram 35] 1 is a set of flow cytometry plots showing the reduction of CAR19-HilD expression following lenalidomide treatment.

[0289] [Diagram 36] Graph showing the level of tumor control in the various treatment groups. Total flux of the ROI is plotted against days after Nalm6 implantation. Early injection of lenalidomide effectively abolished CART expression in mice treated with CART-HilD, resulting in a lack of tumor control in this group. Post-treatment of lenalidomide (day 5 after CART injection) also reduced CART function as shown by the loss of tumor control in this group of mice.

[0290] [Figure 37A]Graphs analyzing CAR expression in CD3+ cells derived from splenocytes. Figure 37A is a graph showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD (Group 1). Figures 37B, 37C, and 37D are graphs showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD and lenalidomide (Groups 2, 3, and 4, respectively). Peaks in Figures 37A-37D represent CD3 expression levels of individual mice. Group 1. CAR19.HilD(5x106). Group 2. CART19-HilD(5x106)+Lena qd. Group 3. CART19-HilD(5x106)+Lena bid. Group 4. CAR19.HilD(5x106)+Lena+5 days. Figure 37E is a graph summarizing the data. [Figure 37B] Graphs analyzing CAR expression in CD3+ cells derived from splenocytes. Figure 37A is a graph showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD (Group 1). Figures 37B, 37C, and 37D are graphs showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD and lenalidomide (Groups 2, 3, and 4, respectively). Peaks in Figures 37A-37D represent CD3 expression levels of individual mice. Group 1. CAR19.HilD(5x106). Group 2. CART19-HilD(5x106)+Lena qd. Group 3. CART19-HilD(5x106)+Lena bid. Group 4. CAR19.HilD(5x106)+Lena+5 days. Figure 37E is a graph summarizing the data. [Figure 37C]Graphs analyzing CAR expression in CD3+ cells derived from splenocytes. Figure 37A is a graph showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD (Group 1). Figures 37B, 37C, and 37D are graphs showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD and lenalidomide (Groups 2, 3, and 4, respectively). Peaks in Figures 37A-37D represent CD3 expression levels of individual mice. Group 1. CAR19.HilD(5x106). Group 2. CART19-HilD(5x106)+Lena qd. Group 3. CART19-HilD(5x106)+Lena bid. Group 4. CAR19.HilD(5x106)+Lena+5 days. Figure 37E is a graph summarizing the data. [Figure 37D] Graphs analyzing CAR expression in CD3+ cells derived from splenocytes. Figure 37A is a graph showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD (Group 1). Figures 37B, 37C, and 37D are graphs showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD and lenalidomide (Groups 2, 3, and 4, respectively). Peaks in Figures 37A-37D represent CD3 expression levels of individual mice. Group 1. CAR19.HilD(5x106). Group 2. CART19-HilD(5x106)+Lena qd. Group 3. CART19-HilD(5x106)+Lena bid. Group 4. CAR19.HilD(5x106)+Lena+5 days. Figure 37E is a graph summarizing the data. [Figure 37E]Graphs analyzing CAR expression in CD3+ cells derived from splenocytes. Figure 37A is a graph showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD (Group 1). Figures 37B, 37C, and 37D are graphs showing CAR expression in CD3+ cells derived from splenocytes of mice treated with CART-HilD and lenalidomide (Groups 2, 3, and 4, respectively). Peaks in Figures 37A-37D represent CD3 expression levels of individual mice. Group 1. CAR19.HilD(5x106). Group 2. CART19-HilD(5x106)+Lena qd. Group 3. CART19-HilD(5x106)+Lena bid. Group 4. CAR19.HilD(5x106)+Lena+5 days. Figure 37E is a graph summarizing the data.

[0291] [Figure 38A] 38A is a graph showing the effect of compound I-112 on the expression and activity of the CAR19-CARB tag. Figure 38A is a Western blot of Jurkat NFAT luciferase (JNL) cells expressing the CAR19-CARB tag treated with various doses of compound I-112 or DMSO for 24 hours, showing the dose-response degradation of the CAR19-CARB tag. Figure 38B is a set of histograms showing flow cytometry analysis of the surface expression of CAR19 in JNL CAR19-CARB tag cells compared to untagged CAR19 cells after treatment with 10 μM compound I-112. Figure 38C is a graph showing the results of a JNL assay in which JNL luciferase cells expressing the CAR19-CARB tag were treated with a dose-response of compound I-112 for 15 hours, followed by co-treatment with either K562 (CD19-) or Nalm6 (CD19+) cells with a luciferase activity readout. [Figure 38B]38A is a graph showing the effect of compound I-112 on the expression and activity of the CAR19-CARB tag. Figure 38A is a Western blot of Jurkat NFAT luciferase (JNL) cells expressing the CAR19-CARB tag treated with various doses of compound I-112 or DMSO for 24 hours, showing the dose-response degradation of the CAR19-CARB tag. Figure 38B is a set of histograms showing flow cytometry analysis of the surface expression of CAR19 in JNL CAR19-CARB tag cells compared to untagged CAR19 cells after treatment with 10 μM compound I-112. Figure 38C is a graph showing the results of a JNL assay in which JNL luciferase cells expressing the CAR19-CARB tag were treated with a dose-response of compound I-112 for 15 hours, followed by co-treatment with either K562 (CD19-) or Nalm6 (CD19+) cells with a luciferase activity readout. [Figure 38C] 38A is a graph showing the effect of compound I-112 on the expression and activity of the CAR19-CARB tag. Figure 38A is a Western blot of Jurkat NFAT luciferase (JNL) cells expressing the CAR19-CARB tag treated with various doses of compound I-112 or DMSO for 24 hours, showing the dose-response degradation of the CAR19-CARB tag. Figure 38B is a set of histograms showing flow cytometry analysis of the surface expression of CAR19 in JNL CAR19-CARB tag cells compared to untagged CAR19 cells after treatment with 10 μM compound I-112. Figure 38C is a graph showing the results of a JNL assay in which JNL luciferase cells expressing the CAR19-CARB tag were treated with a dose-response of compound I-112 for 15 hours, followed by co-treatment with either K562 (CD19-) or Nalm6 (CD19+) cells with a luciferase activity readout.

[0292] [Figure 39]Western blot of HEK293T cells transiently transfected with CARB tag-MITF-FLAG and treated with 10 μM, 1 μM, 0.1 μM, or 0.01 μM of compound I-112 or lenalidomide or DMSO, showing I-112-specific degradation of CARB-tagged MITF.

[0293] [Figure 40A] Graphs analyzing the effect of lenalidomide on BCMACAR19-HilD tag expression and activity. Figure 40A is a set of histograms showing flow cytometry analysis of JNL cells infected with BCMACAR HilD-tag treated with a lenalidomide dose response for 24 hours, showing lenalidomide dose-dependent degradation of BCMACAR. Figure 40B is a graph showing a JNL assay in which Jurkat NFAT luciferase cells expressing BCMA-HilD tag were treated with a lenalidomide dose response for 15 hours, followed by co-treatment with KMS11 cells with luciferase activity readout. [Figure 40B] Graphs analyzing the effect of lenalidomide on BCMACAR19-HilD tag expression and activity. Figure 40A is a set of histograms showing flow cytometry analysis of JNL cells infected with BCMACAR HilD-tag treated with a lenalidomide dose response for 24 hours, showing lenalidomide dose-dependent degradation of BCMACAR. Figure 40B is a graph showing a JNL assay in which Jurkat NFAT luciferase cells expressing BCMA-HilD tag were treated with a lenalidomide dose response for 15 hours, followed by co-treatment with KMS11 cells with luciferase activity readout. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0294] The present disclosure provides, at least in part, a fusion polypeptide comprising a compound of formula (I) (COF1) / CRBN binding polypeptide, a compound of formula (II) (COF2) / CRBN binding polypeptide, or a compound of formula (III) (COF3) / CRBN binding polypeptide for targeted protein inactivation. In some embodiments, the fusion polypeptide comprises one or more COF1 / CRBN, COF2 / CRBN, or COF3 / CRBN binding polypeptides and one or more heterologous polypeptides, such as heterologous mammalian, bacterial, or viral polypeptides, such as one or more polypeptides of interest. The COF1 / CRBN, COF2 / CRBN, or COF3 / CRBN binding polypeptides can be operably linked to the heterologous polypeptides, for example, via a linker. In some embodiments, in the presence of COF1 or COF2 (such as thalidomide and its derivatives (e.g., lenalidomide, pomalidomide, and thalidomide)) or in the presence of COF3 (e.g., compounds disclosed in Table 29), the COF1 / CRBN, COF2 / CRBN, or COF3 / CRBN binding polypeptide increases the degradation, e.g., ubiquitination-mediated degradation, of the fusion polypeptide; and / or alters the level and / or activity of the fusion polypeptide. In some embodiments, the degradation of the fusion polypeptide is ubiquitin-dependent.

[0295] Without being bound by theory, in some embodiments, the COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide provides an amino acid sequence and / or structural motif that results in post-translational modification (e.g., ubiquitination) of the fusion polypeptide in the presence of COF1 or COF2 (such as thalidomide and its derivatives (e.g., lenalidomide, pomalidomide and thalidomide)) or in the presence of COF3 (e.g., compounds disclosed in Table 29) to result in a modified, e.g., ubiquitinated, fusion polypeptide. For example, one or more amino acids, e.g., lysine or methionine, in the fusion polypeptide can be ubiquitinated in the presence of COF1, COF2 or COF3. In some embodiments, the ubiquitinated fusion polypeptide is selectively degraded. In some embodiments, the post-translational modification of the fusion polypeptide increases degradation (e.g., an increased level and / or rate of degradation) of the fusion polypeptide (e.g., all or a portion of a heterologous polypeptide). In some embodiments, the increased level and / or rate of degradation is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 500%, 10-fold, 100-fold, 1,000-fold or more than the level and / or rate of degradation of a reference protein, e.g., a fusion polypeptide in the absence of COF1, COF2 or COF3, a heterologous polypeptide, a fusion of a heterologous polypeptide without a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide, or a fusion of a heterologous polypeptide with a moiety other than a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide.

[0296] Without being bound by theory, degradation of the fusion polypeptide is believed to occur through the following steps: (1) binding of COF1 or COF2 (e.g., thalidomide and its derivatives (e.g., lenalidomide)) or COF3 (e.g., compounds disclosed in Table 29) to one or more subunits of a ubiquitin ligase complex (e.g., an E3 ubiquitin ligase complex), e.g., CUL4, RBX1, DDBI and / or CRBN (also known as CRL4(CRBN)), typically a DDB1-CRBN complex, to form a COF1-ligase or COF2-ligase complex; (2) a COF1-ligase, a COF2-ligase or a COF3-ligase complex binds to and increases the ubiquitination of one or more amino acids, e.g., lysine or methionine, in the fusion polypeptide to form a ubiquitinated fusion polypeptide, e.g., a mono- or polyubiquitinated fusion polypeptide; and (3) The ubiquitinated fusion polypeptide is targeted for degradation, e.g., the fusion polypeptide is selectively targeted for degradation, e.g., to the proteasome. It may include one, two or all of the above.

[0297] In some embodiments, the COF1 / CRBN or COF2 / CRBN binding polypeptide comprises about 10 to about 95 amino acid residues, about 15 to about 90 amino acid residues, about 20 to about 85 amino acid residues, about 25 to about 80 amino acid residues, about 30 to about 75 amino acid residues, about 35 to about 70 amino acid residues, about 40 to about 65 amino acid residues, about 45 to about 65 amino acid residues, about 50 to about 65 amino acid residues, or about 55 to about 65 amino acid residues of IKZF1 (e.g., SEQ ID NO: 20) or IKZF3 (e.g., SEQ ID NO: 19).

[0298] In some embodiments, the COF3 / CRBN binding polypeptide comprises about 10 to about 95 amino acid residues, about 15 to about 90 amino acid residues, about 20 to about 85 amino acid residues, about 25 to about 80 amino acid residues, about 30 to about 75 amino acid residues, about 35 to about 70 amino acid residues, about 40 to about 65 amino acid residues, about 45 to about 65 amino acid residues, about 50 to about 65 amino acid residues, or about 55 to about 65 amino acid residues of IKZF2 (e.g., SEQ ID NO: 21).

[0299] In some embodiments, a COF1 / CRBN or COF2 / CRBN binding polypeptide comprises a β-turn (e.g., the β-turn of IKZF3). In some embodiments, a COF1 / CRBN or COF2 / CRBN binding polypeptide comprises a β-turn (e.g., the β-turn of IKZF3) and an α-helix (e.g., the α-helix of IKZF3). In some embodiments, a COF1 / CRBN or COF2 / CRBN binding polypeptide comprises amino acid residues 136-170 or 136-180 and / or 236-249 of IKZF3 (numbered according to SEQ ID NO: 19), or an amino acid sequence substantially identical thereto (e.g., at least 85, 87, 90, 95, 97, 98, 99 or 100% identical thereto). In some embodiments, the COF1 / CRBN or COF2 / CRBN binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100, or an amino acid sequence substantially identical thereto (e.g., at least 85, 87, 90, 95, 97, 98, 99 or 100% identical thereto).

[0300] In some embodiments, a COF3 / CRBN binding polypeptide comprises a β-turn (e.g., an IKZF2 β-turn). In some embodiments, a COF3 / CRBN binding polypeptide comprises a β-turn (e.g., an IKZF2 β-turn) and an α-helix (e.g., an IKZF2 α-helix). In some embodiments, a COF3 / CRBN binding polypeptide comprises amino acid residues 130-174 and / or 230-243 of IKZF2 (numbered according to SEQ ID NO:21), or an amino acid sequence substantially identical thereto (e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto). In some embodiments, the COF3 / CRBN binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 109, 113, and 114, or an amino acid sequence substantially identical thereto (e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto).

[0301] In some embodiments, a COF1 / CRBN or COF2 / CRBN binding polypeptide comprises a β-turn (e.g., an IKZF1 β-turn). In some embodiments, a COF1 / CRBN or COF2 / CRBN binding polypeptide comprises a β-turn (e.g., an IKZF1 β-turn) and an α-helix (e.g., an IKZF1 α-helix).

[0302] In some embodiments, the heterologous polypeptide of the fusion polypeptide is susceptible to post-translational modification (e.g., ubiquitination at one or more residues) and degradation in the presence of COF1 or COF2 (e.g., thalidomide and its derivatives, such as lenalidomide, pomalidomide and thalidomide) or in the presence of COF3 (e.g., the compounds disclosed in Table 29).

[0303] Optionally, the COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide and the heterologous polypeptide can be operably linked, for example, via a linker, such as a glycine-serine linker (e.g., SEQ ID NOs: 28, 37, 38, 39 or 99). For example, a fusion polypeptide can include three elements: a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide, such as a portion of a degradation amino acid sequence (e.g., a degron), a heterologous polypeptide of interest to be degraded, and a linker separating the two. The heterologous polypeptide can be a cytosolic protein, a nuclear protein, a transmembrane protein (e.g., containing one or more transmembrane domains), or a secreted protein. For example, a heterologous polypeptide of interest can include, e.g., a chimeric antigen receptor (CAR), a CRISPR-associated protein, CD8, CD19, CD22, a transcription factor (e.g., STAT3, STAT5, NF-kappaB, beta-catenin, Notch, GLI, or c-JUN), e.g., as described herein.

[0304] In some embodiments, the fusion polypeptide of the invention further comprises a degradation domain. In some embodiments, the degradation domain has a first state associated with a first level of expression of the fusion polypeptide and a second state associated with a second level of expression of the fusion polypeptide, the second level being increased, for example, in the presence of a stabilizing compound, by at least 2, 3, 4, 5, 10, 20, or 30 times the first level. In some embodiments, the degradation domain is separated from the COF1 / CRBN binding polypeptide and the heterologous polypeptide by a heterologous cleavage site. In some embodiments, the degradation domain is separated from the COF2 / CRBN binding polypeptide and the heterologous polypeptide by a heterologous cleavage site. In some embodiments, the degradation domain is separated from the COF3 / CRBN binding polypeptide and the heterologous polypeptide by a heterologous cleavage site.

[0305] In some embodiments, the fusion polypeptide comprises a first domain and a second domain, the first domain comprises a degradation domain, and the second domain comprises a COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptide and a heterologous polypeptide. In some embodiments, the first domain and the second domain are separated by a heterologous cleavage site. Without being bound by theory, the expression level of the fusion polypeptide may be controlled by a stabilizing compound and COF1, COF2 or COF3. In some embodiments, in the absence of a stabilizing compound, the degradation domain is unable to adopt a proper conformation and is targeted for degradation by an intracellular degradation pathway along with the remainder of the fusion polypeptide. In some embodiments, in the presence of a stabilizing compound, the degradation domain adopts a proper conformation and is less susceptible to degradation by an intracellular degradation pathway. In some embodiments, in the presence of a stabilizing compound, proper folding of the degradation domain exposes the heterologous cleavage site, subject to cleavage of the heterologous cleavage site and removal of the degradation domain from the remainder of the fusion polypeptide. The level of the fusion polypeptide may further be controlled by COF1, COF2 or COF3 as described above.

[0306] In some embodiments, the degradation domain is selected from an estrogen receptor (ER) domain, an FKB protein (FKBP) domain, or a dihydrofolate reductase (DHFR) domain. In some embodiments, the degradation domain is an estrogen receptor (ER) domain and comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 46 or 48, e.g., the degradation domain comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the degradation domain is an estrogen receptor (ER) domain and the stabilizing compound is bazedoxifene or 4-hydroxytamoxifen (4-OHT). In some embodiments, the degradation domain is an FKB protein (FKBP) domain and comprises an amino acid sequence that is at least 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 50, e.g., the degradation domain comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the degradation domain is an FKB protein (FKBP) domain and the stabilizing compound is Shield-1. In some embodiments, the degradation domain is a dihydrofolate reductase (DHFR) domain, e.g., the degradation domain comprises an amino acid sequence that is at least 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 51, e.g., the degradation domain comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the degradation domain is a dihydrofolate reductase (DHFR) domain and the stabilizing compound is trimethoprim.

[0307] Thus, disclosed herein are fusion polypeptides comprising heterologous polypeptides, COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptides and / or degradation domains, such as polypeptides of interest for selective protein degradation, as well as nucleic acid molecules encoding the fusion polypeptides, vectors and cells, such as host cells comprising the above-mentioned fusion polypeptides. The fusion polypeptides and related compositions disclosed herein can be used to activate or inactivate, e.g., degrade, various target proteins for therapeutic purposes, such as controlling secretory, cellular or transmembrane therapies (e.g., CAR therapy), controlling gene expression (e.g., via controlling the expression and / or activity of components of the CRISPR / CAS system), target validation and library screening. Further disclosed are methods for selectively controlling (e.g., degrading) said fusion polypeptides, for example, for treating a subject.

[0308] The compositions and methods disclosed herein provide novel and innovative features over regulatory systems known in the art, including the fact that COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptides act at the protein level (as opposed to mRNA) and cause active degradation of existing and newly made proteins in the cell (as opposed to blocking the production of nascent proteins). In addition, COF1 / CRBN, COF2 / CRBN or COF3 / CRBN binding polypeptides can be short in length, and COF1, COF2 and COF3 are typically low molecular weight.

[0309] Without being bound by theory, as described in Example 16, COF1 or COF2 (e.g., thalidomide and its derivatives (e.g., lenalidomide, pomalidomide and thalidomide)) does not cause or does not substantially cause degradation of a fusion polypeptide comprising a COF3 / CRBN binding polypeptide described herein (e.g., a fusion polypeptide comprising a CARB tag described herein, e.g., a fusion polypeptide comprising a CARB tag comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 109, 113 and 114). In some embodiments, the degradation of a fusion polypeptide comprising a COF3 / CRBN binding polypeptide described herein in the presence of COF1 or COF2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20% or less of the degradation of the fusion polypeptide in the presence of COF3 under the same conditions.

[0310] Similarly, COF3 (e.g., a compound disclosed in Table 29) does not cause or does not substantially cause degradation of a fusion polypeptide comprising a COF1 / CRBN or COF2 / CRBN binding polypeptide described herein (e.g., a fusion polypeptide comprising a HilD tag described herein, e.g., a fusion polypeptide comprising a HilD tag comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 11-15, 40, 41-43, 77, 78, 84-86, and 100). In some embodiments, degradation of a fusion polypeptide comprising a COF1 / CRBN or COF2 / CRBN binding polypeptide described herein in the presence of COF3 is no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20% of the degradation of said fusion polypeptide in the presence of COF1 or COF2 under the same conditions.

[0311] Thus, two target polypeptides, one tagged with a COF1 / CRBN or COF2 / CRBN binding polypeptide (e.g., the HilD tag described herein) and the other tagged with a COF3 / CRBN binding polypeptide (e.g., the CARB tag described herein), can be independently regulated using COF1 or COF2 and COF3. For example, a cell expressing a HilD-tagged protein and a CARB-tagged protein can be engineered to express only the HilD-tagged protein (e.g., by contacting the cell with COF3), to express only the CARB-tagged protein (e.g., by contacting the cell with COF1 or COF2), or to express neither protein (e.g., by contacting the cell with COF1 or COF2 and COF3).

[0312] definition Unless otherwise defined, 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 pertains.

[0313] As used herein, the term "compound of formula (I) (COF1) / CRBN binding polypeptide" refers to a polypeptide that binds to COF1, a polypeptide that binds to a complex of COF1 and CRBN, or a polypeptide that binds to CRBN in the presence of COF1. In some embodiments, a COF1 / CRBN binding polypeptide has a binding activity of at least 10, as measured, for example, by art-recognized methods, e.g., Biacore. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D In some embodiments, the COF1 / CRBN binding polypeptide binds to COF1 with a CRBN affinity of 10 or more, e.g., as measured by art-recognized methods, e.g., Biacore. -3 , 10 -4 , 10 -5 , 10 -6 , 10-7 or 10 -8 Affinity (K D ) and binds to the complex of COF1 and CRBN. In some embodiments, the COF1 / CRBN binding polypeptide has a binding affinity of 10 or more, e.g., as measured by art-recognized methods, e.g., Biacore. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D) and binds to CRBN in the presence of COF1. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF1 / CRBN binding polypeptide can result in increased ubiquitination of the fusion polypeptide. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF1 / CRBN binding polypeptide can result in increased degradation of the fusion polypeptide. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF1 / CRBN binding polypeptide can result in increased inactivation of the fusion polypeptide. In some embodiments, increased ubiquitination, degradation and / or inactivation occurs in the presence of COF1 and one or more components of a ubiquitination ligase complex (e.g., CRBN). In some embodiments, the increase in ubiquitination, degradation and / or inactivation is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 500%, 10-fold, 100-fold, 1,000-fold or more greater than the ubiquitination, degradation and / or inactivation of a reference polypeptide, e.g., a reference fusion polypeptide with a COF1 / CRBN binding polypeptide or a reference polypeptide without a COF1 / CRBN binding polypeptide in the absence of COF1. In some embodiments, degradation of a fusion polypeptide containing a COF1 / CRBN binding polypeptide is ubiquitin-dependent. For example, one or more amino acids, eg, lysine or methionine, in a fusion polypeptide involving a COF1 / CRBN binding polypeptide is ubiquitinated in the presence of COF1.

[0314] As used herein, the term "compound of formula (II) (COF2) / CRBN binding polypeptide" refers to a polypeptide that binds to COF2, a polypeptide that binds to a complex of COF2 and CRBN, or a polypeptide that binds to CRBN in the presence of COF2. In some embodiments, a COF2 / CRBN binding polypeptide has a binding activity of at least 10, as measured, for example, by art-recognized methods, e.g., Biacore. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D In some embodiments, the COF2 / CRBN binding polypeptide has a 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D In some embodiments, the COF2 / CRBN binding polypeptide has a binding affinity of 10 to 150, e.g., as measured by art-recognized methods, e.g., Biacore. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D) and binds to CRBN in the presence of COF2. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF2 / CRBN binding polypeptide can result in increased ubiquitination of the fusion polypeptide. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF2 / CRBN binding polypeptide can result in increased degradation of the fusion polypeptide. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF2 / CRBN binding polypeptide can result in increased inactivation of the fusion polypeptide. In some embodiments, increased ubiquitination, degradation and / or inactivation occurs in the presence of COF2 and one or more components of a ubiquitination ligase complex (e.g., CRBN). In some embodiments, the increase in ubiquitination, degradation and / or inactivation is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 500%, 10-fold, 100-fold, 1,000-fold or more greater than the ubiquitination, degradation and / or inactivation of a reference polypeptide, e.g., a reference fusion polypeptide with a COF2 / CRBN binding polypeptide in the absence of COF2 or a reference polypeptide without a COF2 / CRBN binding polypeptide. In some embodiments, degradation of a fusion polypeptide containing a COF2 / CRBN binding polypeptide is ubiquitin-dependent. For example, one or more amino acids, such as lysine or methionine, in a fusion polypeptide involving a COF2 / CRBN binding polypeptide is ubiquitinated in the presence of COF2.

[0315] As used herein, the term "compound of formula (III) (COF3) / CRBN binding polypeptide" refers to a polypeptide that binds to COF3, a polypeptide that binds to a complex of COF3 and CRBN, or a polypeptide that binds to CRBN in the presence of COF3. In some embodiments, a COF3 / CRBN binding polypeptide has a binding activity of at least 10, as measured, for example, by art-recognized methods, e.g., Biacore. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D In some embodiments, the COF3 / CRBN binding polypeptide has a 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D ) and binds to the COF3 and CRBN complex. In some embodiments, the COF3 / CRBN binding polypeptide has a binding affinity of 10 or more, e.g., as measured by art-recognized methods, e.g., Biacore. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 Affinity (K D) and binds to CRBN in the presence of COF3. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF3 / CRBN binding polypeptide can result in increased ubiquitination of the fusion polypeptide. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF3 / CRBN binding polypeptide can result in increased degradation of the fusion polypeptide. In some embodiments, when present in a fusion polypeptide (e.g., operably linked to a heterologous polypeptide (e.g., a fusion polypeptide as described herein)), the COF3 / CRBN binding polypeptide can result in increased inactivation of the fusion polypeptide. In some embodiments, increased ubiquitination, degradation and / or inactivation occurs in the presence of COF3 and one or more components of a ubiquitination ligase complex (e.g., CRBN). In some embodiments, the increase in ubiquitination, degradation and / or inactivation is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 500%, 10-fold, 100-fold, 1,000-fold or more greater than the ubiquitination, degradation and / or inactivation of a reference polypeptide, e.g., a reference fusion polypeptide with a COF3 / CRBN binding polypeptide in the absence of COF3 or a reference polypeptide without a COF3 / CRBN binding polypeptide. In some embodiments, degradation of a fusion polypeptide containing a COF3 / CRBN binding polypeptide is ubiquitin-dependent. For example, one or more amino acids, such as lysine or methionine, in a fusion polypeptide involving a COF3 / CRBN binding polypeptide is ubiquitinated in the presence of COF3.

[0316] As used herein, "ubiquitination" refers to the addition of a ubiquitin molecule, e.g., a single ubiquitin (mono-ubiquitination) or two or more ubiquitins (e.g., a chain of ubiquitin molecules or poly-ubiquitination). Ubiquitination can be carried out by an enzymatic machinery that includes one or more of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3).

[0317] As used herein, the term "CRBN" refers to the protein encoded by the CRBN gene in humans, or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto). Swiss-Prot Accession Number Q96SW2 provides an exemplary human CRBN amino acid sequence.

[0318] As used herein, "IKZF polypeptide" refers to IKZF or a fragment or variant thereof (eg, an amino acid sequence substantially identical thereto, eg, at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto).

[0319] As used herein, the term "IKZF3" refers to the protein encoded by the IKZF3 gene in humans. Swiss-Prot Accession Number Q9UKT9 provides an exemplary human IKZF3 amino acid sequence. An exemplary human IKZF3 amino acid sequence is provided by SEQ ID NO: 19. The term "IKZF3 polypeptide" refers to IKZF3 or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto).

[0320] As used herein, the term "IKZF1" refers to the protein encoded by the IKZF1 gene in humans. Swiss-Prot Accession Number Q13422 provides an exemplary human IKZF1 amino acid sequence. An exemplary human IKZF1 amino acid sequence is provided by SEQ ID NO: 20. The term "IKZF1 polypeptide" refers to IKZF1 or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto).

[0321] As used herein, the term "IKZF2" refers to the protein encoded by the IKZF2 gene in humans. Swiss-Prot Accession Number Q9UKS7 provides an exemplary human IKZF2 amino acid sequence. An exemplary human IKZF2 amino acid sequence is provided by SEQ ID NO:21. The term "IKZF2 polypeptide" refers to IKZF2 or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto).

[0322] As used herein, the term "IKZF4" refers to the protein encoded by the IKZF4 gene in humans. Swiss-Prot Accession Number Q9H2S9 provides an exemplary human IKZF4 amino acid sequence. An exemplary human IKZF4 amino acid sequence is provided by SEQ ID NO: 22. The term "IKZF4 polypeptide" refers to IKZF4 or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto).

[0323] As used herein, the term "IKZF5" refers to the protein encoded by the IKZF5 gene in humans. Swiss-Prot Accession Number Q9H5V7 provides an exemplary human IKZF5 amino acid sequence. An exemplary human IKZF5 amino acid sequence is provided by SEQ ID NO: 23. The term "IKZF5 polypeptide" refers to IKZF5 or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto).

[0324] As used herein, a "fusion polypeptide" or "chimeric polypeptide" refers to a polypeptide that comprises two or more heterologous amino acid sequences and / or protein domains in a single contiguous polypeptide. In some embodiments, the two or more heterologous protein domains are covalently linked directly or indirectly, for example, via a linker.

[0325] As used herein, the term "estrogen receptor (ER)" refers to a protein encoded by the ESR1 gene in humans. Swiss-Prot Accession Number P03372 provides an exemplary human estrogen receptor (ER) amino acid sequence. An "estrogen receptor (ER) domain" refers to an estrogen receptor or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., at least 85, 87, 90, 95, 97, 98, 99, or 100% identical thereto). Exemplary estrogen receptor (ER) domain amino acid sequences are provided in SEQ ID NOs: 44, 46, and 48. Exemplary estrogen receptor (ER) domain nucleotide sequences are provided in SEQ ID NOs: 45, 47, and 49.

[0326] As used herein, "FKB protein (FKBP) domain" refers to FKBP or a fragment or variant thereof. An exemplary FKB protein (FKBP) domain amino acid sequence is provided by SEQ ID NO:50.

[0327] As used herein, the term "dihydrofolate reductase (DHFR)" refers to the protein encoded by the DHFR gene in humans. Swiss-Prot Accession Number P00374 provides an exemplary dihydrofolate reductase (DHFR) amino acid sequence. "Dihydrofolate reductase (DHFR) domain" refers to DHFR or a fragment or variant thereof. An exemplary dihydrofolate reductase (DHFR) domain amino acid sequence is provided by SEQ ID NO:51.

[0328] As used herein, the term "degradation domain" refers to a domain of a fusion polypeptide that adopts a stable conformation when expressed in the presence of a stabilizing compound. Most degradation domains (and typically any proteins to which they are fused) that do not adopt a stable conformation when expressed in a cell of interest will be degraded by endogenous cellular machinery. Of note, the degradation domain is not a domain of a naturally occurring protein, but is designed to be unstable in the absence of contact with a stabilizing compound. Thus, a degradation domain can be identified by the following characteristics: (1) it is not naturally occurring; (2) its expression is co-translationally or post-translationally controlled by an increase or decrease in the rate of degradation; (3) the rate of degradation is substantially decreased in the presence of a stabilizing compound. In some embodiments, the degradation domain or other domains of the fusion polypeptide are substantially undetectable in or on a cell in the absence of a stabilizing compound. In some embodiments, the degradation domain is in a destabilized state in the absence of a stabilizing compound. In some embodiments, the degradation domain does not self-associate, e.g., does not homodimerize, in the absence of a stabilizing compound. In some embodiments, the degradation domain is fused to a heterologous protease cleavage site, such that in the presence of a stabilizing compound, cleavage of the heterologous protease cleavage site is more efficient than in the absence of the stabilizing compound.

[0329] A degradation domain is not an aggregation domain as defined in PCT Application No. PCT / US2017 / 027778.

[0330] "Stabilizing compound" or "stabilizing compound" means a compound that, when added to a cell expressing a degradation domain, stabilizes the degradation domain and any protein fused to it, reducing the rate at which it is subsequently degraded. Stabilizing compounds or stabilizing compounds can be naturally occurring or synthetic.

[0331] The term "heterologous polypeptide" refers to an amino acid sequence (e.g., a protein domain) that differs (e.g., by at least one amino acid) from a COF1 / CRBN, COF2 / CRBN, or COF3 / CRBN binding polypeptide and is not an active luciferase domain or has a luciferase sequence. In some embodiments, the heterologous polypeptide is not a reporter polypeptide, e.g., luciferase, green fluorescent protein, or b-galactosidase. In some embodiments, the heterologous polypeptide comprises an amino acid sequence from or derived from a mammalian polypeptide, a bacterial polypeptide, a viral polypeptide, a plant polypeptide, a yeast polypeptide, a fungal polypeptide, an archaeal polypeptide, or a fish, e.g., a zebrafish polypeptide. In some embodiments, the heterologous polypeptide comprises a polypeptide in Table 2, e.g., a cytoplasmic and / or nuclear polypeptide, a secreted polypeptide, or a transmembrane polypeptide as set forth in Table 2.

[0332] Furthermore, a "heterologous protease cleavage site" refers to a protease cleavage site that has a different origin than one or more protein domains to which it is fused (e.g., is not naturally fused to at least one of the other referenced domains).

[0333] "Protease" means a protein that cleaves another protein based on the presence of a cleavage site in the protein to be cleaved.

[0334] By "intracellular protease" is meant a protease that is naturally expressed inside the cell of interest.

[0335] By "extracellular protease" is meant a protease that is naturally expressed in an organism (e.g., a mammal) and secreted or exposed outside the cell (e.g., in the blood or on the surface of the skin).

[0336] As used herein, the term "cleavage" refers to the cleavage of covalent bonds, such as in the backbone of a nucleic acid molecule, or the hydrolysis of peptide bonds. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible. Double-strand breaks can result from two different single-strand break events.

[0337] Additional terms are described below.

[0338] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0339] The term "about," when referring to a measurable value, such as an amount, duration over time, and the like, is meant to encompass a ±20% variation, or in some cases a ±10% variation, or in some cases a ±5% variation, or in some cases a ±1% variation, or in some cases a ±0.1% variation from the specified value, as such variations are appropriate for performing the disclosed methods.

[0340] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0341] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (either VL or VH) such as sdAbs, camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments may also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0342] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, which usually determines the class to which the antibody belongs.

[0343] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0344] The term "antigen", "Ag" or "antigenic molecule" refers to a molecule that elicits an immune response. The immune response requires either or both of antibody production or activation of specific immunologically competent cells. In some embodiments, an antigen is any macromolecule, including whole proteins or peptides. In other embodiments, the antigen is derived from recombinant or genomic DNA. Thus, any DNA that contains a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen" as that term is used herein.

[0345] An antigen need not be encoded solely by the full-length nucleotide sequence of a gene. In embodiments, antigens include, but are not limited to, the use of partial nucleotide sequences of two or more genes and these nucleotide sequences arranged in various combinations to encode a polypeptide that elicits a desired immune response. In certain embodiments, antigens need not be encoded by a "gene". In one embodiment, antigens can be synthetically produced or derived from a biological sample, or can be macromolecules other than polypeptides. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or fluids with other biological components. In embodiments, antigens include, for example, carbohydrates (e.g., monosaccharides, disaccharides, oligosaccharides, and polysaccharides).

[0346] The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0347] The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that when present in an immune effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and the generation of an intracellular signal. In some embodiments, the CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and an intracytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. In some embodiments, the set of polypeptides is in the same polypeptide chain (e.g., comprises a chimeric fusion protein). In some embodiments, the set of polypeptides is not contiguous with each other, e.g., in different polypeptide chains. In some embodiments, the set of polypeptides is not contiguous with each other, e.g., in different polypeptide chains. In some embodiments, the set of polypeptides comprises a dimerization switch that can couple the fusion polypeptides to each other in the presence of a dimerization molecule, e.g., can couple the antigen binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains from at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule of the CAR is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain from a costimulatory molecule and a functional signaling domain from a stimulatory molecule.In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains from one or more costimulatory molecules and a functional signaling domain from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains from one or more costimulatory molecules and a functional signaling domain from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, where the leader sequence is optionally cleaved from the antigen binding domain (e.g., scFv) during intracellular processing and localization of the CAR to the cell membrane.

[0348] The term "cancer" refers to a disease characterized by uncontrolled and abnormal cell proliferation. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous and malignant cancers and tumors.

[0349] "CAR molecule" refers to a CAR (e.g., a CAR polypeptide), a nucleic acid encoding a CAR, or both, depending on the context.

[0350] A CAR that comprises an antigen-binding domain (e.g., scFv or TCR) that targets a particular tumor antigen X, such as those described herein, is also referred to as an XCAR. For example, a CAR that comprises an antigen-binding domain that targets CD19 or BCMA is referred to as a CD19CAR or BCMACAR, respectively.

[0351] As used herein, the term "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17, BCM or CD269) is a member of the tumor necrosis receptor (TNFR) family and is expressed primarily on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligands are called B cell activating factor of the TNF family (BAFF) and proliferation-inducing ligand (APRIL). BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. The gene for BCMA is encoded on chromosome 16 and produces a 994 nucleotide-long primary mRNA transcript (NCBI catalog NM_001192.2) that encodes a 184 amino acid protein (...

Claims

1. (i) a CRBN-binding polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3; and (ii) a chimeric antigen receptor (CAR) comprising, in the N-terminal to C-terminal direction, an antigen-binding domain, a transmembrane domain, and one or more intracellular signaling domains. A fusion polypeptide comprising:

2. The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide comprises the amino acid sequence of SEQ ID NO:

40.

3. The fusion polypeptide of claim 1 or 2, wherein the CRBN-binding polypeptide comprises the amino acid sequence of SEQ ID NO:

11.

4. 4. The CRBN-binding polypeptide comprising: (a) a first sequence that comprises the amino acid sequence of SEQ ID NO:5 or that differs from SEQ ID NO:5 by 1, 2, 3, or no more than 4 amino acid residues; and (b) a second sequence comprising the amino acid sequence of SEQ ID NO:11; The fusion polypeptide of any one of claims 1 to 3, comprising:

5. The fusion polypeptide of any of claims 1-4, wherein the CRBN-binding polypeptide and the CA are linked by a peptide bond.

6. The fusion polypeptide of any of claims 1-4, wherein the CAR is directly linked to the CRBN-binding polypeptide.

7. The fusion polypeptide of any of claims 1 to 4, wherein the CRBN-binding polypeptide and the CAR are operably linked via a linker.

8. The fusion polypeptide of claim 7, wherein the linker is a glycine-serine linker or comprises the amino acid sequence of SEQ ID NO:

28.

9. The fusion polypeptide of any of claims 1-8, wherein the CRBN-binding polypeptide is linked to the C-terminus or N-terminus of the CAR.

10. The fusion polypeptide of any one of claims 1 to 9, wherein degradation of the fusion polypeptide in the absence of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof is 70% or less of degradation of the fusion polypeptide in the presence of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof.

11. The fusion polypeptide of any one of claims 1 to 10, wherein the antigen-binding domain is an scFv.

12. The antigen-binding domain is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family members, B cell maturation antigen, Tn antigen (Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2; mesothelin; interleukin IL-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor ( EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor mutated 2 (ELF2M); ephrinB2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta, 9 (LMP2); glycoprotein 100 (gp100); breakpoint cluster region (BCR) and A oncogene polypeptide consisting of Belson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial cell marker 1 (TEM1 / CD248);Tumor endothelial cell marker 7-related (TEM7R); Claudin 6 (CLDN6); Thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); Chromosome X open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta specific 1 (PLAC1); Hexasaccharide moiety of globoH glycoceramide (GloboH); Mammary differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenergic receptor beta 3 (ADRB3 ); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survival; telomerase; prostate carcinoma tumor antigen-1, melanoma antigen 1 recognized by T cells; rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma metastasis breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG ( Transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1);CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2 (mut 12. The fusion polypeptide of any of claims 1 to 11, which binds to an antigen selected from the group consisting of hsp70-2; CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

13. (a) the one or more intracellular signaling domains comprise one or more primary signaling domains; or (b) the one or more intracellular signaling domains comprise one or more costimulatory signaling domains. A fusion polypeptide according to any one of claims 1 to 12.

14. (a) one of the one or more primary signaling domains comprises a CD3-zeta stimulatory domain; or (b) one of the one or more costimulatory signaling domains is an intracellular domain from a costimulatory protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds CD83; The fusion polypeptide of claim 13.

15. The fusion polypeptide of any of claims 1-14, wherein the fusion polypeptide further comprises a degradation domain, said degradation domain separated from the CRBN binding polypeptide and the CAR by a heterologous protease cleavage site.

16. 16. The fusion polypeptide of claim 15, wherein the degradation domain has a first state associated with a first level of expression of the fusion polypeptide and a second state associated with a second level of expression of the fusion polypeptide, the second level being increased from the first level in the presence of a stabilizing compound.

17. The fusion polypeptide of claim 16, wherein the second level is increased at least two-fold over the first level in the presence of a stabilizing compound.

18. The fusion polypeptide of any one of claims 15 to 17, wherein the degradation domain is selected from an estrogen receptor (ER) domain, an FKBP domain, or a dihydrofolate reductase (DHFR) domain.

19. (a) the degradation is an ER domain and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 46 or 48; (b) the cleavage domain is an FKBP domain and comprises an amino acid sequence at least 90% identical to SEQ ID NO: 50; or (c) the degradation domain is a DHFR domain and comprises an amino acid sequence at least 90% identical to SEQ ID NO:51; The fusion polypeptide of claim 18.

20. The stabilizing compound comprises: (a) when the degradation domain is an ER domain, it is bazedoxifene or 4-hydroxytamoxifen (4-OHT) or a pharma- ceutically acceptable salt thereof; (b) when the degradation domain is an FKBP domain, it is Shield-1 or a pharma- ceutically acceptable salt thereof; or (c) when the degradation domain is a DHFR domain, it is trimethoprim or a pharma- ceutically acceptable salt thereof; A fusion polypeptide according to claim 18 or 19.

21. The fusion polypeptide of any of claims 15 to 20, wherein the heterologous protease cleavage site is cleaved by a mammalian intracellular protease.

22. 22. The fusion polypeptide of any of claims 15 to 21, wherein the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, granzyme B, factor XA, enterokinase, genenase, sortase, prescission protease, thrombin, TEV protease and elastase 1.

23. The heterologous protease cleavage site may be selected from the group consisting of the Arg-X-Lys / Arg-Arg consensus motif (wherein X can be any amino acid; SEQ ID NO: 52), the Arg-X-X-X-Lys / Arg-Arg consensus motif (wherein X can be any amino acid; SEQ ID NO: 53), the Arg-Arg-X consensus motif (SEQ ID NO: 54), the Ile-Glu-Pro-Asp-X consensus motif (SEQ ID NO: 55), the Ile-Glu / Asp-Gly-Arg consensus motif (SEQ ID NO: 56), the Asp-Asp-Asp-Asp-Lys consensus motif (SEQ ID NO: 57), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 58), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 59), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 60), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 61), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 62), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 63), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 64), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 65), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 66), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 67), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 68), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 69), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 70), the Pro-Gly-Ala-Al consensus motif (SEQ ID NO: 71), the Pro-Gly-Ala- 23. The fusion polypeptide of any of claims 15 to 22, comprising a sequence comprising a cleavage motif selected from the group consisting of a-His-Tyr (SEQ ID NO:58), Leu-Pro-X-Thr-Gly / Ala consensus motif (SEQ ID NO:59), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO:60), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:61), Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO:62) and [Ala / Gly / Ser / Val]-X (wherein X can be any amino acid; SEQ ID NO:63).

24. The fusion polypeptide of any one of claims 15 to 23, wherein the heterologous protease cleavage site is cleaved by furin.

25. The fusion polypeptide of claim 24, wherein the heterologous protease cleavage site comprises a furin cleavage site selected from the group consisting of RTKR (SEQ ID NO: 123); GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125); GTGAEDPRPSRKRR (SEQ ID NO: 127); LQWLEQQVAKRRTKR (SEQ ID NO: 129); GTGAEDPRPSRKRRSLGG (SEQ ID NO: 131); GTGAEDPRPSRKRRSLG (SEQ ID NO: 133); SLNLTESHNSRKKR (SEQ ID NO: 135); CKINGYPKRGRKRR (SEQ ID NO: 137); and SARNRQKR (SEQ ID NO: 34).

26. The fusion polypeptide of any of claims 15 to 20, wherein the heterologous protease cleavage site is cleaved by a mammalian extracellular protease.

27. 27. The fusion polypeptide of claim 26, wherein the mammalian extracellular protease is selected from the group consisting of factor XA, enterokinase, genenase, sortase, prescission protease, thrombin, TEV protease and elastase 1.

28. Heterologous protease cleavage sites include Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 56), Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 57), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 58), Leu-Pro-X-Thr-Gly / Ala consensus motif (SEQ ID NO: 59), Leu-Glu-Val-Phe-Gln-Gly-P 28. The fusion polypeptide of claim 26 or 27, comprising an amino acid sequence selected from the group consisting of: Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:61), Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO:62) and [Ala / Gly / Ser / Val]-X (wherein X can be any amino acid; SEQ ID NO:63).

29. A nucleic acid molecule encoding the fusion polypeptide of any one of claims 1 to 28.

30. A vector comprising the nucleic acid molecule of claim 29.

31. A viral particle comprising the vector of claim 30.

32. A cell comprising the fusion polypeptide of any one of claims 1 to 28, the nucleic acid molecule of claim 29, the vector of claim 30 or the viral particle of claim 31.

33. 33. The cell of claim 32, wherein the cell is a T cell or a NK cell.

34. The cell of claim 32 or 33, wherein the cell comprises a fusion polypeptide of any of claims 1 to 14 or the cell comprises a nucleic acid molecule encoding the fusion polypeptide of any of claims 1 to 14, and when the cell is contacted with lenalidomide or pomalidomide or a pharmaceutically acceptable salt thereof, degradation of the fusion polypeptide is increased compared to degradation of the fusion polypeptide when the cell is not contacted with lenalidomide or pomalidomide or a pharmaceutically acceptable salt thereof.

35. The cell of claim 32 or 33, wherein the cell comprises a fusion polypeptide of any of claims 15 to 28 or the cell comprises a nucleic acid molecule encoding a fusion polypeptide of any of claims 15 to 28, and wherein in the absence of a stabilizing compound, the fusion polypeptide is degraded by a degradative pathway of the cell.

36. 36. The cell of claim 35, wherein the cell further comprises a protease capable of cleaving the heterologous protease cleavage site.

37. 37. The cell of claim 35 or 36, wherein the cell further comprises a stabilizing compound.

38. The cell of claim 37, wherein when the cell is contacted with both the stabilizing compound and lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, degradation of the fusion polypeptide is increased compared to degradation of the fusion polypeptide when the cell is contacted with only the stabilizing compound and not with lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

39. A pharmaceutical composition comprising a fusion polypeptide of any of claims 1-28 or a cell of any of claims 32-38 and a pharma- ceutically acceptable carrier, excipient or stabilizer.

40. 15. An ex vivo method for degrading a fusion polypeptide, comprising contacting the fusion polypeptide of any one of claims 1 to 14 or a cell containing said fusion polypeptide with lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof.

41. The method of claim 40, wherein in the presence of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, the expression level of the fusion polypeptide is decreased compared to the expression level of the fusion polypeptide in the absence of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof.

42. 1. An ex vivo method for controlling expression of a fusion polypeptide, comprising: (i) contacting a fusion polypeptide of any one of claims 15 to 28 or a cell containing the fusion polypeptide with a stabilizing compound, wherein the expression level of the fusion polypeptide in the presence of the stabilizing compound is increased compared to the expression level of the fusion polypeptide in the absence of the stabilizing compound. method.

43. The method further comprises after step (i): (ii) contacting a fusion polypeptide or a cell comprising said fusion polypeptide with lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, wherein in the presence of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, the expression level of the fusion polypeptide is reduced after step (i) compared to the expression level of the fusion polypeptide before step (ii); 43. The method of claim 42.

44. 1. A method of producing a cell, comprising: (i) the cell is a cell of any of claims 32 to 38, the method comprising providing to the cell a nucleic acid molecule of claim 29, a vector of claim 30 or a viral particle of claim 31; or (ii) a method comprising providing a cell comprising a nucleic acid molecule encoding a fusion polypeptide of any one of claims 1 to 28, and contacting the cell ex vivo with lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof. method.

45. A pharmaceutical composition for treating a disease associated with expression of a tumor antigen in a subject, comprising a cell comprising the fusion polypeptide of any one of claims 1 to 14.

46. The pharmaceutical composition of claim 45, wherein the cells are contacted with lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof ex vivo prior to administration, and in the presence of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, the expression level of the fusion polypeptide is reduced compared to the expression level of the fusion polypeptide before contacting the cells with lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof.

47. 46. ​​The pharmaceutical composition of claim 45, wherein the cells are contacted with lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof ex vivo prior to administration, and wherein the amount of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof in contact with the cells is reduced after contacting the cells with lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof and before administering the cells to a subject.

48. The pharmaceutical composition of any of claims 45 to 47, characterized in that after administration of the pharmaceutical composition, an effective amount of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof is administered, wherein the administration of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof reduces the expression level of the fusion polypeptide compared to the expression level of the fusion polypeptide after administration of the cells and before administration of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof.

49. 49. The pharmaceutical composition of claim 48, wherein administration of lenalidomide or pomalidomide, or a pharma- ceutically acceptable salt thereof, reduces or prevents adverse effects.

50. 50. The pharmaceutical composition of claim 48 or 49, characterized in that after administration of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof, administration of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof is discontinued.

51. 51. The pharmaceutical composition of any of claims 45 to 50, wherein said cells are autologous to said subject.

52. 51. The pharmaceutical composition of any of claims 45 to 50, wherein said cells are allogeneic to said subject.

53. 50. The pharmaceutical composition of claim 48 or 49, wherein the subject is administered 2.5 mg, 5 mg, 10 mg, 15 mg or 25 mg of lenalidomide or a pharma- ceutically acceptable salt thereof per day.

54. 30. A pharmaceutical composition comprising cells comprising the fusion polypeptide of any one of claims 15 to 28 for the treatment of a disease associated with expression of a tumor antigen in a subject, comprising: (i) administering to the subject an effective amount of the cells in combination with a stabilizing compound, thereby treating the disease.

55. Treatment further: (a) after step (i), comprising a step (ii) of discontinuing administration of the stabilized compound, wherein the discontinuation of administration of the stabilized compound is in response to the subject's response to the treatment of step (i); (b) after step (i), comprising a step (iii) of discontinuing administration of the stabilized compound, wherein administration of the stabilized compound reduces or prevents adverse effects; or (c) after step (i), discontinuing administration of the stabilized compound and administering to the subject an effective amount of lenalidomide or pomalidomide, or a pharma- ceutically acceptable salt thereof, step (iv), which reduces or prevents adverse effects or acute toxicity.

55. The pharmaceutical composition of claim 54.

56. 56. The pharmaceutical composition of claim 55, wherein the treatment further comprises the step (v) of discontinuing administration of lenalidomide or pomalidomide or a pharma- ceutically acceptable salt thereof after step (iv).

57. 56. The pharmaceutical composition of claim 55, wherein lenalidomide or a pharma- ceutically acceptable salt thereof is administered in an amount of 2.5 mg, 5 mg, 10 mg, 15 mg or 25 mg per day.

58. The treatment further comprises, after step (ii), (iii), (iv) or (v), a step (vi) of administering an effective amount of a stabilizing compound: (I) administration of the stabilized compound treats or prevents tumor recurrence; or (II) the treatment further comprises, after step (vi), repeating step (iii), (iv), (v) or (vi), thereby treating the disease; 58. A pharmaceutical composition according to claim 56 or 57.

59. 59. The pharmaceutical composition of any of claims 54 to 58, wherein the treatment further comprises the step (viii) of contacting the cells with a stabilizing compound ex vivo, prior to step (i).

60. 59. The pharmaceutical composition of any of claims 54 to 58, wherein the cells are not contacted with the stabilizing compound or lenalidomide or pomalidomide, or a pharma- ceutically acceptable salt thereof, ex vivo prior to administration.

61. The stabilizing compound is: (a) when the degradation domain is an ER domain, it is bazedoxifene or 4-hydroxytamoxifen (4-OHT) or a pharma- ceutically acceptable salt thereof; (b) when the degradation domain is an FKBP domain, it is Shield-1 or a pharma- ceutically acceptable salt thereof; or (c) when the degradation domain is a DHFR domain, it is trimethoprim or a pharma- ceutically acceptable salt thereof; 59. The pharmaceutical composition of any one of claims 54 to 58.

62. A pharmaceutical composition comprising the fusion polypeptide of any of claims 1 to 28, the nucleic acid molecule of claim 29, the vector of claim 30, the viral particle of claim 31 or the cell of any of claims 32 to 38 for use in treating a subject having a disease associated with expression of a tumor antigen.

63. The pharmaceutical composition according to any one of claims 45 to 62, wherein the disease associated with expression of a tumor antigen is cancer.

64. (a) the cancer is mesothelioma, lung cancer, pancreatic cancer, esophageal adenocarcinoma, ovarian cancer, breast cancer, colorectal cancer, bladder cancer, or any combination thereof; or (b) the cancer is a blood cancer; 64. The pharmaceutical composition of claim 63.

65. (i) the hematological cancer is selected from leukemia or lymphoma; (ii) the mesothelioma is malignant pleural mesothelioma or mesothelioma in a subject that has progressed on at least one prior standard of care; (iii) the lung cancer is non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer or large cell lung cancer; (iv) the pancreatic cancer is pancreatic ductal adenocarcinoma or metastatic pancreatic ductal adenocarcinoma (PDA) or pancreatic cancer in a subject that has progressed on at least one prior standard of care; or (v) the ovarian cancer is serous epithelial ovarian cancer or ovarian cancer in a subject that has progressed after at least one prior standard of care regimen; 65. The pharmaceutical composition of claim 64.

66. Cancers include chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B lymphoma, and pulmonary leukemia. DLBCL, DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (mucosa-associated lymphoid tissue type extranodal marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndromes, non-Hodgkin's lymphoma , plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, nodal marginal zone lymphoma, childhood nodal marginal zone lymphoma, primary cutaneous 64. The pharmaceutical composition of claim 63, wherein the tumor is selected from follicular center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML) or unclassifiable lymphoma.